CPT1C and endocannabinoids as hypothalamic players in early stages of obesity development

Abstract

Obesity prevalence in worldwide population is increasing every day due to an imbalance between the energy intake and the energy expenditure. Rich-caloric food is highly palatable and easily accessible, whereas sedentary lifestyle is the majority. This situation makes difficult to maintain energy homeostasis and leads to obesity development. Nowadays we failure to have a treatment for obesity due to a lack of acknowledge in the molecular mechanisms underlying obesity development. Lately, the activation of brown adipose tissue (BAT) thermogenesis into obese patients have emerged as a promising treatment. BAT thermogenesis is controlled from the central nervous system (CNS) upon peripheral signals that are integrated in different nuclei of the hypothalamus. The hypothalamus is the region sensing energy surplus (fats or carbohydrates intake) and consequently sending the information through the sympathetic nervous system (SNS) to activate BAT thermogenesis. Within the hypothalamic nuclei, I would like to highlight the ventromedial nucleus of the hypothalamus (VMH) as an important regulator of BAT thermogenesis (Contreras C, 2017). However, the exact mechanism by which VMH is sensing the peripheral energy intake and controlling BAT activation is not known. Here, we hypothesized that the brain isoform of the carnitine palmitoyltransferase 1 (CPT1s), the CPT1C, could be a crucial factor in the VMH controlling BAT thermogenesis. CPT1C awaked our interest since it was discovered in 2002 by Dr. Zammit group. CPT1C is an intriguing protein since have no catalytic activity and is mainly localized in neurons. Several studies have demonstrate that CPT1C is involved in the regulation of obesity. CPT1C-Knock out (KO) animals are more susceptible to diet-induced obesity (DIO) (Wolfgang MJ, 2006 and 2008) and have altered peripheral glucose and lipid metabolism (Gao XF, 2009). It has been suggested that CPT1C in the hypothalamus is sensing energy deficiency or surplus and consequently it regulates body weight and peripheral metabolism (Wolfgang MJ, 2006 and 2008). However, the molecular mechanism underlying CPT1C control of obesity in not known. Furthermore, CPT1C binds malonyl-CoA (Price NT, 2002). Malonyl-CoA is the physiological inhibitor of other CPT1s isoforms (CPT1A and CPT1B) and fluctuates within the hypothalamus depending on the energy disposition (Tokutake Y, 2010). In a situation of fasting, malonyl-CoA levels decreases whereas in a refeeding or HFD situation it increases in the hypothalamus (Tokutake Y, 2010). In fact, malonyl-CoA have been observed to be a crucial regulator of food intake and energy homeostasis in the hypothalamus (Wolfgang MJ and Lane MD, 2006 and 2011). We hypothesized that CPT1C acts as a malonyl-CoA sensor to proportionate information about the energy status to the cell (Casals N, 2016). Our results demonstrated that CPT1C-KO mice presented an attenuated BAT thermogenesis after 7 days of high-fat diet (HFD) feeding or leptin administration. Restoration of CPT1C expression in the VMH was sufficient to properly activate BAT thermogenesis. Moreover, malonyl-CoA sensing through CPT1C in the VMH was necessary for BAT activation. Malonyl-CoA levels are under the control of AMPK signalling and a correct AMPK signalling in the hypothalamus is necessary for sympathetic activation of BAT thermogenesis (Contreras C, 2017). We demonstrate that CPT1C is a downstream factor in the AMPK mediation of BAT thermogenesis in the VMH. Overall, we have demonstrated that CPT1C is necessary for BAT thermogenesis activation from the VMH after 7 days of HFD intake or acute leptin administration, probably acting downstream the AMPK(VMH)-ACC-Malonyl-CoA axis. The fact that CPT1C is not catalytically active made us hypothesised that exerts its functions by interacting with other proteins. Moreover, the ability of CPT1C to sense malonyl-CoA would proportionate a checkpoint to other proteins depending on the energetic status of the cell. In the glutamate receptors complex (AMPAR) (Brechet A, 2017), CPT1C interacts with ABHD6, an hydrolase of the main endocannabinoid (eCB), the 2-arachydonoyl glycerol (2-AG). This finding was of our interest since the endocannabinoid system (ECS) is involved in the regulation of energy homeostasis from the hypothalamus and is dysregulated in obesity (Ruiz de Azua and Lutz, 2019). Increased eCBs tone in the hypothalamus generally leads to an increase of body weight and to a decrease in energy expenditure. Since the molecular mechanism by which CPT1C controls BAT thermogenesis in the VMH is not known we hypothesized that CPT1C could regulate BAT thermogenesis activation through modifying the eCBs levels in the hypothalamus interacting with ABHD6. For this reason, we studied 2-AG and anandamide (AEA) levels in the hypothalamus of wild-type (WT) and CPT1C-KO mice fed a SD or HFD. We found that CPT1C-KO mice have increased both eCB in the hypothalamus in normal fed conditions compared to WT mice. After 7 days of HFD feeding eCB levels of WT and CPT1C-KO mice were increased compared to their respective SD fed animals. The eCBs phenotype of CPT1C-KO mice leads us to question whether the increase in hypothalamic eCBs levels was the cause of diminished BAT thermogenesis in CPT1C-KO mice and whether CPT1C was able to regulate the eCBs levels through ABHD6. However, the hypothalamic dynamic of eCBs in DIO model and the relation with BAT thermogenesis activation in early stages of obesity (short-term HFD) have been poorly studied, thus we were not able to interpret our results correctly. In addition, we realised that all the studies about hypothalamic eCBs in obesity were done in male but not in female rodents, so we decided to address this topic. At this point, we concurrently studied i) the eCBs dynamics in the hypothalamus during obesity development in the DIO model and whether they correlate with BAT thermogenesis and present sex dimorphism, and ii) whether CPT1C interacts and regulates ABHD6 activity depending on malonyl-CoA sensing. Firstly, concerning hypothalamic eCBs during DIO development, we observed that hypothalamic eCBs substantially increased after 7 days of HFD feeding and progressively decrease to basal levels until 90 days of feeding in both male and female mice. This increase of eCBs at 7 days was accompanied by a higher expression of eCBs synthesis enzymes whereas the expression of the degrading enzymes was not altered. The hypothalamic eCBs profile negatively correlated with body weight gain whereas positively correlated with BAT thermogenesis activation. It was at 7 days of HFD administration when both eCBs levels and BAT thermogenesis activation were the highest. At this time point, mice did not present an obese phenotype so we suggest that the transitory increase of hypothalamic eCBs and BAT thermogenesis counteracts obesity development. To further elucidate the crosstalk between hypothalamic eCB and BAT thermogenesis we acutely activated BAT thermogenesis by central leptin and peripheral β3-adenoreceptors agonist administration and both leaded to an increase of eCB levels in the hypothalamus. In contrast, acute central administration of 2-AG and AEA did not altered BAT thermogenesis. It is generally accepted that the ECS tone increases in the hypothalamus when obesity is stablished and this activates CB1R that inhibits SNS tone leading to a decrease in BAT thermogenesis (Quarta C, 2011). Here for the first time, we describe the opposite process, activation of BAT thermogenesis signals the hypothalamic ECS, overall being important in early stages of obesity. Secondly, regarding the study of CPT1C and ABHD6, we confirmed that CPT1C and ABHD6 were interacting by using two different approaches, the FRET assay and co-immunoprecipitation in HEK-293T obtaining the same results. Besides, this interaction was independent of CPT1C malonyl-CoA sensing. Next, we wondered whether CPT1C could modulate ABHD6 activity thus, we developed a new ABHD6 activity assay based on the 4-MUH fluorogenic substrate. We demonstrated that CPT1C acts as a negative regulator of ABHD6 activity and this phenomenon is dependent on CPT1C malonyl-CoA sensing. When CPT1C is sensing malonyl-CoA, ABHD6 activity decreases, whereas when CPT1C is unable to detect malonyl-CoA levels, ABHD6 activity is restored. These results suggest that CPT1C could act as a metabolic sensor and consequently regulate ABHD6 activity. Moreover, we studied whether CPT1C could modify the cannabinoid receptor 1 (CB1R) intracellular response (measuring the cAMP) via modulating 2-AG degradation by ABHD6. The results indicated that CPT1C is able to modulate 2-AG levels and the intracellular signalling through ABHD6. Finally, we consider whether CPT1C negative regulation of ABHD6 activity was the cause of dysregulated hypothalamic eCBs levels in CPT1C-KO mice and consequently impaired BAT thermogenesis. Moreover, both proteins have been described to play a role in metabolic flexibility in the VMH nuclei of the hypothalamus since disruption of both proteins in this nucleus diminished EE and enhanced DIO (Rodríguez-Rodríguez R, 2019 and Fisette A, 2016). However, our results evidenced that hypothalamic homogenates of WT animals presented less ABHD6 activity compared to CPT1C-KO homogenates, then a decrease in 2-AG hypothalamic levels were expected in CPT1C-KO mice. In contrast, CPT1C-KO mice have both 2-AG and AEA levels increased in the hypothalamus indicating that ABHD6 is not the cause of this eCBs dysregulation. Therefore, after all these results two question remains opened: i) Why CPt1C-KO mice have increased hypothalamic endocannabinoids levels? ii) Which function of ABHD6 is CPT1C regulating? These are two questions that we will try to answer in the near future. Altogether, this thesis has defined for the first time a link between CPT1C and the ECS. Both systems at the hypothalamus are crucial regulators of BAT thermogenesis and DIO. We have demonstrated that CPT1C in the VMH is a key sensor of energy status necessary for the regulation of BAT thermogenesis and body weight gain. On the other hand, we have described a crosstalk between BAT thermogenesis and endocannabinoids in the hypothalamus at early stages of DIO in male and female mice. Finally, we have characterized CPT1C as the first ABHD6 physiological inhibitor. Overall, our results have contributed to a better understanding of CPT1C functions and the molecular mechanisms underlying energy homeostasis.