Knockout studies in mice demonstrated that deletion of the CB1R prevented diet-induced obesity, thereby making this GPCR-linked receptor an attractive target for novel therapeutic approaches. CB1R inverse agonists, such as ibipinabant, AM251 and the clinical analog rimonabant, produce potent anorectic effects in animals. However, these compounds also exert off-target effects independent of the CB1R through unknown mechanisms. Here, we demonstrate that all CB1R inverse agonists tested (e.g., AM251, rimonabant and ibipinabant) induced ERRαdegradation in three different CB1R-negative cultured cell lines. Previous studies have shown that the synthetic nonsteroidal estrogen, diethylstilbestrol, antagonizes ERRαsignaling through its interaction with the ligand-binding domain of ERRα. We therefore hypothesized that diethylstilbestrol and the classical ERRαinverse agonist, XCT790, might have AM251-mimicking actions, whereas selective ERRαagonists might block AM251 signaling. All of these predictions were found to be correct, hence confirming the importance of ERRαdestabilization in the cellular actions of AM251. Additional experiments demonstrated that AM251 promoted the proteolytic degradation of ERRαprotein, with a half-life of 7 hours, without loss of the corresponding mRNA. Moreover, cell pretreatment with the proteasome inhibitor, MG132, blocked AM251-induced ERRαdegradation and resulted in nuclear accumulation of the orphan nuclear receptor, thereby confirming that AM251 induces ERRαprotein degradation. In order to further elucidate the mechanism for such degradation, work is currently underway to identify the putative posttranslational modification(s) responsible for the destabilization of ERRαand subsequent alterations in target gene expression in response to AM251 and related compounds. Previous studies have demonstrated that ERRαprotein is a target for posttranslational modifications such as phosphorylation, acetylation and sumoylation, with such modifications altering subsequent transcriptional activity. These posttranslational modifications are also likely to alter the recruitement of nuclear co-regulatory molecules (e.g., PGC-1αand RIP140) and other DNA-binding proteins to ERRαat the target gene promoter region. Therefore, DNA pull-down assays are being carried out to measure ERRα-DNA binding in solution in the absence and presence of AM251, and mapping the interacting multiprotein complexes by affinity purification and mass spectrometry in a panel of cell culture models treated with CB1R inverse agonists. A similar approach will be used in tissues (e.g., liver, skeletal muscle, adipocytes) of animal models that received peripherally active CB1R inverse agonists. A second aspect of the project focuses on the role of the ERRα/PGC-1αcomplex is regulating energy metabolism and mitochondrial function. We hypothesize that AM251 and related CB1R inverse agonists destabilize this critical complex and thereby may alter mitochondrial biogenesis and respiration. Our preliminary in vitro experiments indicate a decrease in mitochondrial activity in response to AM251. Using confocal microscopy, AM251 treatment decreased ERRαstaining with a corresponding reduction in MitoTracker Red CMXRos staining, suggesting a loss of mitochondrial membrane potential. These findings are supported by initial flow cytometry results demonstrating an increase in mitochondrial membrane depolarization via TMRM staining in AM251-treated cells. Additionally, all CB1R inverse agonists tested decrease nonyl acridine orange staining, suggesting alterations in total mitochondrial mass by these compounds. Studies are currently underway using DNA pull-down assays to determine if such mitochondrial alterations are indeed dependent on the destabilization of the ERRα/PGC-1αcomplex. These changes in mitochondrial biogenesis and membrane potential are particularly interesting since mice deficient in ERRαexhibit defects in fatty acid metabolism and are resistant to diet-induced obesity. In collaboration with Rafael deCabo and Julie Mattison, we performed a global gene expression profiling of middle-aged male Rhesus monkeys fed a high fat-high sugar (HFS) diet for 2 years in the absence or presence of resveratrol, a plant molecule known to promote longevity and improves mitochondrial function. The results showed significant increase in ERRαgene expression in skeletal muscle of HFS-fed animals as compared to controls, but not in liver or adipose tissue. Moreover, resveratrol blocked the HFS-inducible increase in ERRαexpression while counteracting the depletion in PGC-1αmRNA levels by HFS. Subsequent Western blot analyses confirmed these tissue-specific changes, with ERRαprotein expression increasing in skeletal muscle and, yet, decreasing in the liver as a result of the HSF diet. Interestingly, resveratrol supplementation also had tissue-specific effects that restored values to that of control animals. Additional studies in rats have further demonstrated the ability of both diet and age to influence ERRαand PGC-1αexpression in skeletal muscle. Specifically, while muscle PGC-1αexpression decreases with age, both ERRαand PGC-1αare increased as a result of caloric restriction. It is therefore reasonable to conclude that pharmacological interventions aimed at targeting ERRαstability and/or function may prove useful in fighting metabolic diseases and improving redox status, ultimately extending not only lifespan, but healthspan as well.
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