Isabel González-Mariscal

Resveratrol Prevents β-Cell Dedifferentiation in Nonhuman Primates Given a High-Fat/High-Sugar Diet

Eating a “Westernized” diet high in fat and sugar leads to weight gain and numerous health problems, including the development of type 2 diabetes mellitus (T2DM). Rodent studies have shown that resveratrol supplementation reduces blood glucose levels, preserves β-cells in islets of Langerhans, and improves insulin action. Although rodent models are helpful for understanding β-cell biology and certain aspects of T2DM pathology, they fail to reproduce the complexity of the human disease as well as that of nonhuman primates. Rhesus monkeys were fed a standard diet (SD), or a high-fat/high-sugar diet in combination with either placebo (HFS) or resveratrol (HFS+Resv) for 24 months, and pancreata were examined before overt dysglycemia occurred. Increased glucose-stimulated insulin secretion and insulin resistance occurred in both HFS and HFS+Resv diets compared with SD. Although islet size was unaffected, there was a significant decrease in β-cells and an increase in α-cells containing glucagon and glucagon-like peptide 1 with HFS diets. Islets from HFS+Resv monkeys were morphologically similar to SD. HFS diets also resulted in decreased expression of essential β-cell transcription factors forkhead box O1 (FOXO1), NKX6–1, NKX2–2, and PDX1, which did not occur with resveratrol supplementation. Similar changes were observed in human islets where the effects of resveratrol were mediated through Sirtuin 1. These findings have implications for the management of humans with insulin resistance, prediabetes, and diabetes.

Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism

Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism.

The Biarylpyrazole Compound AM251 Alters Mitochondrial Physiology via Proteolytic Degradation of ERRα

The orphan nuclear receptor estrogen-related receptor alpha (ERRα) directs the transcription of nuclear genes involved in energy homeostasis control and the regulation of mitochondrial mass and function. A crucial role for controlling ERRα-mediated target gene expression has been ascribed to the biarylpyrazole compound 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251) through direct binding to and destabilization of ERRα protein. Here, we provide evidence that structurally related AM251 analogs also have negative impacts on ERRα protein levels in a cell-type-dependent manner while having no deleterious actions on ERRγ. We show that these off-target cellular effects of AM251 are mediated by proteasomal degradation of nuclear ERRα. Cell treatment with the nuclear export inhibitor leptomycin B did not prevent AM251-induced destabilization of ERRα protein, whereas proteasome inhibition with MG132 stabilized and maintained its DNA-binding function, indicative of ERRα being a target of nuclear proteasomal complexes. NativePAGE analysis revealed that ERRα formed a ∼220-kDa multiprotein nuclear complex that was devoid of ERRγ and the coregulator peroxisome proliferator-activated receptor γ coactivator-1. AM251 induced SUMO-2,3 incorporation in ERRα in conjunction with increased protein kinase C activity, whose activation by phorbol ester also promoted ERRα protein loss. Down-regulation of ERRα by AM251 or small interfering RNA led to increased mitochondria biogenesis while negatively impacting mitochondrial membrane potential. These results reveal a novel molecular mechanism by which AM251 and related compounds alter mitochondrial physiology through destabilization of ERRα.

Incretin secretion in humans is under the influence of cannabinoid receptors

The mechanisms regulating incretin secretion are not fully known. Human obesity is associated with altered incretin secretion and elevated endocannabinoid levels. Since cannabinoid receptors (CBRs) are expressed on incretin-secreting cells in rodents, we hypothesized that endocannabinoids are involved in the regulation of incretin secretion. We compared plasma glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) responses during oral glucose tolerance test (OGTT) in 20 lean and 20 obese participants from the Baltimore Longitudinal Study of Aging (BLSA). Next, we recruited 20 healthy men to evaluate GIP and GLP-1 responses during OGTT after administering placebo or nabilone (CBR agonist) in a randomized, double-blind, crossover fashion. Compared with the BLSA lean group, the BLSA obese group had significantly higher fasting and post-OGTT GIP levels, but similar fasting GLP-1 and significantly lower post-OGTT GLP-1 levels. In the nabilone vs. placebo study, when compared with placebo, nabilone resulted in significantly elevated post-dose fasting GIP levels and post-OGTT GIP levels, but no change in post-dose fasting GLP-1 levels together with significantly lower post-OGTT GLP-1 levels. Glucose levels were not different with both interventions. We conclude that elevated GIP levels in obesity are likely a consequence of increased endocannabinoid levels. CBRs exert tonic control over GIP secretion, which may have a homeostatic effect in suppressing GLP-1 secretion. This raises the possibility that gut hormones are influenced by endocannabinoids.

Targeted proteomics of cannabinoid receptor CB1 and the CB1b isoform

HIGHLIGHTSSRM‐MS method for the identification and differentiation of CB1 and CB1b.Application of the method to CHO cells transduced with CB1 or CB1b. ABSTRACT Cannabinoid receptors (CBR), including CB1 and CB2 have been therapeutic targets for a number of conditions. Recently, splice variants of the CB1R have been identified in humans. The isoforms differ in their N‐terminus sequence and pharmacological activity relative to the CB1R, as a result, the differentiation between the CB1 receptor and its isoform is required. As a result, a selected reaction monitoring mass spectrometry (SRM‐MS) method was developed for the quantitation of CB1 and the CB1b isoform in CHO cells transduced with CB1 and CB1b. The SRM‐MS protocol was assessed with isotopically labeled peptide standards and had high reproducibility of intra‐day assay (CVs from 1.9 to 4.3% for CB1 and 0.5 to 5.9% for CB1b) and inter‐day assay (CVs from 1.2 to 5.2% for CB1 and 1.2 to 6.1% for CB1b).

Endocannabinoids in Islets of Langerhans: The Ugly, the Bad and the Good Facts

The endocannabinoid system (ECS) regulates cellular homeostasis and whole-body metabolism. There is an autonomous ECS in the endocrine pancreas, including the cannabinoid 1 receptor (CB1R) that is present in β-cells. Here, we discuss conflicts that have arisen with regard to the function(s) of the ECs in the endocrine pancreas and that have caused confusion when defining the role of the ECS in islets of Langerhans, especially the role(s) of CB1R in β-cells. We also discuss the latest data published concerning the ECS in islets. CB1R in particular is not simply a negative modulator of insulin secretion as it is also involved in intra-islet inflammation during high fat-high sugar intake and it is a negative regulator of β-cell viability and turnover. We also discuss the feasibility of using CB1R as a target for the treatment of diabetes.

Identification of novel mouse and rat CB1R isoforms and in silico modeling of human CB1R for peripheral cannabinoid therapeutics

Targeting peripheral CB1R is desirable for the treatment of metabolic syndromes without adverse neuropsychiatric effects. We previously reported a human hCB1b isoform that is selectively enriched in pancreatic beta-cells and hepatocytes, providing a potential peripheral therapeutic hCB1R target. It is unknown whether there are peripherally enriched mouse and rat CB1R (mCB1 and rCB1, respectively) isoforms. In this study, we found no evidence of peripherally enriched rodent CB1 isoforms; however, some mCB1R isoforms are absent in peripheral tissues. We show that the mouse Cnr1 gene contains six exons that are transcribed from a single promoter. We found that mCB1A is a spliced variant of extended exon 1 and protein-coding exon 6; mCB1B is a novel spliced variant containing unspliced exon 1, intron 1, and exon 2, which is then spliced to exon 6; and mCB1C is a spliced variant including all 6 exons. Using RNAscope in situ hybridization, we show that the isoforms mCB1A and mCB1B are expressed at a cellular level and colocalized in GABAergic neurons in the hippocampus and cortex. RT-qPCR reveals that mCB1A and mCB1B are enriched in the brain, while mCB1B is not expressed in the pancreas or the liver. Rat rCB1R isoforms are differentially expressed in primary cultured neurons, astrocytes, and microglia. We also investigated modulation of Cnr1 expression by insulin in vivo and carried out in silico modeling of CB1R with JD5037, a peripherally restricted CB1R inverse agonist, using the published crystal structure of hCB1R. The results provide models for future CB1R peripheral targeting.

Abstract 5516: (R,R’)-4’-methoxy-1-naphthylfenoterol Inhibits GPR55 signaling and the modulation of motility in human cancer cells.

(R,R’)-4’-methoxy-1-naphthylfenoterol (MNF) promotes growth inhibition and apoptosis of human HepG2 hepatocarcinoma cells via cannabinoid receptor (CBR) activation. The synthetic CB1R inverse agonist, AM251, has been shown to block the anti-mitogenic effect of MNF in these cells; however, AM251 is also an agonist of the recently deorphanized, lipid-sensing receptor, GPR55, whose upregulation contributes to carcinogenesis. Here, we investigated the role of GPR55 in MNF signaling in human HepG2 and PANC-1 cancer cell lines in culture, with a focus on internalization of the fluorescent ligand Tocrifluor 1117 (T1117), reorganization of actin cytoskeleton, and cell motility as measured by scratch assay. Control experiments indicated that GPR55 knockdown by RNA interference markedly reduced cellular uptake of T1117, a process that was sensitive to MNF inhibition. GPR55 internalization mediated by the atypical cannabinoid O-1602 was blocked by MNF in GPR55-expressing HEK293 cells. Pretreatment of HepG2 and PANC-1 cells with MNF significantly abrogated the induction of ERK phosphorylation in response to AM251, O-1602 and fetal bovine serum, known to contain bioactive lipids. Moreover, MNF exerted a coordinated regulation of AM251 and O-1602 inducible processes, including cell motility. This study shows for the first time that MNF impairs GPR55-mediated signaling and, therefore, may have therapeutic potential for the management of cancer. This work was supported entirely by the Intramural Research Program of the NIA, NIH. Citation Format: Rajib K. Paul, Artur Wnorowski, Isabel Gonzalez-Mariscal, Ruin Moaddel, Fred E. Indig, Irving W. Wainer, Michel Bernier. (R,R’)-4’-methoxy-1-naphthylfenoterol Inhibits GPR55 signaling and the modulation of motility in human cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5516. doi:10.1158/1538-7445.AM2013-5516