CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release

Endocannabinoid signaling has been implicated in modulating insulin release from β cells of the endocrine pancreas. β Cells express CB₁ cannabinoid receptors (CB₁Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB₁R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB₁Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB₁Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes.     

Tumor associated macrophages and neutrophils in tumor progression

Tumor‐associated macrophages (TAMs) are a key component of the tumor microenvironment and orchestrate various aspects of cancer. Diversity and plasticity are hallmarks of cells of the monocyte–macrophage lineage. In response to distinct signals macrophages undergo M1 (classical) or M2 (alternative) activation, which represent extremes of a continuum in a spectrum of activation states. Metabolic adaptation is a key component of macrophage plasticity and polarization, instrumental to their function in homeostasis, immunity and inflammation. Generally, TAMs acquire an M2‐like phenotype that plays important roles in many aspects of tumor growth and progression. There is now evidence that also neutrophils can be driven towards distinct phenotypes in response to microenvironmental signals. The identification of mechanisms and molecules associated with macrophage and neutrophil plasticity and polarized activation provides a basis for new diagnostic and therapeutic strategies. J. Cell. Physiol. 228: 1404–1412, 2013. © 2012 Wiley Periodicals, Inc.     

Fluoro‐Sorafenib (Regorafenib) effects on hepatoma cells: Growth inhibition,quiescence, and recovery

To evaluate the growth‐inhibitory properties of the potent multi‐kinase antagonist Regorafenib (Fluoro‐Sorafenib), which was synthesized as a more potent Sorafenib, a Raf inhibitor and to determine whether similar mechanisms were involved, human hepatoma cell lines were grown in the presence or absence of Regorafanib and examined for growth inhibition. Western blots were performed for Raf targets, apoptosis, and autophagy. Regorafenib inhibited growth of human Hep3B, PLC/PRF/5, and HepG2 cells in a concentration‐ and time‐dependent manner. Multiple signaling pathways were altered, including MAP kinases phospho‐ERK and phospho‐JNK and its target phospho‐c‐Jun. There was evidence for apoptosis by FACS, cleavage of caspases and increased Bax levels; as well as induction of autophagy, as judged by increased Beclin‐1 and LC3 (II) levels. Prolonged drug exposure resulted in cell quiescence. Full growth recovery occurred after drug removal, unlike with doxorubicin chemotherapy. Regorafenib is a potent inhibitor of cell growth. Cells surviving Regorafenib treatment remain viable, but quiescent and capable of regrowth following drug removal. The reversibility of tumor cell growth suppression after drug removal may have clinical implications. J. Cell. Physiol. 228: 292–297, 2013. © 2012 Wiley Periodicals, Inc.     

Unraveling amyloid toxicity pathway in NIH3T3 cells by a combined proteomic and 1H‐NMR metabonomic approach

A range of debilitating human diseases is known to be associated with the formation of stable highly organized protein aggregates known as amyloid fibrils. The early prefibrillar aggregates behave as cytotoxic agents and their toxicity appears to result from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increase in free Ca²⁺ that lead to apoptotic or necrotic cell death. However, specific signaling pathways that underlie amyloid pathogenicity remain still unclear. This work aimed to clarify cell impairment induced by amyloid aggregated. To this end, we used a combined proteomic and one‐dimensional ¹H‐NMR approach on NIH‐3T3 cells exposed to prefibrillar aggregates from the amyloidogenic apomyoglobin mutant W7FW14F. The results indicated that cell exposure to prefibrillar aggregates induces changes of the expression level of proteins and metabolites involved in stress response. The majority of the proteins and metabolites detected are reported to be related to oxidative stress, perturbation of calcium homeostasis, apoptotic and survival pathways, and membrane damage. In conclusion, the combined proteomic and ¹H‐NMR metabonomic approach, described in this study, contributes to unveil novel proteins and metabolites that could take part to the general framework of the toxicity induced by amyloid aggregates. These findings offer new insights in therapeutic and diagnostic opportunities. J. Cell. Physiol. 228: 1359–1367, 2013. © 2012 Wiley Periodicals, Inc.     

CHF5074 restores visual memory ability and pre‐synaptic cortical acetylcholine release in pre‐plaque Tg2576 mice

CHF5074, a new microglial modulator, attenuates memory deficit in Alzheimer's disease transgenic mice. In this study, the effect of an acute or subacute CHF5074 treatment on in vivo novel object recognition test and on [³H]Acetylcholine (ACh) and GABA release in pre‐plaque (7‐month‐old) Tg2576 mice have been compared with those induced by the γ‐secretase inhibitor LY450139 (semagacestat). Vehicle‐treated Tg2576 mice displayed an impairment of recognition memory compared with wild‐type animals. This impairment was recovered in transgenic animals acutely treated with CHF5074 (30 mg/kg), while LY450139 (1, 3, 10 mg/kg) was ineffective. In frontal cortex synaptosomes from vehicle‐treated Tg2576 mice, K⁺‐evoked [³H]ACh release was lower than that measured in wild‐type mice. This reduction was absent in transgenic animals subacutely treated with CHF5074 (30 mg/kg daily for 8 days), while it was slightly, not significantly, amplified by LY450139 (3 mg/kg daily for 8 days). There were no differences between the groups on spontaneous [³H]ACh release as well as spontaneous and K⁺‐evoked GABA release. These results suggest that CHF5074 has beneficial effects on visual memory and cortical cholinergic dysfunctions in pre‐plaque Tg2576 mice. Together with previous findings, these data suggest that CHF5074 could be a possible candidate for early Alzheimer's disease therapeutic regimens.     

BDNF prevents NMDA‐induced toxicity in models of Huntington's disease: the effects are genotype specific and adenosine A2A receptor is involved

NMDA receptor‐mediated excitotoxicity is thought to play a pivotal role in the pathogenesis of Huntington's disease (HD). The neurotrophin brain‐derived neurotrophic factor (BDNF), which is also highly involved in HD and whose effects are modulated by adenosine A_2ARs, influences the activity and expression of striatal NMDA receptors. In electrophysiology experiments, we investigated the role of BDNF toward NMDA‐induced effects in HD models, and the possible involvement of A_2ARs. In corticostriatal slices from wild‐type mice and age‐matched symptomatic R6/2 mice (a model of HD), NMDA application (75 μM) induced a transient or a permanent (i.e., toxic) reduction of field potential amplitude, respectively. BDNF (10 ng/mL) potentiated NMDA effects in wild‐type, while it protected from NMDA toxicity in R6/2 mice. Both effects of BDNF were prevented by A_2AR blockade. The protective effect of BDNF against NMDA‐induced toxicity was reproduced in a cellular model of HD. These findings may have very important implications for the neuroprotective potential of BDNF and A_2AR ligands in HD.