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Effects of glycated insulin on adipogenic differentiation. A. Piccoli1, T. Bisogno1,2, M. Maccarrone1,3, P. Pozzilli1,4, N. Napoli1,5, R. Strollo1. 1Universita Campus Bio-Medico di Roma, Rome, Italy; 2National Research Council, Pozzuoli, Italy; 3European Center for Brain Research (CERC)/Santa Lucia Foundation IRCCS, Rome, Italy; 4Queen Mary, University of London, London, UK; 5Washington University in St Louis, St Louis, MO, USA Hyperglycaemia and chronic low-grade inflammation associated with obesity and diabetes are a main source of reactive oxidants (ROS). High levels of ROS may generate oxidative post-translational modification (oxPTMs) that may impair proteins biological functions. We have shown that insulin could also be a target of these modifications that include glycation, oxidation and chlorination. Adipose tissue is a primary target of this hormone and a key player in insulin resistance (IR): evidence from animal studies show that the glycation of insulin or other proteins result in a significant impairment of biological function of adipose tissue, affecting adipokines expression and insulin sensitivity. However, how oxPTMs such as glycation interfere with insulin function and their involvement in IR and type 2 diabetes (T2D) is still unknown. We hypothesize that insulin glycation may be involved in insulin resistance and in the pathogenesis of T2D. Therefore, the aim of this study was to assess the possible effects of glycated vs. native insulin on adipocyte differentiation. Human recombinant insulin was glycated in vitro following our established protocol. Insulin glycation was monitored through PAGE. Human preadipocytes (HPAd), a primary cell line from subcutaneous adipose tissue, and adipose-derived stem cells (ASCs), isolated from tissues of lean and obese subjects (BMI30), were differentiated with standard adipogenic medium in presence of native or glycated insulin. Cellular differentiation was assessed by gene and protein expression analyses of adipogenic markers (adiponectin, peroxisome proliferator-activated receptor gamma -Ppar?-, fatty acid binding protein 4 -Fabp4-, glucose transporter 4 -Glut4-) and of receptors for adiponectin (AdipoR1) and for insulin (InsR), through real time RT-PCR and immunoblot respectively. Our preliminary results showed that preadipocytes (both HPAd and ASCs) exposed to glycated insulin had impaired differentiation capacities: Oil Red O-staining showed that these cells had lower number of lipid droplets compared to those exposed to native insulin, suggesting an impaired adipocyte differentiation. Considering gene expression, we observed an overall downregulation of adipogenic markers, with a significant decrease of adiponectin both in leans- and in obese-derived cells (p=0.007 and p=0.017 respectively), lower levels of Glut4 and a significant lower expression of Fabp4 (p=0.002) in cells from leans differentiated with glycated insulin. Immunoblotting showed a similar pattern to that observed with gene expression analysis but, regarding insulin receptor, protein levels of the receptor presented a significant down regulation in cells treated with glycated insulin, with a p value of 0.0266 in leans and p=0.011 in obese subjects. Conversely, gene expression of insulin receptor did not present any difference between the native and glycated insulin. In conclusion, our data suggest that glycated insulin could affect adipocytes differentiation and impair insulin receptor expression, acting at translational/post-translational level or increasing its degradation. Its role and mechanisms that lead to these effects should be investigated as a possible mediator in the pathogenesis of T2D.

In this study, we investigated the mechanism of S100B neurotoxicity and the effect of cannabinoids, in C6 cells treated with 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP) and co-cultured with differentiated PC12 cells. MPTP concentration- and time-dependently increased S100B density in C6 cells. This effect was followed by increased C6 cell proliferation and decreased cell viability of co-cultured PC12 cells. An antibody against S100B, given to PC12 cells before co-culture, led to their survival. Treatment with arachidonyl-2-chloroethylamide, a CB1 agonist, significantly inhibited MPTP-induced S100B density in C6 cells and protected co-cultured PC12 cells from cell death. Because MPTP selectively increased the levels of anandamide in C6 cells, the involvement of the endocannabinoid system was investigated by using selective inhibitors of endocannabinoid inactivation (cellular re-uptake or enzymatic hydrolysis) and selective cannabinoid CB1 and CB2 receptor antagonists and by silencing the CB1 receptor. Our data suggest that selective activation of CB1 receptors by either exogenous or endogenous cannabinoids might afford neuroprotection in MPTP-induced neurotoxicity also by controlling S100B up-regulation in activated glial cells.

During transit through the epididymis, spermatozoa are normally kept immotile and do not attain the ability to become motile until they reach the caudal epididymis. This study was undertaken to determine whether endocannabinoids play a role in the epididymis and in particular in suppressing the ability of spermatozoa to become motile. We show that the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are high in mouse spermatozoa isolated from the caput (head) of the epididymis, where these cells do not move (or possess sluggish and irregular motility) and decrease dramatically in spermatozoa isolated from the cauda (tail). The subsequent gradient regulates, via autocrine communication, the activity of cannabinoid receptor CNR1 (previously known as CB1) present on the sperm cell membrane and induces caudal spermatozoa to acquire the potential to become motile (‘‘start-up’’). Accordingly, the genetic or pharmacological inactivation of CNR1 increases number of motile spermatozoa in caput. Also, blockers of endocannabinoid cellular uptake inhibit the potential to move of spermatozoa and destroy the 2-AG gradient throughout the epididymis. This gradient-regulated mechanism may encourage further research for future therapies related to male infertility.

Palmitoylethanolamide (PEA) is an anti-inflammatory mediator that enhances the activation by anandamide (AEA) of cannabinoid receptors and transient receptor potential vanilloid type-1 (TRPV1) channels, and directly activates peroxisome proliferator-activated receptor-alpha (PPAR-alpha). In mice, 2,4-dinitrofluorobenzene (DNFB)-induced contact allergic dermatitis (CAD) in inflamed ears is partly mediated by the chemokine Monocyte Chemotactic Protein-2 (MCP-2) and accompanied by elevation of AEA levels. No datum is available on PEA regulation and role in CAD.We examined whether PEA is produced during DNFB-induced CAD, and if it has any direct protective action in keratinocytes in vitro.Eight- to ten-week-old female C57BL/6J wild-type and CB(1)/CB(2) double knock-out mice were used to measure PEA levels and the expression of TRPV1, PPAR-alpha receptors and enzymes responsible for PEA biosynthesis and degradation. Human keratinocytes (HaCaT) cells were stimulated with polyinosinic polycytidylic acid [poly-(I:C)], and the expression and release of MCP-2 were measured in the presence of PEA and antagonists of its proposed receptors.2,4-Dinitrofluorobenzene increased ear skin PEA levels and up-regulated TRPV1, PPAR-alpha and a PEA-biosynthesizing enzyme in ear keratinocytes. In HaCaT cells, stimulation with poly-(I:C) elevated the levels of both PEA and AEA, and exogenous PEA (10 microM) inhibited poly-(I:C)-induced expression and release of MCP-2 in a way reversed by antagonism at TRPV1, but not PPAR-alpha. PEA (5-10 mg/kg, intraperitoneal) also inhibited DNFB-induced ear inflammation in mice in vivo, in a way attenuated by TRPV1 antagonism.We suggest that PEA is an endogenous protective agent against DNFB-induced keratinocyte inflammation and could be considered for therapeutic use against CAD.

Diet plays a central role in maintaining health throughout life and a controlled food intake is associated to a reduced risk of certain diseases. A proper diet should include vitamins, minerals, carbohydrates, proteins, and fats that have to be optimally balanced in order to exert their physiological functions. The endogenous ligands of type-1 and type-2 cannabinoid receptors, N-arachidonoyl-ethanolamine and 2-arachidonoylglycerol, are arachidonic acid (AA) derivatives whose levels are regulated by the activity of metabolic enzymes, as well as by AA availability. Since the only sources of AA in mammals are diet and the enzymatic production in the liver from shorter-chain essential fatty acids like linoleic acid, it is realistic to hypothesize that endocannabinoid levels might be modulated by fatty acid composition of food. Therefore, in this review we summarize literature data indicating that endocannabinoid levels, and hence their activity at cannabinoid receptors, might be modulated by food composition. We focused our attention on dietary fatty acid content, and on type and esterified form of fatty acids in the different diets. © 2014 International Union of Biochemistry and Molecular Biology.

In the ventrolateral periaqueductal gray (PAG), activation of excitatory output neurons projecting monosynaptically to OFF cells in the rostral ventromedial medulla (RVM) causes antinociceptive responses and is under the control of cannabinoid receptor type-1 (CB1) and vanilloid transient receptor potential vanilloid type 1 (TRPV1) receptors. We studied in healthy rats the effect of elevation of PAG endocannabinoid [anandamide and 2-arachidonoylglycerol (2-AG)] levels produced by intra-PAG injections of the inhibitor of fatty acid amide hydrolase URB597 [cyclohexylcarbamic acid-3'-carbamoyl-biphenyl-3-yl ester] on 1) nociception in the “plantar test” and 2) spontaneous and tail-flick-related activities of RVM neurons. Depending on the dose or time elapsed since administration, URB597 (0.5–2.5 nmol/rat) either suppressed or increased thermal nociception via TRPV1 or CB1 receptors, respectively. TRPV1 or cannabinoid receptor agonists capsaicin (6 nmol) and (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate [WIN55,212-2 (4 nmol)] also suppressed or enhanced nociception, respectively. URB597 dose dependently enhanced PAG anandamide and 2-AG levels, with probable subsequent activation of TRPV1/CB1 receptors and only CB1 receptors, respectively. The TRPV1-mediated antinociception and CB1-mediated nociception caused by URB597 correlated with enhanced or reduced activity of RVM OFF cells, suggesting that these effects occur via stimulation or inhibition of excitatory PAG output neurons, respectively. Accordingly, several ventrolateral PAG neurons were found by immunohistochemistry to coexpress TRPV1 and CB1 receptors. Finally, at the highest doses tested, URB597 (4 nmol/rat) and, as previously reported, WIN55,212-2 (25–100 nmol) also caused CB1-mediated analgesia, correlating with stimulation (possibly disinhibition) of RVM OFF cells. Thus, endocannabinoids affect the descending pathways of pain control by acting at either CB1 or TRPV1 receptors in healthy rats.

We have previously reported the development of CB-25 and CB-52, two ligands of CB1 and CB2 cannabinoid receptors. We assessed here their functional activity.The effect of the two compounds on forskolin-induced cAMP formation in intact cells or GTP-gamma-S binding to cell membranes, and their action on nociception in vivo was determined.CB-25 enhanced forskolin-induced cAMP formation in N18TG2 cells (EC50 approximately 20 nM, max. stimulation = 48%), behaving as an inverse CB1 agonist, but it stimulated GTP-gamma-S binding to mouse brain membranes, behaving as a partial CB1 agonist (EC50 =100 nM, max. stimulation = 48%). At human CB1 receptors, CB-25 inhibited cAMP formation in hCB1-CHO cells (EC50 = 1600 nM, max. inhibition = 68% of CP-55,940 effect). CB-52 inhibited forskolin-induced cAMP formation by N18TG2 cells (IC50 = 450 nM, max. inhibition = 40%) and hCB1-CHO cells (EC50 = 2600 nM, max. inhibition = 62% of CP-55,940 effect), and stimulated GTP-gamma-S binding to mouse brain membranes (EC50 = 11 nM, max. stimulation approximately 16%). Both CB-25 and CB-52 showed no activity in all assays of CB2-coupled functional activity and antagonized CP55940-induced stimulation of GTP-gamma-S binding to hCB2-CHO cell membranes. In vivo, both compounds, administered i.p., produced dose-dependent nociception in the plantar test carried out in healthy rats, and antagonised the anti-nociceptive effect of i.p. WIN55,212-2. In the formalin test in mice, however, the compounds counteracted both phases of formalin-induced nociception.CB-25 and CB-52 behave in vitro mostly as CB1 partial agonists and CB2 neutral antagonists, whereas their activity in vivo might depend on the tonic activity of cannabinoid receptors.

1. We have studied the effect of cannabinoid agonists (CP 55,940 and cannabinol) on intestinal motility in a model of intestinal inflammation (induced by oral croton oil in mice) and measured cannabinoid receptor expression, endocannabinoids (anandamide and 2-arachidonylglycerol) and anandamide amidohydrolase activity both in physiological and pathophysiological states. 2. CP 55,940 (0.03 - 10 nmol mouse(-1)) and cannabinol (10 - 3000 nmol mouse(-1)) were more active in delaying intestinal motility in croton oil-treated mice than in control mice. These inhibitory effects were counteracted by the selective cannabinoid CB(1) receptor antagonist SR141716A (16 nmol mouse(-1)). SR141716A (1 - 300 nmol mouse(-1)), administered alone, increased intestinal motility to the same extent in both control and croton oil-treated mice. 3. Croton oil-induced intestinal inflammation was associated with an increased expression of CB(1) receptor, an unprecedented example of up-regulation of cannabinoid receptors during inflammation. 4. High levels of anandamide and 2-arachidonylglycerol were detected in the small intestine, although no differences were observed between control and croton oil-treated mice; by contrast anandamide amidohydrolase activity increased 2 fold in the inflamed small intestine. 5. It is concluded that inflammation of the gut increases the potency of cannabinoid agonists possibly by 'up-regulating' CB(1) receptor expression; in addition, endocannabinoids, whose turnover is increased in inflamed gut, might tonically inhibit intestinal motility.

Cannabidiol (CBD) and Delta(9)-tetrahydrocannabinol (THC) interact with transient receptor potential (TRP) channels and enzymes of the endocannabinoid system. EXPERIMENTAL APPROACH The effects of 11 pure cannabinoids and botanical extracts [botanical drug substance (BDS)] from Cannabis varieties selected to contain a more abundant cannabinoid, on TRPV1, TRPV2, TRPM8, TRPA1, human recombinant diacylglycerol lipase a (DAGL alpha), rat brain fatty acid amide hydrolase (FAAH), COS cell monoacylglycerol lipase (MAGL), human recombinant N-acylethanolamine acid amide hydrolase (NAAA) and anandamide cellular uptake (ACU) by RBL-2H3 cells, were studied using fluorescence-based calcium assays in transfected cells and radiolabelled substrate-based enzymatic assays. Cannabinol (CBN), cannabichromene (CBC), the acids (CBDA, CBGA, THCA) and propyl homologues (CBDV, CBGV, THCV) of CBD, cannabigerol (CBG) and THC, and tetrahydrocannabivarin acid (THCVA) were also tested. KEY RESULTS CBD, CBG, CBGV and THCV stimulated and desensitized human TRPV1. CBC, CBD and CBN were potent rat TRPA1 agonists and desensitizers, but THCV-BDS was the most potent compound at this target. CBG-BDS and THCV-BDS were the most potent rat TRPM8 antagonists. All non-acid cannabinoids, except CBC and CBN, potently activated and desensitized rat TRPV2. CBDV and all the acids inhibited DAGL alpha. Some BDS, but not the pure compounds, inhibited MAGL. CBD was the only compound to inhibit FAAH, whereas the BDS of CBC > CBG > CBGV inhibited NAAA. CBC = CBG > CBD inhibited ACU, as did the BDS of THCVA, CBGV, CBDA and THCA, but the latter extracts were more potent inhibitors. CONCLUSIONS AND IMPLICATIONS These results are relevant to the analgesic, anti-inflammatory and anti-cancer effects of cannabinoids and Cannabis extracts.

During transit through the epididymis, spermatozoa are normally kept immotile and do not attain the ability to become motile until they reach the caudal epididymis. This study was undertaken to determine whether endocannabinoids play a role in the epididymis and in particular in suppressing the ability of spermatozoa to become motile. We show that the levels of the endocannabinoid, 2-arachidonoylglycerol (2-AG), are high in mouse spermatozoa isolated from the caput (head) of the epididymis, where these cells do not move (or possess sluggish and irregular motility), and decrease dramatically in spermatozoa isolated from the cauda (tail). The subsequent gradient regulates, via autocrine communication, the activity of cannabinoid receptor CNR1 (previously known as CB1) present on the sperm cell membrane and induces caudal spermatozoa to acquire the potential to become motile ("start up"). Accordingly, the genetic or pharmacological inactivation of CNR1 increases number of motile spermatozoa in caput. Also blockers of endocannabinoid cellular uptake inhibit the potential to move of spermatozoa and destroy the 2-AG gradient throughout the epididymis. This gradient-regulated mechanism may encourage further research for future therapies related to male infertility.