The endocannabinoid system plays an important role in the intake of palatable food. For example, endocannabinoid signaling in the upper small-intestinal epithelium is increased (i) in rats after tasting dietary fats, which promotes intake of fats, and (ii) in a mouse model of diet-induced obesity, which promotes overeating via impaired nutrient-induced gut–brain satiation signaling. We now utilized a combination of genetic, pharmacological, and behavioral approaches to identify roles for cannabinoid CB1Rs in upper small-intestinal epithelium in preferences for a western-style diet (WD, high-fat/sucrose) versus a standard rodent diet (SD, low-fat/no sucrose). Mice were maintained on SD in automated feeding chambers. During testing, mice were given simultaneous access to SD and WD, and intakes were recorded. Mice displayed large preferences for the WD, which were inhibited by systemic pretreatment with the cannabinoid CB1R antagonist/inverse agonist, AM251, for up to 3 h. We next used our novel intestinal epithelium-specific conditional cannabinoid CB1R-deficient mice (IntCB1−/−) to investigate if intestinal CB1Rs are necessary for WD preferences. Similar to AM251 treatment, preferences for WD were largely absent in IntCB1−/− mice when compared to control mice for up to 6 h. Together, these data suggest that CB1Rs in the murine intestinal epithelium are required for acute WD preferences.
The endocannabinoid system is expressed in cells throughout the body and controls a variety of physiological and pathophysiological functions. We describe robust and reproducible UPLC-MS/MS-based methods for analyzing metabolism of the endocannabinoids, 2-arachidonoyl-sn-glycerol and arachidonoyl ethanolamide, and related monoacylglycerols (MAGs) and fatty acid ethanolamides (FAEs), respectively, in mouse mucosal tissues (i.e., intestine and lung). These methods are optimized for analysis of activity of the MAG biosynthetic enzyme, diacylglycerol lipase (DGL), and MAG degradative enzymes, monoacylglycerol lipase (MGL) and alpha/beta hydrolase domain containing-6 (ABHD6). Moreover, we describe a novel UPLC-MS/MS-based method for analyzing activity of the FAE degradative enzyme, fatty acid amide hydrolase (FAAH), that does not require use of radioactive substrates. In addition, we describe in vivo pharmacological methods to inhibit MAG biosynthesis selectively in the mouse small-intestinal epithelium. These methods will be useful for profiling endocannabinoid metabolism in rodent mucosal tissues in health and disease.
Sickle cell disease (SCD) is characterized by multiple comorbidities including pain. SCD patients often use cannabinoids to alleviate pain, but their psychoactive effects and social stigma impose major challenges. Strategies to elevate endogenous cannabinoids (eCBs) are devoid of such challenges, but pharmacologic approaches showed adverse-effects in clinical trials. Therefore, we examined the potential of non-pharmacologic integrative approaches to elevate eCBs. Enriched high-energy diet has been shown to increase levels of eCBs (Argueta et al., Front Physiol 2019) and when combined with companionship reduced hyperalgesia in sickle mice (Tran et al., Blood 2016). We hypothesized that enriched diet and companionship would enhance eCBs without adverse effects and reduce hyperalgesia by inhibiting peripheral and central pro-nociceptive mechanisms. We fed male homozygous-BERK (sickle) mice, regular Rodent Diet (RD; 2018, Harlan) or customized high calorie enriched Sickle Mouse Diet (SD; 59M3, TestDiet), housed with or without a female companion (C+ or C-, respectively) for 3-weeks. RD/SD contain 18.6/26.4% protein, 6.2/11.1% fat, 24/27.5% carbohydrates and 18/26% kcal/g, respectively; and SD contains higher minerals, vitamins and ω-3 fatty acids compared to RD. Control HbAA-BERK and sickle mice were divided into 4 groups: [i] R/C-, RD, no companion, [ii] S/C-, SD without companion, [iii] R/C+, RD with companion, and [iv] S/C+, SD with companion. After 3-weeks of treatment, spinal cord eCBs were analyzed using targeted lipid quantitation with liquid chromatography mass spectrometry (LCMS). We observed a 20% decrease in palmitoylethanolamide (PEA), in sickle mice compared to control mice, in R/C- group (p<0.05). Further, we observed increased spinal PEA in S/C+ compared to R/C- sickle mice (~40%, p<0.05), which was concomitant with reduced mechanical, heat, and cold hyperalgesia in S/C+ sickle mice group (~80%, p<0.001; ~60%, p<0.01; & ~30%, p<0.001, respectively). Therefore, sickle diet and companionship enhances endogenous spinal PEA which has an inhibitory effect on hyperalgesia in sickle mice. Treatment of control and sickle mice in R/C- group with PEA (i.p. 20 mg/kg/day) led to acute (1 hour) reduction of mechanical- (~40%, p<0.01) and cold-hyperalgesia (~40%, p<0.001) in sickle mice compared to pre-treatment, which was sustained during 3 day treatment, but had no effect on control mice which do not have hyperalgesia. PEA inhibits substance P (SP)-induced mast cell activity, and sickle mice show increased spinal SP, neuronal sensitization, peripheral nerve injury and mast cell activation (Tran et al., Blood 2017). Pain in SCD is both neuropathic and inflammatory. We examined if PEA inhibited the mechanisms that underlie spinal nerve repair by neurite outgrowth inhibitor, NOGO-A/reticulon-4, which regulates nerve regeneration via Rho Kinase (ROCK) signaling. NOGO-A contributes to inflammatory pain and hyperalgesia following spinal cord injury via NOGO receptor 1 in spinal cord. We observed that spinal NOGO-A expression and ROCK activity are upregulated (20% & 100%, respectively) in sickle mice compared to control mice (all R/C-), which were inhibited upon 3-day treatment with PEA. We validated ROCK activity downstream of NOGO-A using SH-SY5Y neuroblastoma cells, simulating a sickle microenvironment with hemin (40 µM) and TNFα (1 ng/ml)(H+T). ROCK activity increased in H+T-treated SH-SY5Y cells compared to vehicle (~30%, p=0.05). In parallel, we analyzed the effect of PEA on extracellular traps (ET) in cutaneous mast cells from sickle mice induced by H+T in vitro. PEA treatment inhibited ET formation and extravasation of nuclear contents in H+T induced mast cells. Thus, PEA has the potential to attenuate neuropathic and inflammatory pain by inhibiting neuronal NOGO-A/ROCK pathway and mast cell activation in a sickle milieu. PEA has analgesic and anti-inflammatory effects on chronic pain in several clinical conditions. Therefore, our data suggest that diet and pleasure have the potential to upregulate pro-analgesic PEA that inhibits NOGO-A signaling and mast cell activation, leading to attenuation of hyperalgesia in sickle mice. Disclosures Gupta: Grifols: Research Funding; Cyclerion: Research Funding; 1910 Genetics: Research Funding; Novartis: Honoraria; Tautona Group: Honoraria; CSL Behring: Honoraria.
Sickle cell disease (SCD) is characterized by chronic pain and bouts of extreme acute pain from vasoocclusive crises (VOC). Sickle pain has both neuropathic and inflammatory features (Tran et al., Blood 2017). Mechanisms underlying neural injury remain unknown in SCD. Neurite outgrowth inhibitor (NOGO-A/reticulon-4) and its receptor NGR1 contribute to pain, neuronal damage, and inhibition of neurite outgrowth (Hu et al., FASEB J 2019). We examined if NOGO-A pathway is activated in a sickle microenvironment and if its inhibition will ameliorate hyperalgesia in BERK sickle mice. We used Rho kinase activity (ROCK) downstream of NGR1 as a readout of activation of NOGO-A/NGR1 pathway. We observed increased expression of NOGO-A (~260%, p<0.05) and NGR1 (~180%, p<0.05) in the dorsal root ganglia, and increased NOGO-A and ROCK activity in spinal cords of sickle mice compared to control mice expressing normal human hemoglobin A. Earlier, we found that an endogenous cannabinoid, palmitoylethanolamide (PEA) inhibits spinal NOGO-A expression and ROCK activity in sickle mice (Argueta et al., ASH 2020 #225). We hypothesized that sickle microenvironment with cell-free heme and inflammation activates NOGO-A/NGR1-ROCK pathway leading to nerve injury and pain, and inhibition of this pathway will ameliorate hyperalgesia in sickle mice. Using terminally differentiated rat pheochromocytoma (PC12) cells, we simulated a sickle microenvironment with hemin (40 µM) and TNFα (1 ng/ml) (H+T). H+T elevated ROCK activity compared to vehicle (~40%, p<0.05). PEA (30 uM) and 2 µM NEP (1-40), a competitive antagonist of NGR1, attenuated H+T-induced ROCK activity (both p<0.01); co-treatment had no additive effect, indicating a common pathway. As well, siRNA (10 nM) knockdown of NGR1 reversed H+T-induced ROCK activity (p<0.001), which was equally effective with 30 µM PEA co-treatment. Functionally, treatment with 30 µM PEA or 2 µM NEP (1-40) enhanced neurite outgrowth in H+T-treated PC-12 cells (~120%, p<0.001). NEP (1-40) at 5 mg/kg reduced mechanical and cold (both ~50%, p<0.001) hyperalgesia in sickle mice compared to baseline (BL) and/or vehicle treatment. Together, these data demonstrate that NOGO-A/NGR1 pathway activation may underlie nerve injury, and inhibition of this pathway with a NGR1 antagonist or PEA promotes neurite outgrowth and reduces hyperalgesia in a sickle microenvironment. We next examined the contribution of exogenously administered and endogenously produced PEA in ameliorating hyperalgesia. Mass spectrometry revealed that spinal PEA is reduced in female (p<0.05) and male (p<0.001) sickle mice compared to age/sex-matched control mice. Exogenous PEA (i.p. 20 mg/kg/d) reduced cold avoidance over a 3-day treatment period, showing significantly more time in the cold chamber compared to BL or vehicle at 1 h, 24 h, and 72 h (p<0.05). The analgesic effect of PEA was maintained for 9 days of treatment without developing tolerance. We next increased endogenous PEA by inhibiting its degradative enzyme, N-acylethanolamine acid amidase, with ARN19702 (i.p. 3, 10, & 30 mg/kg/d), which reduced mechanical (~50%, p<0.001) and cold hyperalgesia (~40%, p<0.001) over 72 hours in a dose-dependent manner. Since hypoxia and ischemia reperfusion injury contribute to acute VOC pain, we incited hypoxia-reoxygenation (HR; 3 h @ 8% O 2, 92% N 2, followed by 1 h @ normoxia) to simulate acute VOC pain. We observed that 5-day pretreatment with PEA (i.p. 20 mg/kg/d) before HR prevented mechanical and cold hyperalgesia following HR in sickle mice. Moreover, treatment with PEA after HR incitement significantly reduced hyperalgesia for 24 h after HR compared to BL (~30%, p<0.001) and vehicle treated (~66%, p<0.001) sickle mice. NGR1 antagonism reduces spinal microglial injury/activation. Heme and TNFα have been shown to cause microglial injury in vitro, while spinal microglial activation has been demonstrated in sickle mice (Lei et al., Antioxid Redox Signal 2021). Thus, NGR1/ROCK cascade may contribute to both neuronal injury and inflammation in the central nervous system leading to neuropathic pain. Our data suggest that PEA and targeting NOGO-A pathway may prevent/reduce chronic and acute hyperalgesia in sickle mice. We speculate that interventions targeting NOGO-A pathway may prevent/reduce neuropathic pain and that PEA has the translational potential for the treatment of chronic and acute pain in SCD. Disclosures Gupta: Tautona Group: Consultancy, Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; 1910 Genetics: Other: Grantee; Grifols: Other: Grantee; SCDAA: Membership on an entity's Board of Directors or advisory committees; CSL Behring LLC: Honoraria; NIH: Other: Grantee; University of Minnestoa Foundation: Other: Philanthropic Funding; Southern California Institute for Research and Education Foundation: Other: Philanthropic Funding; Cyclerion: Research Funding; UCI Foundation: Other: Philanthropic Funding.
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