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Enkephalins are opioid peptides that modulate analgesia, reward, and stress. In vivo detection of enkephalins remains difficult due to transient and low endogenous concentrations and inherent sequence similarity. To begin to address this, we previously developed a system combining in vivo optogenetics with microdialysis and a highly sensitive mass spectrometry-based assay to measure opioid peptide release in freely moving rodents (Al-Hasani et al., 2018, eLife). Here, we show improved detection resolution and stabilization of enkephalin detection, which allowed us to investigate enkephalin release during acute stress. We present an analytical method for real-time, simultaneous detection of Met- and Leu-enkephalin (Met-Enk and Leu-Enk) in the mouse nucleus accumbens shell (NAcSh) after acute stress. We confirm that acute stress activates enkephalinergic neurons in the NAcSh using fiber photometry and that this leads to the release of Met- and Leu-Enk. We also demonstrate the dynamics of Met- and Leu-Enk release as well as how they correlate to one another in the ventral NAc shell, which was previously difficult due to the use of approaches that relied on mRNA transcript levels rather than posttranslational products. This approach increases spatiotemporal resolution, optimizes the detection of Met-Enk through methionine oxidation, and provides novel insight into the relationship between Met- and Leu-Enk following stress.

ObjectiveThe enteric nervous system (ENS) plays a key role in controlling the gut-brain axis under normal and pathological conditions, such as type 2 diabetes. The discovery of intestinal actors, such as enterosynes, able to modulate the ENS-induced duodenal contraction is considered an innovative approach. Among all the intestinal factors, the understanding of the role of gut microbes in controlling glycaemia is still developed. We studied whether the modulation of gut microbiota by prebiotics could permit the identification of novel enterosynes.DesignWe measured the effects of prebiotics on the production of bioactive lipids in the intestine and tested the identified lipid on ENS-induced contraction and glucose metabolism. Then, we studied the signalling pathways involved and compared the results obtained in mice to human.ResultsWe found that modulating the gut microbiota with prebiotics modifies the actions of enteric neurons, thereby controlling duodenal contraction and subsequently attenuating hyperglycaemia in diabetic mice. We discovered that the signalling pathway involved in these effects depends on the synthesis of a bioactive lipid 12-hydroxyeicosatetraenoic acid (12-HETE) and the presence of mu-opioid receptors (MOR) on enteric neurons. Using pharmacological approaches, we demonstrated the key role of the MOR receptors and proliferator-activated receptor γ for the effects of 12-HETE. These findings are supported by human data showing a decreased expression of the proenkephalin and MOR messanger RNAs in the duodenum of patients with diabetic.ConclusionsUsing a prebiotic approach, we identified enkephalin and 12-HETE as new enterosynes with potential real beneficial and safety impact in diabetic human.

Enkephalins are endogenous opioid pentapeptides that play a role in neurotransmission and pain modulation in vertebrates. However, the distribution pattern of enkephalinergic neurons in the brains of reptiles has been understudied. This study reports the organization of the methionine-enkephalin (M-ENK) and leucine-enkephalin (L-ENK) neuronal systems in the central nervous system of the gecko Hemidactylus frenatus using an immunofluorescence labeling method. Although M-ENK and L-ENK-immunoreactive (ir) fibers extended throughout the pallial and subpallial subdivisions, including the olfactory bulbs, M-ENK and L-ENK-ir cells were found only in the dorsal septal nucleus. Enkephalinergic perikarya and fibers were highly concentrated in the periventricular and lateral preoptic areas, as well as in the anterior and lateral subdivisions of the hypothalamus, while enkephalinergic innervation was observed in the hypothalamic periventricular nucleus, infundibular recess nucleus and median eminence. The dense accumulation of enkephalinergic content was noticed in the pars distalis of the hypophysis. In the thalamus, the nucleus rotundus and the dorsolateral, medial, and medial posterior thalamic nuclei contained M-ENK and L-ENK-ir fibers, whereas clusters of M-ENK and L-ENK-ir neurons were observed in the pretectum, mesencephalon, and rhombencephalon. The enkephalinergic fibers were also seen in the area X around the central canal, as well as the dorsal and ventral horns. The widespread distribution of enkephalin-containing neurons within the central nervous system implies that enkephalins regulate a variety of functions in the gecko, including sensory, behavioral, hypophysiotropic, and neuroendocrine functions.

Regulatory T (Treg) cells, expressing CD25 (interleukin-2 receptor α chain) and Foxp3 transcription factor, maintain immunological selftolerance and suppress various immune responses. Herewe report a feature of skin Treg cells expanded by ultraviolet B (UVB) exposure. We found that skin Treg cells possessing a healing function are expanded by UVB exposure with the expression of an endogenous opioid precursor, proenkephalin (PENK). Upon UVB exposure, skin Treg cells were expanded with a unique TCR repertoire. Also, they highly expressed a distinctive set of genes enriched in “wound healing involved in inflammatory responses” and the “neuropeptide signaling pathway,” as indicated by the high expression of Penk. We found that not only was PENK expression at the protein level detected in the UVB-expanded skin Treg (UVB-skin Treg) cells, but that a PENK-derived neuropeptide, methionine enkephalin (Met-ENK), from Treg cells promoted the outgrowth of epidermal keratinocytes in an ex vivo skin explant assay. Notably, UVB-skin Treg cells also promoted wound healing in an in vivo wound closure assay. In addition, UVB-skin Treg cells produced amphiregulin (AREG), which plays a key role in Treg-mediated tissue repair. Identification of a unique function of PENK+ UVB-skin Treg cells provides a mechanism for maintaining skin homeostasis.

-Met-enkephalin is a neuropeptide known to exert protective effects in various experimental models of autoimmune and inflammatory diseases. These effects are mediated through opioid receptors and can be abolished by the opioid receptor antagonist naltrexone. Investigation of peptide enantiomerism and the incorporation of d-amino acids are crucial for designing novel peptides with altered structural and biological properties compared with their native l-forms. Since no data are currently available on the properties or biological activity of the d-Met-enkephalin enantiomer, we evaluated its hepatoprotective potential in a mouse model of acetaminophen-induced hepatotoxicity. Male CBA mice were treated with d-Met-enkephalin, and hepatoprotection was assessed by measuring plasma alanine aminotransferase (ALT) and aspartate amino-transferase (AST) activities, along with histological liver necrosis scores. The peptide’s secondary structure and antisense peptide binding were analysed using circular dichroism and fluorescence spectroscopy, respectively. -Met-enkephalin demonstrated dose-dependent hepatoprotective effects within the range of 0.5–20 mg kg–1, with maximal protection observed at 5 mg kg , a dose comparable to that of the l-enantiomer (7.5 mg kg ). This preservation of biological activity may be attributed to the presence of the achiral amino acid glycine at positions 2 and 3, which maintains the functional conformation of the d-enantiomer. The role of opioid receptor involvement was further examined through direct receptor blockade using naltrexone and indirect inhibition with the antisense peptide IPPKY.

Azithromycin is recommended for the treatment of uncomplicated urogenital chlamydia infection although the standard 1gram dose sometimes fails to eradicate the infection (treatment failure). One hypothesis proposed for treatment failure has been insufficient levels of the antibiotic at the site of infection. We developed an assay using liquid chromatography and tandem mass spectrometry (LC-MS/MS) to measure azithromycin concentration in high-vaginal swabs and monitor how concentration changes over time following routine azithromycin treatment. Azithromycin concentrations were measured in two groups of women either within the first 24h of taking a 1g dose (N = 11) or over 9 days (N = 10). Azithromycin concentrations were normalised to an internal standard (leucine enkephalin), and the bulk lipid species phosphatidylcholine [PC(34:1)], using an Agilent 6490 triple quadrupole instrument in positive ionisation mode. The abundances of azithromycin, PC(34:1), and leu-enkephalin were determined by multiple reaction monitoring and absolute levels of azithromycin estimated using standard curves prepared on vaginal specimens. Vaginal azithromycin concentrations of women were rapidly obtained after 5h post-treatment (mean concentration = 1031mcg/mg of lipid, range = 173-2693mcg/mg). In women followed for 9 days, peak concentrations were highest after day 2 (mean concentration = 2206mcg/mg, range = 721-5791mcg/mg), and remained high for at least 9 days with a mean concentration of 384mcg/mg (range = 139-1024mcg/mg) on day 9. Our study confirmed that a single 1g dose of azithromycin is rapidly absorbed and remains in the vagina at relatively high levels for at least a week, suggesting that poor antibiotic absorption is unlikely to be an explanation for treatment failure.

Background T cell-derived opioids play a key role in the control of inflammatory pain. However, the nature of opioids produced by T cells is still matter of debate in mice. Whereas β-endorphin has been found in T lymphocytes by using antibody-based methods, messenger RNA (mRNA) quantification shows mainly mRNA encoding for enkephalins. The objective of the study is to elucidate the nature of T cell-derived opioids responsible for analgesia and clarify discrepancy of the results at the protein and genetic levels. Methods CD4 + T lymphocytes were isolated from wild-type and enkephalin-deficient mice. mRNA encoding for β-endorphin and enkephalin was quantified by RT-qPCR. The binding of commercially available polyclonal anti-endorphin antibodies to lymphocytes from wild-type or enkephalin knockout mice was assessed by cytofluorometry. Opioid-mediated analgesic properties of T lymphocytes from wild-type and enkephalin-deficient mice were compared in a model of inflammation-induced somatic pain by measuring sensitivity to mechanical stimuli using calibrated von Frey filaments. Results CD4 + T lymphocytes expressed high level of mRNA encoding for enkephalins but not for β-endorphin in mice. Anti-β-endorphin polyclonal IgG antibodies are specific for β-endorphin but cross-react with enkephalins. Anti-β-endorphin polyclonal antibodies bound to wild-type but not enkephalin-deficient CD4 + T lymphocytes. Endogenous regulation of inflammatory pain by wild-type T lymphocytes was completely abolished when T lymphocytes were deficient in enkephalins. Pain behavior of immune-deficient (i.e., without B and T lymphocytes) mice was superimposable to that of mice transferred with enkephalin-deficient lymphocytes. Conclusions Rabbit polyclonal anti-β-endorphin serum IgG bind to CD4 + T lymphocytes because of their cross-reactivity towards enkephalins. Thus, staining of T lymphocytes by anti-β-endorphin polyclonal IgG reported in most of studies in mice is because of their binding to enkephalins. In mice, CD4 + T lymphocytes completely lose their analgesic opioid-mediated activity when lacking enkephalins.

Negative affect is critical for conferring vulnerability to opiate addiction as reflected by the high comorbidity of opiate abuse with major depressive disorder (MDD). Rodent models implicate amygdala prodynorphin (Pdyn) as a mediator of negative affect; however, evidence of PDYN involvement in human negative affect is limited. Here, we found reduced PDYN mRNA expression in the postmortem human amygdala nucleus of the periamygdaloid cortex (PAC) in both heroin abusers and MDD subjects. Similar to humans, rats that chronically self-administered heroin had reduced Pdyn mRNA expression in the PAC at a time point associated with a negative affective state. Using the in vivo functional imaging technology DREAMM (DREADD-assisted metabolic mapping, where DREADD indicates designer receptors exclusively activated by designer drugs), we found that selective inhibition of Pdyn-expressing neurons in the rat PAC increased metabolic activity in the extended amygdala, which is a key substrate of the extrahypothalamic brain stress system. In parallel, PAC-specific Pdyn inhibition provoked negative affect-related physiological and behavioral changes. Altogether, our translational study supports a functional role for impaired Pdyn in the PAC in opiate abuse through activation of the stress and negative affect neurocircuitry implicated in addiction vulnerability.

Pre-proenkephalin 1 (Penk1) is a pro-neuropeptide that belongs to the typical opioid peptide’s family, having analgesic properties. We previously found Penk1 to be the most downregulated gene in a whole gene profiling analysis performed in osteoblasts subjected to microgravity as a model of mechanical unloading. In this work, Penk1 downregulation was confirmed in the bones of two in vivo models of mechanical unloading: tail-suspended and botulinum toxin A (botox)-injected mice. Consistently, in the sera from healthy volunteers subjected to bed rest, we observed an inverse correlation between PENK1 and bed rest duration. These results prompted us to investigate a role for this factor in bone. Penk1 was highly expressed in mouse bone, but its global deletion failed to impact bone metabolism in vivo. Indeed, Penk1 knock out (Penk1−/−) mice did not show an overt bone phenotype compared to the WT littermates. Conversely, in vitro Penk1 gene expression progressively increased during osteoblast differentiation and its transient silencing in mature osteoblasts by siRNAs upregulated the transcription of the Sost1 gene encoding sclerostin, and decreased Wnt3a and Col1a1 mRNAs, suggesting an altered osteoblast activity due to an impairment of the Wnt pathway. In line with this, osteoblasts treated with the Penk1 encoded peptide, Met-enkephalin, showed an increase of Osx and Col1a1 mRNAs and enhanced nodule mineralization. Interestingly, primary osteoblasts isolated from Penk1−/− mice showed lower metabolic activity, ALP activity, and nodule mineralization, as well as a lower number of CFU-F compared to osteoblasts isolated from WT mice, suggesting that, unlike the transient inhibition, the chronic Penk1 deletion affects both osteoblast differentiation and activity. Taken together, these results highlight a role for Penk1 in the regulation of the response of the bone to mechanical unloading, potentially acting on osteoblast differentiation and activity in a cell-autonomous manner.

G-protein-coupled receptors (GPCRs) are key signaling proteins that mostly function as monomers, but for several receptors constitutive dimer formation has been described and in some cases is essential for function. Using single-molecule microscopy combined with super-resolution techniques on intact cells, we describe here a dynamic monomer–dimer equilibrium of µ-opioid receptors (µORs), where dimer formation is driven by specific agonists. The agonist DAMGO, but not morphine, induces dimer formation in a process that correlates both temporally and in its agonist- and phosphorylation-dependence with β-arrestin2 binding to the receptors. This dimerization is independent from, but may precede, µOR internalization. These data suggest a new level of GPCR regulation that links dimer formation to specific agonists and their downstream signals. Single-molecule and super-resolution approaches define a monomer–dimer equilibrium of µ-opioid receptors and show that receptors form agonist-induced dimers coincident with β-arrestin2 binding to receptors.