C1q/TNF-related protein 12 (CTRP12) is a secreted regulator of glucose and lipid metabolism. It circulates in plasma as a full-length protein or as a cleaved isoform generated by furin/PCSK3 cleavage. These isoforms preferentially activate different signaling pathways, and their ratio in plasma is altered in obesity and diabetes. Here, we show that three conserved asparagine residues (Asn-39, Asn-287, and Asn-297) play important roles in modulating CTRP12 cleavage, secretion, and stability. Mass spectrometry analysis provided direct evidence of Asn-39 glycosylation. When N-linked glycosylation was inhibited by tunicamycin or abolished by the N39Q, N39A, or T41A mutation, CTRP12 cleavage was enhanced. Complex-type N-glycans on CTRP12 blocked cleavage by the Golgi-localized furin. In N-acetylglucosaminyltransferase I (GnTI)-deficient cells that could not form hybrid and complex-type N-glycans in the Golgi, CTRP12 cleavage was enhanced, and re-expressing GnTI reduced cleavage. Replacing the nonglycosylated Asn-297 with glutamine or alanine also increased CTRP12 cleavage. Both Asn-39 and Asn-297 contributed independently to CTRP12 cleavage: maximum cleavage was observed in the double mutant. In addition, CTRP12 cleavage was abolished in furin-deficient cells and restored by furin re-expression. Replacing the nonglycosylated Asn-287 with glutamine or alanine resulted in protein misfolding and aggregation, leading to retention in the endoplasmic reticulum. Cycloheximide chase analyses indicated reduced protein stability for N39Q, T41A, and N297Q mutants. Lastly, we show that increasing the flux through the hexosamine biosynthesis pathway by exogenous glucosamine, known to disrupt protein glycosylation, also promoted CTRP12 cleavage. Combined, these data highlight glycosylation-dependent and -independent mechanisms regulating CTRP12 cleavage, secretion, and protein stability.

2019-02-01·European journal of pharmacology3区 · 医学

The antihyperalgesic effect of docosahexaenoic acid in streptozotocin-induced neuropathic pain in the rat involves the opioidergic system

3区 · 医学

Article

作者: Landa-Juárez, Arizai Yolia ; Ortiz, Mario I ; Castañeda-Hernández, Gilberto ; Chávez-Piña, Aracely Evangelina ; Pérez-Severiano, Francisca

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid that has shown an antinociceptive effect in multiple pain models, such as inflammatory and neuropathic pain by chronic constriction injury in rats; however, its mechanism of action is still not well-understood. Reports suggest that DHA activates opioid signaling, but there is no information on this from a model of neuropathic pain. As a result, the aims of this study were (1) to determine the antihyperalgesic and antiallodynic effect of peripheral DHA administration, and (2) to evaluate the participation of the opioid receptors in the antihyperalgesic effect of DHA on streptozotocin-induced neuropathic pain in the rat. Female Wistar rats were injected with streptozotocin (50 mg/kg, i.p.) to induce hyperglycemia. The formalin, Hargreaves, and von Frey filaments tests were used to assess the nociceptive activity. Intraplantar administration of DHA (100-1000 μg/paw) or gabapentin (562-1778 μg/paw) decreased formalin-evoked hyperalgesia in diabetic rats, in a dose-dependent manner. Furthermore, DHA (562 μg/paw) and gabapentin (1000 μg/paw) reduced thermal hyperalgesia and allodynia. Local peripheral administration of naloxone (non-selective opioid receptor antagonist; 100 μg/paw), naltrindole (selective δ receptor antagonist; 1 μg/paw), and CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, μ receptor antagonist; 20 μg/paw) prevented formalin-evoked hyperalgesia in diabetic rats but not by GNTI (guanidinonaltrindole, κ receptor antagonist;1 µg/paw). It is suggested that peripheral DHA shows an antihyperalgesic effect in neuropathic pain in the rat. Furthermore, δ and μ receptors are involved in the antihyperalgesic peripheral effect of DHA in diabetic rats.

2018-01-01·International journal of medical sciences3区 · 医学

Manual acupuncture relieves bile acid-induced itch in mice: the role of microglia and TNF-α

3区 · 医学

ArticleOA

作者: Lee, Yu-Chen ; Hung, Shih-Ya ; Chung, Hsin-Yi ; Lin, Chia-Hsien ; Chen, Yi-Hung ; Lin, Pei-Lin ; Chu, Yu-Ting ; MacDonald, Iona ; Luo, Sih-Ting

Pruritus, or itch, is a frequent complaint amongst patients with cholestatic hepatobiliary disease and is difficult to manage, with many patients refractory to currently available antipruritic treatments. In this study, we examined whether manual acupuncture (MA) at particular acupoints represses deoxycholic acid (DCA)-induced scratching behavior and microglial activation and compared these effects with those induced by another pruritogen, 5'-guanidinonaltrindole (GNTI, a kappa opioid receptor antagonist). MA at Hegu (LI4) and Quchi (LI11) acupoints significantly attenuated DCA- and GNTI-induced scratching, whereas no such effects were observed at the bilateral Zusanli acupoints (ST36). Interestingly, GNTI-induced scratching was reduced similarly by both MA and electroacupuncture (EA) at the LI4 and LI11 acupoints. MA at non-acupoints did not affect scratching behavior. Intraperitoneal injection of minocycline (a microglial inhibitor) reduced GNTI- and DCA-induced scratching behavior. In Western blot analysis, subcutaneous DCA injection to the back of the neck increased spinal cord expression of ionized calcium-binding adapter molecule 1 (Iba1) and tumor necrosis factor-alpha (TNF-α) as compared with saline injection, while MA at LI4 and LI11 reduced these DCA-induced changes. Immunofluorescence confocal microcopy revealed that DCA-induced Iba1-positive cells with thicker processes emanated from the enlarged cell bodies, while this effect was attenuated by pretreatment with MA. It is concluded that microglia and TNF-α play important roles in the itching sensation and MA reduces DCA-induced scratching behavior by alleviating spinal microglial activation. MA may be an effective treatment for cholestatic pruritus.

100 项与 GNTI (University of Minnesota) 相关的药物交易

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