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Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer’s disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

Methylphenidate (MPH) and methamphetamine (METH) are the current treatments of choice for attention deficit/hyperactivity disorder. We previously reported that METH induces the release of dopamine (DA) and of the neurotransmitter candidate L-3,4-dihydroxyphenylalanine (L-DOPA). In contrast, we here found that MPH increased the DA release while it did not affect the L-DOPA release from the dorsolateral striatum. Nevertheless, MPH-induced hyperlocomotion was reduced in Gpr143 (L-DOPA receptor) gene-deficient (Gpr143−/y) mice. The rewarding effect and increased c-fos expression induced by MPH were also attenuated in Gpr143−/y mice. Together, these findings suggest that GPR143 is involved in the acute and chronic actions of MPH.

Pulmonary hypertension (PH) is a severe and progressive disease that causes elevated right ventricular systolic pressure, right ventricular hypertrophy and ultimately right heart failure. However, the underlying pathophysiologic mechanisms are poorly understood. We previously showed that 3,4-l-dihydroxylphenyalanine (DOPA) sensitizes vasomotor response to sympathetic tone via coupling between the adrenergic receptor alpha1 (ADRA1) and a G protein-coupled receptor 143 (GPR143), a DOPA receptor. We investigated whether DOPA similarly enhances ADRA1-mediated contraction in pulmonary arteries isolated from rats, and whether GPR143 is involved in the PH pathogenesis. Pretreating the isolated pulmonary arteries with DOPA 1 μM enhanced vasoconstriction in response to phenylephrine, an ADRA1 agonist, but not to U-46619, a thromboxane A2 agonist or endothelin-1. We generated Gpr143 gene-deficient (Gpr143-/y) rats, and confirmed that DOPA did not augment phenylephrine-induced contractile response in Gpr143-/y rat pulmonary arteries. We utilized a rat model of monocrotaline (MCT)-induced PH. In the MCT model, the right ventricular systolic pressure was attenuated in the Gpr143-/y rats than in WT rats. Phenylephrine-induced cell migration and proliferation were also suppressed in Gpr143-/y pulmonary artery smooth muscle cells than in WT cells. Our result suggests that GPR143 is involved in the PH pathogenesis in the rat models of PH.

Collapsin response mediator proteins (CRMPs) are key for brain development and function. Here, we link CRMP1 to a neurodevelopmental disorder. We report heterozygous de novo variants in the CRMP1 gene in three unrelated individuals with muscular hypotonia, intellectual disability, and/or autism spectrum disorder. Based on in silico analysis these variants are predicted to affect the CRMP1 structure. We further analyzed the effect of the variants on the protein structure/levels and cellular processes. We showed that the human CRMP1 variants impact the oligomerization of CRMP1 proteins. Moreover, overexpression of the CRMP1 variants affect neurite outgrowth of murine cortical neurons. While altered CRMP1 levels have been reported in psychiatric diseases, genetic variants in CRMP1 gene have never been linked to human disease. We report for the first-time variants in the CRMP1 gene and emphasize its key role in brain development and function by linking directly to a human neurodevelopmental disease.

In amyotrophic lateral sclerosis (ALS), neurodegeneration is characterized by distal axonopathy that begins at the distal axons, including the neuromuscular junctions, and progresses proximally in a “dying back” manner prior to the degeneration of cell bodies. However, the molecular mechanism for distal axonopathy in ALS has not been fully addressed. Semaphorin 3A (Sema3A), a repulsive axon guidance molecule that phosphorylates collapsin response mediator proteins (CRMPs), is known to be highly expressed in Schwann cells near distal axons in a mouse model of ALS. To clarify the involvement of Sema3A–CRMP signaling in the axonal pathogenesis of ALS, we investigated the expression of phosphorylated CRMP1 (pCRMP1) in the spinal cords of 35 patients with sporadic ALS and seven disease controls. In ALS patients, we found that pCRMP1 accumulated in the proximal axons and co-localized with phosphorylated neurofilaments (pNFs), which are a major protein constituent of spheroids. Interestingly, the pCRMP1:pNF ratio of the fluorescence signal in spheroid immunostaining was inversely correlated with disease duration in 18 evaluable ALS patients, indicating that the accumulation of pCRMP1 may precede that of pNFs in spheroids or promote ALS progression. In addition, overexpression of a phospho-mimicking CRMP1 mutant inhibited axonal outgrowth in Neuro2A cells. Taken together, these results indicate that pCRMP1 may be involved in the pathogenesis of axonopathy in ALS, leading to spheroid formation through the proximal progression of axonopathy.

Recent studies have shown that mutation at Ser522 causes inhibition of collapsin response mediator protein 2 (CRMP2) phosphorylation and induces axon elongation and partial recovery of the lost sensorimotor function after spinal cord injury (SCI). We aimed to reveal the intracellular mechanism in axotomized neurons in the CRMP2 knock-in (CRMP2KI) mouse model by performing transcriptome analysis in mouse sensorimotor cortex using micro-dissection punching system. Prior to that, we analyzed the structural pathophysiology in axotomized or neighboring neurons after SCI and found that somatic atrophy and dendritic spine reduction in sensorimotor cortex were suppressed in CRMP2KI mice. Further analysis of the transcriptome has aided in the identification of four hemoglobin genes Hba-a1, Hba-a2, Hbb-bs, and Hbb-bt that are significantly upregulated in wild-type mice with concomitant upregulation of genes involved in the oxidative phosphorylation and ribosomal pathways after SCI. However, we observed substantial upregulation in channel activity genes and downregulation of genes regulating vesicles, synaptic function, glial cell differentiation in CRMP2KI mice. Moreover, the transcriptome profile of CRMP2KI mice has been discussed wherein energy metabolism and neuronal pathways were found to be differentially regulated. Our results showed that CRMP2KI mice displayed improved SCI pathophysiology not only via microtubule stabilization in neurons, but also possibly via the whole metabolic system in the central nervous system, response changes in glial cells, and synapses. Taken together, we reveal new insights on SCI pathophysiology and the regenerative mechanism of central nervous system by the inhibition of CRMP2 phosphorylation at Ser522. All these experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Waseda University, Japan (2017-A027 approved on March 21, 2017; 2018-A003 approved on March 25, 2018; 2019-A026 approved on March 25, 2019).

l-3,4-dihydroxyphenylalanine (l-DOPA) is a candidate neurotransmitter. l-DOPA is released by nicotine through nicotinic receptors. Recently, G-protein coupled receptor GPR143, was identified as a receptor for l-DOPA. In this study, genetic association studies between GPR143 genetic polymorphisms and smoking behaviors revealed that the single-nucleotide polymorphism rs6640499, in the GPR143 gene, was associated with traits of smoking behaviors in Japanese individuals. In Gpr143 gene-deficient mice, nicotine-induced hypolocomotion and rewarding effect were attenuated compared to those in wild-type mice. Our findings suggest the involvement of GPR143 in the smoking behaviors.

Neurite outgrowth is a complex differentiation process regulated by external and/or internal mechanisms. Among external mechanisms, G-protein coupled receptors (GPCRs) have been implicated in this process, but the pathways involved are not fully understood. L-3,4-dihydroxyphenylalanine (l-DOPA) is considered to be inert by itself, and to relieve Parkinson's disease through its conversion to dopamine. We have proposed that l-DOPA acts as a neurotransmitter. GPR143, the gene product of ocular albinism 1 (OA1), was identified as a receptor for l-DOPA. OA1 is an X-linked disorder characterized by all typical visual anomalies associated with hypopigmentation and optic misrouting, resulting in severe reduction of visual acuity. However, the molecular basis for this phenotype remains unknown. To study the function of GPR143, we investigated the phenotypic effect of overexpression of GPR143 in pheochromocytoma (PC12) cells treated with nerve growth factor. Overexpression of mouse GPR143 inhibited neurite outgrowth, and the effect was mitigated by l-DOPA cyclohexylester, an antagonist for l-DOPA. Furthermore, knockdown of G-protein Gα13 attenuated mouse GPR143 induced inhibition of neurite outgrowth. Human wild-type (wt) GPR143 also inhibited neurite outgrowth, but its mutants did not mimic the effect of wt GPR143. Our results provide a mechanism for axon guidance phenotype in ocular albinism 1. Keywords: GPR143, Neurite outgrowth, Pheochromocytoma cell, l-DOPA, Ocular albinism 1

Collapsin response mediator proteins (CRMPs), which consist of five homologous cytosolic proteins, are one of the major phosphoproteins in the developing nervous system. The prominent feature of the CRMP family proteins is a new class of microtubule-associated proteins that play important roles in the whole process of developing the nervous system, such as axon guidance, synapse maturation, cell migration, and even in adult brain function. The CRMP C-terminal region is subjected to posttranslational modifications such as phosphorylation, which, in turn, regulates the interaction between the CRMPs and various kinds of proteins including receptors, ion channels, cytoskeletal proteins, and motor proteins. The gene-knockout of the CRMP family proteins produces different phenotypes, thereby showing distinct roles of all CRMP family proteins. Also, the phenotypic analysis of a non-phosphorylated form of CRMP2-knockin mouse model, and studies of pharmacological responses to CRMP-related drugs suggest that the phosphorylation/dephosphorylation process plays a pivotal role in pathophysiology in neuronal development, regeneration, and neurodegenerative disorders, thus showing CRMPs as promising target molecules for therapeutic intervention.

Pancreatic intraepithelial neoplasia (PanIN), the most common premalignant lesion of the pancreas, is a histologically well-defined precursor to invasive pancreatic ductal adenocarcinoma (PDAC). However, the molecular mechanisms underlying the progression of PanINs have not been fully elucidated. Previously, we demonstrated that the expression of collapsin response mediator protein 4 (CRMP4) in PDAC was associated with poor prognosis. The expression of CRMP4 was also augmented in a pancreatitis mouse model. However, the role of CRMP4 in the progression of PanIN lesions remains uncertain. In the present study, we examined the relationship between CRMP4 expression and progression of PanIN lesions using genetically engineered mouse models. PanIN lesions were induced by peritoneal injection of the cholecystokinin analog caerulein in LSL-KRASG12D; Pdx1-Cre (KC-Crmp4 wild-type, WT) mice and LSL-KRASG12D; Pdx1-Cre; Crmp4−/− (KC-Crmp4 knockout, KO) mice. We analyzed pancreatic tissue sections from these mice and evaluated PanIN grade by hematoxylin and eosin staining. CRMP4 expression was examined and the cellular components assessed by immunohistochemistry using antibodies against CRMP4, CD3, and α-smooth muscle actin (SMA). The incidence of high-grade PanIN in KC-Crmp4 WT mice was higher than that in KC-Crmp4 KO animals. CRMP4 was expressed not only in epithelial cells but also in αSMA-positive cells in stromal areas of PanIN lesions. The CRMP4 expression in stromal areas correlated with PanIN grade in WT mice. These results suggested that the expression of CRMP4 in stromal cells may underlie the incidence or progression of PanIN.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates local translation in dendrites and spines for synaptic plasticity. In axons, FMRP is implicated in axonal extension and axon guidance. We previously demonstrated the involvement of FMRP in growth cone collapse via a translation-dependent response to Semaphorin-3A (Sema3A), a repulsive axon guidance factor. In the case of attractive axon guidance factors, RNA-binding proteins such as zipcode binding protein 1 (ZBP1) accumulate towards the stimulated side of growth cones for local translation. However, it remains unclear how Sema3A effects FMRP localization in growth cones. Here, we show that levels of FMRP in growth cones of hippocampal neurons decreased after Sema3A stimulation. This decrease in FMRP was suppressed by the ubiquitin-activating enzyme E1 enzyme inhibitor PYR-41 and proteasome inhibitor MG132, suggesting that the ubiquitin-proteasome pathway is involved in Sema3A-induced FMRP degradation in growth cones. Moreover, the E1 enzyme or proteasome inhibitor suppressed Sema3A-induced increases in microtubule-associated protein 1B (MAP1B) in growth cones, suggesting that the ubiquitin-proteasome pathway promotes local translation of MAP1B, whose translation is mediated by FMRP. These inhibitors also blocked the Sema3A-induced growth cone collapse. Collectively, our results suggest that Sema3A promotes degradation of FMRP in growth cones through the ubiquitin-proteasome pathway, leading to growth cone collapse via local translation of MAP1B. These findings reveal a new mechanism of axon guidance regulation: degradation of the translational suppressor FMRP via the ubiquitin-proteasome pathway.

Alzheimer’s disease (AD) is an incurable neurodegenerative disease characterized by memory loss and neurotoxic amyloid beta (Aβ) plaques accumulation. Numerous pharmacological interventions targeting Aβ plaques accumulation have failed to alleviate AD. Also, the pathological alterations in AD start years before the onset of clinical symptoms. To identify proteins at play during the early stage of AD, we conducted proteomic analysis of the hippocampus of young AppNL-F mice model of AD at the preclinical phase of the disease. This was followed by interactome ranking of the proteome into hubs that were further validated in vivo using immunoblot analysis. We also performed double-immunolabeling of these hub proteins and Aβ to quantify colocalization.Behavioral analysis revealed no significant difference in memory performance between 8-month-old AppNL-F and control mice. The upregulation and downregulation of several proteins were observed in the AppNL-F mice compared to control. These proteins corresponded to pathways and processes related to Aβ clearance, inflammatory-immune response, transport, mitochondrial metabolism, and glial cell proliferation. Interactome analysis revealed several proteins including DLGP5, DDX49, CCDC85A, ADCY6, HEPACAM, HCN3, PPT1 and TNPO1 as essential proteins in the AppNL-F interactome. Validation by immunoblot confirmed the over-expression of these proteins except HCN3 in the early-stage AD mice hippocampus. Immunolabeling revealed a significant increase in ADCY6/Aβ and HEPACAM/Aβ colocalized puncta in AppNL-F mice compared to WT. These data suggest that these proteins may be involved in the early stage of AD. Our work suggests new targets and biomarkers for AD diagnosis and therapeutic intervention.

l-3,4-Dihydroxyphenylalanine (l-DOPA) is the most effective therapeutic agent for Parkinson’s disease (PD). l-DOPA is traditionally believed to be an inert amino acid that exerts actions and effectiveness in PD through its conversion to dopamine. In contrast to this generally accepted idea, l-DOPA is proposed to be a neurotransmitter. Recently, GPR143 (OA1), the gene product of ocular albinism 1 was identified as a receptor candidate for l-DOPA. GPR143 is widely expressed in the central and peripheral nervous system. GPR143 immunoreactivity was colocalized with phosphorylated α-synuclein in Lewy bodies in PD brains. GPR143 may contribute to the therapeutic effectiveness of l-DOPA and might be related to pathogenesis of PD.

Through its conversion to dopamine by aromatic l-amino acid decarboxylase (AADC), l-3,4-dihydroxyphenylalanine (l-DOPA) replenishes depleted brain dopamine in Parkinson's disease patients. We recently identified GPR143 as a candidate receptor for l-DOPA. In this study, we investigated the behavioral actions of l-DOPA in wild type (wt) and Gpr143-deficient mice. l-DOPA dose-dependently (10–100 mg/kg, i.p.) induced ptosis under treatment with 3-hydroxybenzylhydrazine, a centrally acting AADC inhibitor. This effect was not mimicked by 3-O-methyldopa. l-DOPA-induced ptosis in Gpr143-deficient mice to a similar extent as in wt mice. These results suggest that l-DOPA induces ptosis in a GPR143-independent fashion in mice.

Induced pluripotent stem (iPS) cells are promising tools to investigate disease mechanism and develop new drugs. Intraneuronal transport, which is fundamental for neuronal survival and function, is vulnerable to various pharmacological and chemical agents and is disrupted in some neurodegenerative disorders. We applied a quantification method for axonal transport by counting CM-DiI–labeled particles traveling along the neurite, which allowed us to monitor and quantitate, for the first time, intraneuronal transport in human neurons differentiated from iPS cells (iCell neurons). We evaluated the acute effects of several anti-neoplastic agents that have been previously shown to affect intraneuronal transport. Vincristine, paclitaxel and oxaliplatin decreased the number of moving particle along neurites. Cisplatin, however, produced no effect on intraneuronal transport, which is in contrast to our previous report indicating that it inhibits transport in chick dorsal root ganglion neurons. Our system may be a useful method for assessing intraneuronal transport and neurotoxicity in human iPS neurons.

AIM: To assess the efficacy of topical Semaphorin-3A (SEMA3A) in the treatment of allergic conjunctivitis. METHODS: Experimental allergic conjunctivitis (EAC) mice model induced by short ragweed pollen (SRW) in 4-week-old of BALB/c mice, mice were evaluated using haematoxylin and eosin (H&E) staining, immunofluorescence and light microscope photographs. Early phase took the samples in 24h after instillation and late phase took the samples between 4 to 14d after the start of treatment. The study use of topical SEMA3A (10 U, 100 U, 1000 U) eye drops and subconjunctival injection of SEMA3A with same concentration. For comparison, five types of allergy eyedrops were quantified using clinical characteristics. RESULTS: Clinical score of composite ocular symptoms of the mice treated with SEMA3A were significantly decreased both in the immediate phase and the late phase compared to those treated with commercial ophthalmic formulations and non-treatment mice. SEMA3A treatment attenuates infiltration of eosinophils entering into conjunctiva in EAC mice. The score of eosinophil infiltration in the conjunctiva of SEMA3A 1000 U-treated group were significantly lower than low-concentration of SEMA3A treated groups and non-treated group. SEMA3A treatment also suppressed T-cell proliferation in vitro and decreased serum total IgE levels in EAC mice. Moreover, Treatment of SEMA3A suppressed Th2-related cytokines (IL-5, IL-13 and IL-4) and pro-inflammatory cytokines (IFN-γ, IL-17 and TNF-α) release, but increased regulatory cytokine IL-10 concentration in the conjunctiva of EAC mice. CONCLUSIONS: SEMA3A as a biological agent, showed the beneficial activity in ocular allergic processes with the less damage to the intraocular tissue. It is expected that SEMA3A may be contributed in patients with a more severe spectrum of refractory ocular allergic diseases including allergic conjunctivitis in the near future.

l-3,4-Dihydroxyphenylalanine (DOPA) is the metabolic precursor of dopamine, and the single most effective agent in the treatment of Parkinson’s disease. One problem with DOPA therapy for Parkinson’s disease is its cardiovascular side effects including hypotension and syncope, the underlying mechanisms of which are largely unknown. We proposed that DOPA is a neurotransmitter in the central nervous system, but specific receptors for DOPA had not been identified. Recently, the gene product of ocular albinism 1 (OA1) was shown to possess DOPA-binding activity. It was unknown, however, whether or not OA1 is responsible for the actions of DOPA itself. Immunohistochemical examination revealed that OA1 was expressed in the nucleus tractus solitarii (NTS). OA1-positive cells adjacent to tyrosine hydroxylase–positive cell bodies and nerve fibers were detected in the depressor sites of the NTS. OA1 knockdown using oa1specific shRNA-adenovirus vectors in the NTS reduced the expression levels of OA1 in the NTS. The prior injection of the shRNA against OA1 suppressed the depressor and bradycardic responses to DOPA but not to glutamate in the NTS of anesthetized rats. Thus OA-1 is a functional receptor of DOPA in the NTS, which warrants reexamination of the mechanisms for the therapeutic and untoward actions of DOPA. Keywords:: DOPA, neurotransmitter, nucleus tractus solitarii, GPCR, baroreceptor reflex

Proper density and morphology of dendritic spines are important for higher brain functions such as learning and memory. However, our knowledge about molecular mechanisms that regulate the development and maintenance of dendritic spines is limited. We recently reported that cyclin-dependent kinase 5 (Cdk5) is required for the development and maintenance of dendritic spines of cortical neurons in the mouse brain. Previous in vitro studies have suggested the involvement of Cdk5 substrates in the formation of dendritic spines; however, their role in spine development has not been tested in vivo. Here, we demonstrate that Cdk5 phosphorylates collapsin response mediator protein 2 (CRMP2) in the dendritic spines of cultured hippocampal neurons and in vivo in the mouse brain. When we eliminated CRMP2 phosphorylation in CRMP2KI/KI mice, the densities of dendritic spines significantly decreased in hippocampal CA1 pyramidal neurons in the mouse brain. These results indicate that phosphorylation of CRMP2 by Cdk5 is important for dendritic spine development in cortical neurons in the mouse hippocampus.

Axonal transport plays a crucial role in neuronal morphogenesis, survival and function. Despite its importance, however, the molecular mechanisms of axonal transport remain mostly unknown because a simple and quantitative assay system for monitoring this cellular process has been lacking. In order to better characterize the mechanisms involved in axonal transport, we formulate a novel computer-assisted monitoring system of axonal transport. Potential uses of this system and implications for future studies will be discussed.

Abstract.: Sneezing and persistent itching of the nasal mucosa are distressing symptoms of allergic rhinitis (AR). Recent studies have revealed that hyperinnervation of sensory neurons in the nasal turbinate is one of the underlying causes of sneezing and itching. Since Semaphorin-3A (Sema3A) has been previously shown to restrict innervation of sensory neurons, it is presumed that reduced Sema3A expression in the nasal mucosa might contribute to the hypersensitivity. Analysis of the mouse model of ovalbumin-sensitized AR demonstrated a decreased expression of Sema3A in the nasal epithelium, which was accompanied by an increased nerve fiber density in the lamina propria of the turbinate. In rescue experiments, intranasal administration of recombinant Sema3A in the AR model mice alleviated sneezing and nasal rubbing symptoms. In addition, histological examinations also revealed that nerve fiber density was decreased in the lamina propria of the Sema3A-treated nasal turbinate. These results suggest that the nasal hypersensitivity of AR may be attributed to reduction of Sema3A expression and intranasal administration of Sema3A may provide a novel approach to alleviate the allergic symptoms for AR treatment. Keywords:: allergic rhinitis, itch, nasal mucosa, protein gene-product 9.5 (PGP9.5), Semaphorin-3A

Axonal transport plays a crucial role in neuronal morphogenesis, survival, and function. Despite its importance, however, the molecular mechanisms of axonal transport remain mostly unknown because a simple and quantitative assay system for axonal transport has been lacking. In order to better characterize the molecular mechanisms involved in axonal transport, we here developed a computer-assisted monitoring system. Using lipophilic fluorochrome chloromethylbenzamido dialkylcarbocyanine (CM-DiI) as a labeling dye, we have successfully labeled membranous organelles in cultured chick dorsal root ganglia neurons. We confirmed that sodium azide, an ATPase inhibitor, and nocodazole, a microtubule-destabilizing agent, markedly suppressed anterograde and retrograde axonal transport of CM-DiI–labeled particles. We further tested the effects of several anti-neoplastic drugs on axonal transport. Paclitaxel, vincristine, cisplatin, and oxaliplatin, all of which are known to be neurotoxic and to cause neurological symptoms, suppressed anterograde and retrograde axonal transport. Another series of anti-neoplastic drugs, including methotrexate and 5-fluorouracil, did not affect the axonal transport. This is the first report of an automated monitoring system for axonal transport. This system will be useful for toxicity assays, characterizing axonal transport, or screening drugs that may modify neuronal functions. Keywords:: axonal transport, automated system, chloromethylbenzamido dialkylcarbocyanine (CM-DiI), anti-cancer drug, dorsal root ganglia

The precise role of AMP-activated protein kinase (AMPK), a target of metformin, in pancreatic β cells remains controversial, even though metformin was recently shown to enhance the expression of incretin receptors (GLP-1 and GIP receptors) in pancreatic β cells. In this study, we investigated the effect of AMPK in the regulation of incretin receptors expression in pancreatic islets. The phosphorylation of AMPK in the mouse islets was decreased by increasing glucose concentrations. We showed the expression of incretin receptors in bell-shaped response to glucose. Expression of the incretin receptors in the isolated islets showed higher levels under a medium glucose concentration (11.1 mM) than that under a low glucose concentration (2.8 mM), but was suppressed under a high glucose concentration (22.2 mM). Both treatment with an AMPK inhibitor and DN-AMPK expression produced a significant increase of the incretin receptors expression under a low glucose concentration. By contrast, in hyperglycemic db/db islets, the enhancing effect of the AMPK inhibitor on the expression of incretin receptors was diminished under a low glucose concentration. Taken together, AMPK is involved in the regulation of incretin receptors expression in pancreatic islets under a low glucose concentration.