Although amino acids are dietary nutrients that evoke the secretion of glucagon-like peptide 1 (GLP-1) from intestinal L cells, the precise molecular mechanism(s) by which amino acids regulate GLP-1 ...secretion from intestinal L cells remains unknown. Here, we show that the G protein-coupled receptor (GPCR), family C group 6 subtype A (GPRC6A), is involved in amino acid-induced GLP-1 secretion from the intestinal L cell line GLUTag. Application of l-ornithine caused an increase in intracellular Ca2+ concentration (Ca2+i) in GLUTag cells. Application of a GPRC6A receptor antagonist, a phospholipase C inhibitor, or an IP3 receptor antagonist significantly suppressed the l-ornithine-induced Ca2+i increase. We found that the increase in Ca2+i stimulated by l-ornithine correlated with GLP-1 secretion and that l-ornithine stimulation increased exocytosis in a dose-dependent manner. Furthermore, depletion of endogenous GPRC6A by a specific small interfering RNA (siRNA) inhibited the l-ornithine-induced Ca2+i increase and GLP-1 secretion. Taken together, these findings suggest that the GPRC6A receptor functions as an amino acid sensor in GLUTag cells that promotes GLP-1 secretion.
Background: The molecular mechanisms underlying GLP-1 secretion induced by amino acids from intestinal L cells are not fully understood.
Results: The l-amino acid-sensing GPRC6A receptor is expressed in the clonal L cell GLUTag. Activation of GPRC6A by l-ornithine evoked GLP-1 secretion.
Conclusion: GLUTag cells respond to amino acids via the GPRC6A receptor.
Significance: A new pathway for GLP-1 secretion induced by amino acids in GLUTag cells was identified.
CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) ...mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20–33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as
EGFP
,
mCherry
,
Cre
, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.
Intracellular cAMP and Ca(2+) are important second messengers that regulate insulin secretion in pancreatic β-cells; however, the molecular mechanism underlying their mutual interaction for ...exocytosis is not fully understood. In the present study, we investigated the interplay between intracellular cAMP and Ca(2+) concentrations (cAMP(i) and Ca(2+)(i) respectively) in the pancreatic β-cell line MIN6 using total internal reflection fluorescence microscopy. For measuring cAMP(i), we developed a genetically encoded yellow fluorescent biosensor for cAMP Flamindo (fluorescent cAMP indicator), which changes fluorescence intensity with cAMP binding. Application of high-KCl or glucose to MIN6 cells induced the elevation of cAMP(i) and exocytosis. Furthermore, application of an L-type Ca(2+) channel agonist or ionomycin to induce extracellular Ca(2+) influx evoked the elevation of cAMP(i), whereas application of carbachol or thapsigargin, which mobilize Ca(2+) from internal stores, did not evoke the elevation of cAMP(i). We performed RT (reverse transcription)-PCR analysis and found that Ca(2+)-sensitive Adcy1 (adenylate cyclase 1) was expressed in MIN6 cells. Knockdown of endogenous ADCY1 by small interference RNA significantly suppressed glucose-induced exocytosis and the elevation of both cAMP(i) and Ca(2+)(i). Taken together, the findings of the present study demonstrate that ADCY1 plays an important role in the control of pancreatic β-cell cAMP homoeostasis and insulin secretion.
•Neuritogenesis in primary culture of mouse cortical neurons was examined.•Neuritogenesis was impaired by exposure to NaAsO2 concentrations of 1μM or above.•NaAsO2 exposure to the same concentrations ...also decreased GluA1 level.•GluA1/2 overexpression blocked the inhibition of neurite elongation by NaAsO2.
There has been broad concern that arsenic in the environment exerts neurotoxicity. To determine the mechanism by which arsenic disrupts neuronal development, primary cultured neurons obtained from the cerebral cortex of mouse embryos were exposed to sodium arsenite (NaAsO2) at concentrations between 0 and 2μM from days 2 to 4 in vitro and cell survival, neurite outgrowth and expression of glutamate AMPA receptor subunits were assessed at day 4 in vitro. Cell survival was significantly decreased by exposure to 2μM NaAsO2, whereas 0.5μM NaAsO2 increased cell survival instead. The assessment of neurite outgrowth showed that total neurite length was significantly suppressed by 1μM and 2μM NaAsO2, indicating that the lower concentration of NaAsO2 impairs neuritogenesis before inducing cell death. Immunoblot analysis of AMPA receptor subunit expression showed that the protein level of GluA1, a specific subunit of the AMPA receptor, was significantly decreased by 1μM and 2μM NaAsO2. When immunocytochemistry was used to confirm this effect by staining for GluA1 expression in neuropeptide Y neurons, most of which contain GluA1, GluA1 expression in neuropeptide Y neurons was found to be significantly suppressed by 1μM and 2μM NaAsO2 but to be increased at the concentration of 0.5μM. Finally, to determine whether neurons could be rescued from the NaAsO2-induced impairment of neuritogenesis by compensatory overexpression of GluA1, we used primary cultures of neurons transfected with a plasmid vector to overexpress either GluA1 or GluA2, and the results showed that GluA1/2 overexpression protected against the deleterious effects of NaAsO2 on neurite outgrowth. These results suggest that the NaAsO2 concentration inducing neurite suppression is lower than the concentration that induces cell death and is the same as the concentration that suppresses GluA1 expression. Consequently, the suppression of GluA1 expression by NaAsO2 seems at least partly responsible for neurite suppression induced by NaAsO2.
Since the fusion pore of the secretory vesicle is resealed before complete dilation during 'kiss-and-run' exocytosis, their cargoes are not completely released. Although the transient fusion pore is ...kept open for several seconds, the precise mechanisms that control fusion pore maintenance, and their physiological significance, are not well understood. Using dual-colour TIRF (total internal reflection fluorescence) microscopy in neuroendocrine PC12 cells, we show that myosin II regulates the fusion pore dynamics during kiss-and-run exocytosis. The release kinetics of mRFP (monomeric red fluorescent protein)-tagged tPA (tissue plasminogen activator) and Venus-tagged BDNF (brain-derived neurotrophic factor), which show slower release kinetics than NPY (neuropeptide Y)-mRFP and insulin-mRFP, were prolonged by the overexpression of a wild-type form of the RLC (myosin II regulatory light chain). In contrast, overexpression of a dominant-negative form of RLC shortened the release kinetics. Using spH (synapto-pHluorin), a green fluorescent protein-based pH sensor inside the vesicles, we confirmed that the modulation of the release kinetics by myosin II is due to changes in the duration of fusion pore opening. In addition, we revealed that the amount of hormone released into the extracellular space upon stimulation was increased by overexpression of wild-type RLC. We propose that the duration of fusion pore opening is regulated by myosin II to control the amount of hormone released from a single vesicle.
Obesity is often associated with aging. However, the mechanism of age-related obesity is unknown. The melanocortin-4 receptor (MC4R) mediates leptin-melanocortin anti-obesity signaling in the ...hypothalamus. Here, we discovered that MC4R-bearing primary cilia of hypothalamic neurons progressively shorten with age in rats, correlating with age-dependent metabolic decline and increased adiposity. This “age-related ciliopathy” is promoted by overnutrition-induced upregulation of leptin-melanocortin signaling and inhibited or reversed by dietary restriction or the knockdown of ciliogenesis-associated kinase 1 (CILK1). Forced shortening of MC4R-bearing cilia in hypothalamic neurons by genetic approaches impaired neuronal sensitivity to melanocortin and resulted in decreased brown fat thermogenesis and energy expenditure and increased appetite, finally developing obesity and leptin resistance. Therefore, despite its acute anti-obesity effect, chronic leptin-melanocortin signaling increases susceptibility to obesity by promoting the age-related shortening of MC4R-bearing cilia. This study provides a crucial mechanism for age-related obesity, which increases the risk of metabolic syndrome.
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•MC4R-bearing primary cilia (MC4R+ cilia) of hypothalamic neurons shorten with age•Shortened MC4R+ cilia blunt satiety signals, leading to obesity and leptin resistance•Chronic leptin-melanocortin signaling promotes obesogenic shortening of MC4R+ cilia•Dietary restriction or Cilk1 knockdown reverses or inhibits MC4R+ ciliary shortening
The melanocortin-4 receptor (MC4R) mediates leptin-melanocortin anti-obesity signaling in the hypothalamus. Oya et al. discovered that MC4R-bearing primary cilia of hypothalamic neurons progressively shorten with age to develop age-related obesity and leptin resistance. This “age-related ciliopathy” is promoted by chronic leptin-melanocortin signaling and inhibited by dietary restriction.
•A vesicular nucleotide transporter (VNUT) transports ATP into vesicles.•VNUT was expressed in astrocytes and localized on secretory lysosome.•VNUT was participated in ATP storage in secretory ...lysosomes.•VNUT-associated lysosomes partially fused to the plasma membrane after exocytosis.•VNUT was participated in ATP release from lysosomes in astrocytes.
Recent studies have suggested that astrocytes release gliotransmitters (i.e., ATP, L-glutamate, D-serine, and peptide hormones) and participate actively in synaptic functioning. Although ATP release from astrocytes modulates the activity of neurons, the mechanisms regulating the ATP release from astrocytes and the source of ATP in astrocytes are not well understood. Recently a vesicular nucleotide transporter (VNUT)/solute carrier family 17, member 9 (SLC17A9) has been identified as a mediator of the active accumulation of ATP into vesicles. Here we show by immunocytochemical analysis under confocal microscope and live cell imaging under total internal reflection fluorescence microscope that lysosome-associated VNUT is responsible for ATP release in astrocytes. VNUT was expressed in both primary cultured cortical astrocytes and glioma cell line C6 cells, and mainly localized on lysosome in the cells. We found that VNUT-associated secretory lysosomes do not fully collapse into the plasma membrane after lysosomal exocytosis. We also found that inhibition of VNUT function by Evans Blue decreased ATP uptake into secretory lysosomes. Depletion and inhibition of endogenous VNUT by small interference RNA and Evans Blue, respectively decreased the amount of ATP release from the cells, whereas overexpression of VNUT increased it. Taken together, these findings indicate that the participation of VNUT in ATP storage in secretory lysosomes during lysosomal exocytosis of ATP from astrocytes.
Amino acids such as l‐glutamate and l‐arginine are potent stimuli for insulin secretion from pancreatic β‐cells. However, the precise molecular mechanisms of amino acid‐induced insulin secretion have ...only partly understood. In this study, we show here that mouse pancreatic β‐cell line MIN6 cells expressed amino acid taste receptor (heterodimer of type 1 taste G protein‐coupled receptor member 1 and member 3; Tas1R1 and Tas1R3, respectively). We found that administration of l‐glutamate or l‐arginine to MIN6 cells caused the increase in free intracellular concentrations of both inositol‐1,4,5‐triphosphate (IP3) and Ca2+, and umami substance inocinate enhanced the effects of l‐glutamate. Effects of amino acids on intracellular IP3 and Ca2+ concentration were diminished by application of lactisole, a Tas1R3 receptor antagonist. Furthermore, we investigated the effect of amino acids on the insulin release from MIN6 cells by both ELISA and total internal reflection fluorescence microscopy. Application of l‐glutamate or l‐arginine significantly increased the release of insulin, whereas inhibited by the application of lactisole. Based on these findings, we propose that heterodimer of Tas1R1 and Tas1R3 is the fundamental receptor for the sensing amino acids and regulates the amino acid‐induced insulin secretion in pancreatic β‐cells.
J. Neurochem. (2010) 114, 171-181. The double C2 (Doc2) family is characterized by an N-terminal Munc13-1-interacting domain and C-terminal tandem C2 domains, and it comprises three isoforms, Doc2α, ...Doc2β, and Doc2γ, in humans and mice. Doc2α, the best-characterized, brain-specific isoform, exhibits Ca²⁺-dependent phospholipid-binding activity through its C2A domain, and the Ca²⁺-binding activity is thought to be important for the regulation of Ca²⁺-dependent exocytosis. In contrast to the C2A domain, however, nothing is known about the physiological functions of the C2B domain in regulated exocytosis. In this study, we demonstrated by a mutation analysis that the polybasic sequence in the C2B domain of Doc2α (306 KKSKHKTCVKKK 317) is required for binding of syntaxin-1a/synaptosome-associated protein of 25 kDa (SNAP-25) heterodimer. We also investigated the effect of Lys-to-Gln (named KQ) mutations in the polybasic sequence of the C2B domain on vesicle dynamics by total internal reflection fluorescence microscopy in PC12 cells. A Doc2α(KQ) mutant, which lacks binding activity toward syntaxin-1a/SNAP-25 heterodimer, significantly decreased the number of plasma membrane-docked vesicles before stimulation and strongly inhibited high-KCl-induced exocytosis from the plasma membrane-docked vesicles. These results indicate that the polybasic sequence in the C2B domain functions as a binding site for syntaxin-1a/SNAP-25 heterodimer and controls the number of 'readily releasable' vesicles in neuroendocrine cells.
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