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1.
Cell Metab ; 35(1): 166-183.e11, 2023 01 03.
Article in English | MEDLINE | ID: mdl-36599300

ABSTRACT

Microproteins (MPs) are a potentially rich source of uncharacterized metabolic regulators. Here, we use ribosome profiling (Ribo-seq) to curate 3,877 unannotated MP-encoding small ORFs (smORFs) in primary brown, white, and beige mouse adipocytes. Of these, we validated 85 MPs by proteomics, including 33 circulating MPs in mouse plasma. Analyses of MP-encoding mRNAs under different physiological conditions (high-fat diet) revealed that numerous MPs are regulated in adipose tissue in vivo and are co-expressed with established metabolic genes. Furthermore, Ribo-seq provided evidence for the translation of Gm8773, which encodes a secreted MP that is homologous to human and chicken FAM237B. Gm8773 is highly expressed in the arcuate nucleus of the hypothalamus, and intracerebroventricular administration of recombinant mFAM237B showed orexigenic activity in obese mice. Together, these data highlight the value of this adipocyte MP database in identifying MPs with roles in fundamental metabolic and physiological processes such as feeding.


Subject(s)
Adipocytes, White , Adipose Tissue, Brown , Humans , Animals , Mice , Adipocytes, White/metabolism , Adipose Tissue, Brown/metabolism , Open Reading Frames/genetics , Adipose Tissue, White/metabolism , Adipocytes, Brown/metabolism , Micropeptides
2.
JCI Insight ; 7(17)2022 09 08.
Article in English | MEDLINE | ID: mdl-35917179

ABSTRACT

In rodent models of type 2 diabetes (T2D), central administration of FGF1 normalizes elevated blood glucose levels in a manner that is sustained for weeks or months. Increased activity of NPY/AgRP neurons in the hypothalamic arcuate nucleus (ARC) is implicated in the pathogenesis of hyperglycemia in these animals, and the ARC is a key brain area for the antidiabetic action of FGF1. We therefore sought to determine whether FGF1 inhibits NPY/AgRP neurons and, if so, whether this inhibitory effect is sufficiently durable to offer a feasible explanation for sustained diabetes remission induced by central administration of FGF1. Here, we show that FGF1 inhibited ARC NPY/AgRP neuron activity, both after intracerebroventricular injection in vivo and when applied ex vivo in a slice preparation; we also showed that the underlying mechanism involved increased input from presynaptic GABAergic neurons. Following central administration, the inhibitory effect of FGF1 on NPY/AgRP neurons was also highly durable, lasting for at least 2 weeks. To our knowledge, no precedent for such a prolonged inhibitory effect exists. Future studies are warranted to determine whether NPY/AgRP neuron inhibition contributes to the sustained antidiabetic action elicited by intracerebroventricular FGF1 injection in rodent models of T2D.


Subject(s)
Diabetes Mellitus, Type 2 , Fibroblast Growth Factor 1 , Agouti-Related Protein/pharmacology , Animals , Diabetes Mellitus, Type 2/drug therapy , Fibroblast Growth Factor 1/pharmacology , Hypoglycemic Agents/pharmacology , Neurons
3.
Peptides ; 136: 170444, 2021 02.
Article in English | MEDLINE | ID: mdl-33245952

ABSTRACT

VGF is a peptide precursor expressed in neuroendocrine cells that is suggested to play a role in the regulation of energy homeostasis. VGF is proteolytically cleaved to yield multiple bioactive peptides. However, the specific actions of VGF-derived peptides on energy homeostasis remain unclear. The aim of the present work was to investigate the role of VGF-derived peptides in energy homeostasis and explore the pharmacological actions of VGF-derived peptides on body weight in preclinical animal models. VGF-derived peptides (NERP-1, NERP-2, PGH-NH2, PGH-OH, NERP-4, TLQP-21, TLQP-30, TLQP-62, HHPD-41, AQEE-30, and LQEQ-19) were synthesized and screened for their ability to affect neuronal activity in vitro on hypothalamic brain slices and modulate food intake and energy expenditure after acute central administration in vivo. In addition, the effects of NERP-1, NERP-2, PGH-NH2, TLQP-21, TLQP-62, and HHPD-41 on energy homeostasis were studied after chronic central infusion. NERP-1, PGH-NH2, HHPD-41, and TLQP-62 increased the functional activity of hypothalamic neuronal networks. However, none of the peptides altered energy homeostasis after either acute or chronic ICV administration. The present data do not support the potential use of the tested VGF-derived peptides as novel anti-obesity drug candidates.


Subject(s)
Anti-Obesity Agents/pharmacology , Neuropeptides/genetics , Neuropeptides/pharmacology , Obesity/drug therapy , Animals , Body Weight/drug effects , Disease Models, Animal , Energy Metabolism/drug effects , Humans , Hypothalamus/drug effects , Hypothalamus/metabolism , Hypothalamus/pathology , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Obesity/genetics , Obesity/pathology , Peptide Fragments/genetics , Peptide Fragments/pharmacology , Rats
4.
Nat Commun ; 11(1): 4458, 2020 09 07.
Article in English | MEDLINE | ID: mdl-32895383

ABSTRACT

In rodent models of type 2 diabetes (T2D), sustained remission of hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1), and the mediobasal hypothalamus (MBH) was recently implicated as the brain area responsible for this effect. To better understand the cellular response to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes from the hypothalamus of diabetic Lepob/ob mice obtained on Days 1 and 5 after icv injection of either FGF1 or vehicle. A wide range of transcriptional responses to FGF1 was observed across diverse hypothalamic cell types, with glial cell types responding much more robustly than neurons at both time points. Tanycytes and ependymal cells were the most FGF1-responsive cell type at Day 1, but astrocytes and oligodendrocyte lineage cells subsequently became more responsive. Based on histochemical and ultrastructural evidence of enhanced cell-cell interactions between astrocytes and Agrp neurons (key components of the melanocortin system), we performed a series of studies showing that intact melanocortin signaling is required for the sustained antidiabetic action of FGF1. These data collectively suggest that hypothalamic glial cells are leading targets for the effects of FGF1 and that sustained diabetes remission is dependent on intact melanocortin signaling.


Subject(s)
Diabetes Mellitus, Experimental/diet therapy , Diabetes Mellitus, Type 2/drug therapy , Fibroblast Growth Factor 1/administration & dosage , Hypoglycemic Agents/administration & dosage , Hypothalamus/drug effects , Recombinant Proteins/administration & dosage , Agouti-Related Protein/metabolism , Animals , Astrocytes/drug effects , Astrocytes/metabolism , Blood Glucose/analysis , Cell Communication , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/etiology , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/etiology , Diabetes Mellitus, Type 2/pathology , Diet, High-Fat/adverse effects , Dietary Sucrose/administration & dosage , Dietary Sucrose/adverse effects , Humans , Hypothalamus/cytology , Hypothalamus/pathology , Injections, Intraventricular , Leptin/genetics , Male , Melanocortins/metabolism , Melanocyte-Stimulating Hormones/administration & dosage , Mice , Mice, Knockout , Neurons/drug effects , Neurons/metabolism , Oligodendroglia/drug effects , Oligodendroglia/metabolism , RNA-Seq , Receptor, Melanocortin, Type 4/genetics , Receptors, Melanocortin/antagonists & inhibitors , Receptors, Melanocortin/metabolism , Remission Induction/methods , Signal Transduction/drug effects , Single-Cell Analysis , Stereotaxic Techniques , Transcriptome/drug effects
5.
JCI Insight ; 5(6)2020 03 26.
Article in English | MEDLINE | ID: mdl-32213703

ABSTRACT

Semaglutide, a glucagon-like peptide 1 (GLP-1) analog, induces weight loss, lowers glucose levels, and reduces cardiovascular risk in patients with diabetes. Mechanistic preclinical studies suggest weight loss is mediated through GLP-1 receptors (GLP-1Rs) in the brain. The findings presented here show that semaglutide modulated food preference, reduced food intake, and caused weight loss without decreasing energy expenditure. Semaglutide directly accessed the brainstem, septal nucleus, and hypothalamus but did not cross the blood-brain barrier; it interacted with the brain through the circumventricular organs and several select sites adjacent to the ventricles. Semaglutide induced central c-Fos activation in 10 brain areas, including hindbrain areas directly targeted by semaglutide, and secondary areas without direct GLP-1R interaction, such as the lateral parabrachial nucleus. Automated analysis of semaglutide access, c-Fos activity, GLP-1R distribution, and brain connectivity revealed that activation may involve meal termination controlled by neurons in the lateral parabrachial nucleus. Transcriptomic analysis of microdissected brain areas from semaglutide-treated rats showed upregulation of prolactin-releasing hormone and tyrosine hydroxylase in the area postrema. We suggest semaglutide lowers body weight by direct interaction with diverse GLP-1R populations and by directly and indirectly affecting the activity of neural pathways involved in food intake, reward, and energy expenditure.


Subject(s)
Body Weight/drug effects , Brain/drug effects , Glucagon-Like Peptides/pharmacology , Neural Pathways/drug effects , Animals , Eating/drug effects , Energy Metabolism/drug effects , Glucagon-Like Peptide-1 Receptor/drug effects , Mice , Rats
6.
J Comp Neurol ; 527(12): 2069-2085, 2019 08 15.
Article in English | MEDLINE | ID: mdl-30809795

ABSTRACT

Central activation of fibroblast growth factor (FGF) receptors regulates peripheral glucose homeostasis and reduces food intake in preclinical models of obesity and diabetes. The current work was undertaken to advance our understanding of the receptor expression, as sites of ligand action by FGF19, FGF21, and FGF1 in the mammalian brain remains unresolved. Recent advances in automated RNAscope in situ hybridization and droplet digital PCR (ddPCR) technology allowed us to interrogate central FGFR/beta klotho (Klb) system at the cellular level in the mouse, with relevant comparisons to nonhuman primate and human brain. FGFR1-3 gene expression was broadly distributed throughout the CNS in Mus musculus, with FGFR1 exhibiting the greatest heterogeneity. FGFR4 expression localized only in the medial habenula and subcommissural organ of mice. Likewise, Klb mRNA was restricted to the suprachiasmatic nucleus (SCh) and select midbrain and hindbrain nuclei. ddPCR in the rodent hypothalamus confirmed that, although expression levels are indeed low for Klb, there is nonetheless a bonafide subpopulation of Klb+ cells in the hypothalamus. In NHP and human midbrain and hindbrain, Klb + cells are quite rare, as is expression of FGFR4. Collectively, these data provide the most robust central map of the FGFR/Klb system to date and highlight central regions that may be of critical importance to assess central ligand effects with pharmacological dosing, such as the putative interactions between the endocrine FGFs and FGFR1/Klb, or FGF19 with FGFR4.


Subject(s)
Brain Mapping/methods , Brain/metabolism , Fibroblast Growth Factors/metabolism , Glucuronidase/metabolism , In Situ Hybridization/methods , Animals , Fibroblast Growth Factors/analysis , Glucuronidase/analysis , Humans , Klotho Proteins , Macaca fascicularis , Male , Mice , Mice, Inbred C57BL
7.
eNeuro ; 4(1)2017.
Article in English | MEDLINE | ID: mdl-28144621

ABSTRACT

Kisspeptin (Kiss1) neurons in the hypothalamic arcuate nucleus (ARC) are key components of the hypothalamic-pituitary-gonadal axis, as they regulate the basal pulsatile release of gonadotropin releasing hormone (GnRH). ARC Kiss1 action is dependent on energy status, and unmasking metabolic factors responsible for modulating ARC Kiss1 neurons is of great importance. One possible factor is glucagon-like peptide 1 (GLP-1), an anorexigenic neuropeptide produced by brainstem preproglucagon neurons. Because GLP fiber projections and the GLP-1 receptor (GLP-1R) are abundant in the ARC, we hypothesized that GLP-1R signaling could modulate ARC Kiss1 action. Using ovariectomized mice, we found that GLP-producing fibers come in close apposition with ARC Kiss1 neurons; these neurons also contain Glp1r mRNA. Electrophysiological recordings revealed that liraglutide (a long-acting GLP-1R agonist) increased action potential firing and caused a direct membrane depolarization of ARC Kiss1 cells in brain slices. We determined that brainstem preproglucagon mRNA is decreased after a 48-h fast in mice, a negative energy state in which ARC Kiss1 expression and downstream GnRH/luteinizing hormone (LH) release are potently suppressed. However, activation of GLP-1R signaling in fasted mice with liraglutide was not sufficient to prevent LH inhibition. Furthermore, chronic central infusions of the GLP-1R antagonist, exendin(9-39), in ad libitum-fed mice did not alter ARC Kiss1 mRNA or plasma LH. As a whole, these data identify a novel interaction of the GLP-1 system with ARC Kiss1 neurons but indicate that CNS GLP-1R signaling alone is not critical for the maintenance of LH during fasting or normal feeding.


Subject(s)
Arcuate Nucleus of Hypothalamus/metabolism , Fasting/metabolism , Glucagon-Like Peptide-1 Receptor/metabolism , Kisspeptins/metabolism , Luteinizing Hormone/blood , Neurons/metabolism , Animals , Arcuate Nucleus of Hypothalamus/cytology , Arcuate Nucleus of Hypothalamus/drug effects , Brain Stem/cytology , Brain Stem/drug effects , Brain Stem/metabolism , Drug Implants , Eating/physiology , Estradiol/administration & dosage , Estrogens/administration & dosage , Female , Glucagon-Like Peptide-1 Receptor/agonists , Glucagon-Like Peptide-1 Receptor/antagonists & inhibitors , Glucagon-Like Peptides/metabolism , Luteinizing Hormone/antagonists & inhibitors , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice, Inbred C57BL , Neurons/cytology , Neurons/drug effects , Ovariectomy , RNA, Messenger/metabolism , Signal Transduction/drug effects , Tissue Culture Techniques
8.
Mol Metab ; 5(3): 198-209, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26977392

ABSTRACT

OBJECTIVE: Humans and animals exposed to undernutrition (UN) during development often experience accelerated "catch-up" growth when food supplies are plentiful. Little is known about the mechanisms regulating early growth rates. We previously reported that actions of leptin and presynaptic inputs to orexigenic NPY/AgRP/GABA (NAG) neurons in the arcuate nucleus of the hypothalamus are almost exclusively excitatory during the lactation period, since neuronal and humoral inhibitory systems do not develop until after weaning. Moreover, we identified a critical step that regulates the maturation of electrophysiological responses of NAG neurons at weaning - the onset of genes encoding ATP-dependent potassium (KATP) channel subunits. We explored the possibility that UN promotes subsequent catch-up growth, in part, by delaying the maturation of negative feedback systems to neuronal circuits driving food intake. METHODS: We used the large litter (LL) size model to study the impacts of postnatal UN followed by catch-up growth. We evaluated the maturation of presynaptic and postsynaptic inhibitory systems in NAG neurons using a combination of electrophysiological and molecular criteria, in conjunction with leptin's ability to suppress fasting-induced hyperphagia. RESULTS: The onset of KATP channel subunit expression and function, the switch in leptin's effect on NAG neurons, the ingrowth of inhibitory inputs to NAG neurons, and the development of homeostatic feedback to feeding circuits were delayed in LL offspring relative to controls. The development of functional KATP channels and the establishment of leptin-mediated suppression of food intake in the peri-weaning period were tightly linked and were not initiated until growth and adiposity of LL offspring caught up to controls. CONCLUSIONS: Our data support the idea that initiation of KATP channel subunit expression in NAG neurons serves as a molecular gatekeeper for the maturation of homeostatic feeding circuits.

9.
J Neurosci ; 35(22): 8558-69, 2015 Jun 03.
Article in English | MEDLINE | ID: mdl-26041922

ABSTRACT

Neurons coexpressing neuropeptide Y, agouti-related peptide, and GABA (NAG) play an important role in ingestive behavior and are located in the arcuate nucleus of the hypothalamus. NAG neurons receive both GABAergic and glutamatergic synaptic inputs, however, the developmental time course of synaptic input organization of NAG neurons in mice is unknown. In this study, we show that these neurons have low numbers of GABAergic synapses and that GABA is inhibitory to NAG neurons during early postnatal period. In contrast, glutamatergic inputs onto NAG neurons are relatively abundant by P13 and are comparatively similar to the levels observed in the adult. As mice reach adulthood (9-10 weeks), GABAergic tone onto NAG neurons increases. At this age, NAG neurons received similar numbers of inhibitory and EPSCs. To further differentiate age-associated changes in synaptic distribution, 17- to 18-week-old lean and diet-induced obesity (DIO) mice were studied. Surprisingly, NAG neurons from lean adult mice exhibit a reduction in the GABAergic synapses compared with younger adults. Conversely, DIO mice display reductions in the number of GABAergic and glutamatergic inputs onto NAG neurons. Based on these experiments, we propose that synaptic distribution in NAG neurons is continuously restructuring throughout development to accommodate the animals' energy requirements.


Subject(s)
Arcuate Nucleus of Hypothalamus/cytology , Arcuate Nucleus of Hypothalamus/growth & development , Neurons/physiology , Synapses/physiology , 2-Amino-5-phosphonovalerate/pharmacology , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Age Factors , Animals , Animals, Newborn , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/genetics , Female , Inhibitory Postsynaptic Potentials/drug effects , Inhibitory Postsynaptic Potentials/genetics , Lysine/analogs & derivatives , Lysine/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neuropeptide Y/genetics , Neuropeptide Y/metabolism , Sodium Channel Blockers/pharmacology , Synapses/drug effects , Synapses/genetics , Tetrodotoxin/pharmacology , Vesicular Inhibitory Amino Acid Transport Proteins/metabolism , gamma-Aminobutyric Acid/pharmacology
10.
J Clin Invest ; 124(10): 4473-88, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25202980

ABSTRACT

Liraglutide is a glucagon-like peptide-1 (GLP-1) analog marketed for the treatment of type 2 diabetes. Besides lowering blood glucose, liraglutide also reduces body weight. It is not fully understood how liraglutide induces weight loss or to what degree liraglutide acts directly in the brain. Here, we determined that liraglutide does not activate GLP-1-producing neurons in the hindbrain, and liraglutide-dependent body weight reduction in rats was independent of GLP-1 receptors (GLP-1Rs) in the vagus nerve, area postrema, and paraventricular nucleus. Peripheral injection of fluorescently labeled liraglutide in mice revealed the presence of the drug in the circumventricular organs. Moreover, labeled liraglutide bound neurons within the arcuate nucleus (ARC) and other discrete sites in the hypothalamus. GLP-1R was necessary for liraglutide uptake in the brain, as liraglutide binding was not seen in Glp1r(-/-) mice. In the ARC, liraglutide was internalized in neurons expressing proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART). Electrophysiological measurements of murine brain slices revealed that GLP-1 directly stimulates POMC/CART neurons and indirectly inhibits neurotransmission in neurons expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP) via GABA-dependent signaling. Collectively, our findings indicate that the GLP-1R on POMC/CART-expressing ARC neurons likely mediates liraglutide-induced weight loss.


Subject(s)
Glucagon-Like Peptide 1/analogs & derivatives , Receptors, Glucagon/metabolism , Weight Loss/drug effects , Animals , Arcuate Nucleus of Hypothalamus/drug effects , Body Weight/drug effects , Disease Models, Animal , Electrophysiology , Glucagon-Like Peptide 1/pharmacology , Glucagon-Like Peptide-1 Receptor , Hypothalamus/metabolism , Liraglutide , Male , Mice , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Paraventricular Hypothalamic Nucleus/metabolism , Pro-Opiomelanocortin/metabolism , Rats , Rats, Sprague-Dawley , Vagus Nerve/metabolism
11.
J Neurosci ; 34(30): 9982-94, 2014 Jul 23.
Article in English | MEDLINE | ID: mdl-25057200

ABSTRACT

Leptin is well known for its role in the regulation of energy homeostasis in adults, a mechanism that at least partially results from the inhibition of the activity of NPY/AgRP/GABA neurons (NAG) in the arcuate nucleus of the hypothalamus (ARH). During early postnatal development in the rodent, leptin promotes axonal outgrowth from ARH neurons, and preautonomic NAG neurons are particularly responsive to leptin's trophic effects. To begin to understand how leptin could simultaneously promote axonal outgrowth from and inhibit the activity of NAG neurons, we characterized the electrochemical effects of leptin on NAG neurons in mice during early development. Here, we show that NAG neurons do indeed express a functional leptin receptor throughout the early postnatal period in the mouse; however, at postnatal days 13-15, leptin causes membrane depolarization in NAG neurons, rather than the expected hyperpolarization. Leptin action on NAG neurons transitions from stimulatory to inhibitory in the periweaning period, in parallel with the acquisition of functional ATP-sensitive potassium channels. These findings are consistent with the idea that leptin provides an orexigenic drive through the NAG system to help rapidly growing pups meet their energy requirements.


Subject(s)
Arcuate Nucleus of Hypothalamus/growth & development , Leptin/physiology , Neurons/physiology , Receptors, Leptin/physiology , Signal Transduction/physiology , Animals , Animals, Newborn , Male , Mice , Mice, Transgenic , Receptors, Leptin/biosynthesis
12.
PLoS One ; 7(1): e30032, 2012.
Article in English | MEDLINE | ID: mdl-22253866

ABSTRACT

In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields.


Subject(s)
Receptor, Adenosine A2B/metabolism , Taste Buds/metabolism , Taste/physiology , Animals , Antibody Specificity/immunology , Epithelium/metabolism , Gene Expression Regulation , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Inbred C57BL , Receptor, Adenosine A2B/genetics , Reverse Transcriptase Polymerase Chain Reaction , Taste Perception , beta-Galactosidase/metabolism
13.
Am J Physiol Cell Physiol ; 300(4): C860-71, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21106690

ABSTRACT

Diabetes is a profound disease that results in a severe lack of regulation of systemic salt and water balance. From our earlier work on the endocrine regulation of salt taste at the level of the epithelial sodium channel (ENaC), we have begun to investigate the ability of insulin to alter ENaC function with patch-clamp recording on isolated mouse taste receptor cells (TRCs). In fungiform and vallate TRCs that exhibit functional ENaC currents (e.g., amiloride-sensitive Na(+) influx), insulin (5-20 nM) caused a significant increase in Na(+) influx at -80 mV (EC(50) = 7.53 nM). The insulin-enhanced currents were inhibited by amiloride (30 µM). Similarly, in ratiometric Na(+) imaging using SBFI, insulin treatment (20 nM) enhanced Na(+) movement in TRCs, consistent with its action in electrophysiological assays. The ability of insulin to regulate ENaC function is dependent on the enzyme phosphoinositide 3-kinase since treatment with the inhibitor LY294002 (10 µM) abolished insulin-induced changes in ENaC. To test the role of insulin in the regulation of salt taste, we have characterized behavioral responses to NaCl using a mouse model of acute hyperinsulinemia. Insulin-treated mice show significant avoidance of NaCl at lower concentrations than the control group. Interestingly, these differences between groups were abolished when amiloride (100 µM) was added into NaCl solutions, suggesting that insulin was regulating ENaC. Our results are consistent with a role for insulin in maintaining functional expression of ENaC in mouse TRCs.


Subject(s)
Epithelial Sodium Channels/metabolism , Insulin/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Receptors, G-Protein-Coupled/metabolism , Taste Buds/cytology , Taste Buds/drug effects , Amiloride/metabolism , Animals , Behavior, Animal/drug effects , Epithelial Sodium Channels/genetics , Insulin Receptor Substrate Proteins/genetics , Insulin Receptor Substrate Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Patch-Clamp Techniques , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositols/chemistry , Phosphatidylinositols/metabolism , Receptor, Insulin/metabolism , Receptors, G-Protein-Coupled/genetics , Sodium/metabolism , Sodium Channel Blockers/metabolism , Sodium, Dietary , Taste Buds/metabolism
14.
Chem Senses ; 32(5): 411-21, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17339611

ABSTRACT

In order to gain insight into the molecular mechanisms that allow taste cells to respond to changes in their osmotic environment, we have used primarily immunocytochemical and molecular approaches to look for evidence of the presence of aquaporin-like water channels in taste cells. Labeling of isolated taste buds from the fungiform, foliate, and vallate papillae in rat tongue with antibodies against several of the aquaporins (AQPs) revealed the presence of AQP1, AQP2, and AQP5 in taste cells from these areas. AQP3 antibodies failed to label isolated taste buds from any of the papillae. There was an apparent difference in the regional localization of AQP labeling within the taste bud. Antibodies against AQP1 and AQP2 labeled predominantly the basolateral membrane, whereas the AQP5 label was clearly evident on both the apical and basolateral membranes of cells within the taste bud. Double labeling revealed that AQP1 and AQP2 labeled many, but not all, of the same taste cells. Similar double-labeling experiments with anti-AQP2 and anti-AQP5 clearly showed that AQP5 was expressed on or near the apical membranes whereas AQP2 was absent from this area. The presence of these 3 types of AQPs in taste buds but not in non-taste bud-containing epithelia was confirmed using reverse transcription-polymerase chain reaction. Experiments using patch clamp recording showed that the AQP inhibitor, tetraethylammonium, significantly reduced hypoosmotic-induced currents in rat taste cells. We hypothesize that the AQPs may play roles both in the water movement underlying compensatory mechanisms for changes in extracellular osmolarity and, in the case of AQP5 in particular, in the gustatory response to water.


Subject(s)
Aquaporins/metabolism , Taste Buds/metabolism , Animals , Immunohistochemistry , Male , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction
15.
J Water Health ; 4 Suppl 1: 35-40, 2006.
Article in English | MEDLINE | ID: mdl-16493898

ABSTRACT

The sense of taste plays critical roles in nutrient identification and toxin avoidance. The ability to respond to hypoosmotic stimuli in mammalian taste receptor cells may reflect the importance of osmotic sensing by the gustatory system. Transduction for hypoosmotic stimuli involves water influx through aquaporins followed by activation of volume-regulated anion channels. The ability of these transduction elements to be regulated by natriferic hormones at the mRNA and protein level in other transporting epithelia suggest that the gustatory system may respond to extrinsic signals related to the restoration of salt and water balance. Plasticity in the peripheral gustatory system is consistent with the activity in the taste system being reflective of underlying nutritional status. Clearly, more research is needed to determine the link between nutrition, taste and the control of food and water intake.


Subject(s)
Drinking , Mouth , Taste , Humans , United States
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