Electrophysiological responses to sugars and amino acids in the nucleus of the solitary tract of type 1 taste receptor double-knockout mice

Strong evidence supports a major role for heterodimers of the type 1 taste receptor (T1R) family in the taste transduction of sugars (T1R2+T1R3) and amino acids (T1R1+T1R3), but there are also neural and behavioral data supporting T1R-independent mechanisms. Most neural evidence for alternate mechanisms comes from whole nerve recordings in mice with deletion of a single T1R family member, limiting conclusions about the functional significance and T1R independence of the remaining responses. To clarify these issues, we recorded single-unit taste responses from the nucleus of the solitary tract in T1R double-knockout (double-KO) mice lacking functional T1R1+T1R3 [KO1+3] or T1R2+T1R3 [KO2+3] receptors and their wild-type background strains [WT; C57BL/6J (B6), 129X1/SvJ (S129)]. In both double-KO strains, responses to sugars and a moderate concentration of an monosodium glutamate + amiloride + inosine 5′-monophosphate cocktail (0.1 M, i.e., umami) were profoundly depressed, whereas a panel of 0.6 M amino acids were mostly unaffected. Strikingly, in contrast to WT mice, no double-KO neurons responded selectively to sugars and umami, precluding segregation of this group of stimuli from those representing other taste qualities in a multidimensional scaling analysis. Nevertheless, residual sugar responses, mainly elicited by monosaccharides, persisted as small “sideband” responses in double-KOs. Thus other receptors may convey limited information about sugars to the central nervous system, but T1Rs appear critical for coding the distinct perceptual features of sugar and umami stimuli. The persistence of amino acid responses supports previous proposals of alternate receptors, but because these stimuli affected multiple neuron types, further investigations are necessary. NEW & NOTEWORTHY The type 1 taste receptor (T1R) family is crucial for transducing sugars and amino acids, but there is evidence for T1R-independent mechanisms. In this study, single-unit recordings from the nucleus of the solitary tract in T1R double-knockout mice lacking T1R1+T1R3 or T1R2+T1R3 receptors revealed greatly reduced umami synergism and sugar responses. Nevertheless, residual sugar responses persisted, mainly elicited by monosaccharides and evident as “sidebands” in neurons activated more vigorously by other qualities.

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Multiple sweet receptors and transduction pathways revealed in knockout mice by temperature dependence and gurmarin sensitivity

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.91018.2008 ◽

2009 ◽

Vol 296 (4) ◽

pp. R960-R971 ◽

Cited By ~ 57

Author(s):

Tadahiro Ohkuri ◽

Keiko Yasumatsu ◽

Nao Horio ◽

Masafumi Jyotaki ◽

Robert F. Margolskee ◽

...

Keyword(s):

Temperature Dependence ◽

Knockout Mice ◽

Taste Receptor ◽

Sweet Taste ◽

Receptor Type ◽

Temperature Sensitive ◽

Chorda Tympani ◽

Temperature Dependent ◽

Mouse Taste

Sweet taste transduction involves taste receptor type 1, member 2 (T1R2), taste receptor type 1, member 3 (T1R3), gustducin, and TRPM5. Because knockout (KO) mice lacking T1R3, gustducin's Gα subunit (Gαgust), or TRPM5 exhibited greatly reduced, but not abolished responses of the chorda tympani (CT) nerve to sweet compounds, it is likely that multiple sweet transduction pathways exist. That gurmarin (Gur), a sweet taste inhibitor, inhibits some but not all mouse CT responses to sweet compounds supports the existence of multiple sweet pathways. Here, we investigated Gur inhibition of CT responses to sweet compounds as a function of temperature in KO mice lacking T1R3, Gαgust, or TRPM5. In T1R3-KO mice, responses to sucrose and glucose were Gur sensitive (GS) and displayed a temperature-dependent increase (TDI). In Gαgust-KO mice, responses to sucrose and glucose were Gur-insensitive (GI) and showed a TDI. In TRPM5-KO mice, responses to glucose were GS and showed a TDI. All three KO mice exhibited no detectable responses to SC45647, and their responses to saccharin displayed neither GS nor a TDI. For all three KO mice, the lingual application of pronase, another sweet response inhibitor, almost fully abolished responses to sucrose and glucose but did not affect responses to saccharin. These results provide evidence for 1) the existence of multiple transduction pathways underlying responses to sugars: a T1R3-independent GS pathway for sucrose and glucose, and a TRPM5-independent temperature sensitive GS pathway for glucose; 2) the requirement for Gαgust in GS sweet taste responses; and 3) the existence of a sweet independent pathway for saccharin, in mouse taste cells on the anterior tongue.

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Inhibition of Endocytosis of Clathrin-Mediated Angiotensin II Receptor Type 1 in Podocytes Augments Glomerular Injury

Journal of the American Society of Nephrology ◽

10.1681/asn.2019010053 ◽

2019 ◽

Vol 30 (12) ◽

pp. 2307-2320 ◽

Cited By ~ 4

Author(s):

Kazunori Inoue ◽

Xuefei Tian ◽

Heino Velazquez ◽

Keita Soda ◽

Zhen Wang ◽

...

Keyword(s):

Angiotensin Ii ◽

Knockout Mice ◽

Renin Angiotensin System ◽

Membrane Dynamics ◽

Receptor Type ◽

Angiotensin Ii Receptor ◽

Double Knockout ◽

Angiotensin System ◽

Renin Angiotensin

BackgroundInhibition of the renin-angiotensin system remains a cornerstone in reducing proteinuria and progression of kidney failure, effects believed to be the result of reduction in BP and glomerular hyperfiltration. However, studies have yielded conflicting results on whether podocyte-specific angiotensin II (AngII) signaling directly induces podocyte injury. Previous research has found that after AngII stimulation, β-arrestin–bound angiotensin II receptor type 1 (AT1R) is internalized in a clathrin- and dynamin-dependent manner, and that Dynamin1 and Dynamin2 double-knockout mice exhibit impaired clathrin-mediated endocytosis.MethodsWe used podocyte-specific Dyn double-knockout mice to examine AngII-stimulated AT1R internalization and signaling in primary podocytes and controls. We also examined the in vivo effect of AngII in these double-knockout mice through renin-angiotensin system blockers and through deletion of Agtr1a (which encodes the predominant AT1R isoform expressed in kidney, AT1aR). We tested calcium influx, Rac1 activation, and lamellipodial extension in control and primary podocytes of Dnm double-knockout mice treated with AngII.ResultsWe confirmed augmented AngII-stimulated AT1R signaling in primary Dnm double-knockout podocytes resulting from arrest of clathrin-coated pit turnover. Genetic ablation of podocyte Agtr1a in Dnm double-knockout mice demonstrated improved albuminuria and kidney function compared with the double-knockout mice. Isolation of podocytes from Dnm double-knockout mice revealed abnormal membrane dynamics, with increased Rac1 activation and lamellipodial extension, which was attenuated in Dnm double-knockout podocytes lacking AT1aR.ConclusionsOur results indicate that inhibiting aberrant podocyte-associated AT1aR signaling pathways has a protective effect in maintaining the integrity of the glomerular filtration barrier.

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First evidence for the presence of amino acid sensing mechanisms in the fish gastrointestinal tract

Scientific Reports ◽

10.1038/s41598-021-84303-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jessica Calo ◽

Ayelén M. Blanco ◽

Sara Comesaña ◽

Marta Conde-Sieira ◽

Sofia Morais ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Gastrointestinal Tract ◽

Intracellular Signaling ◽

Metabotropic Glutamate Receptors ◽

Taste Receptor ◽

Intragastric Administration ◽

Amino Acid Receptors ◽

Amino Acid Sensing

AbstractThis study aimed to characterize amino acid sensing systems in the gastrointestinal tract (GIT) of the carnivorous fish model species rainbow trout. We observed that the trout GIT expresses mRNAs encoding some amino acid receptors described in mammals [calcium-sensing receptor (CaSR), G protein-coupled receptor family C group 6 member A (GPRC6A), and taste receptors type 1 members 1 and 2 (T1r1, T1r2)], while others [taste receptor type 1 member 3 (T1r3) and metabotropic glutamate receptors 1 and 4 (mGlur1, mGlur4)] could not be found. Then, we characterized the response of such receptors, as well as that of intracellular signaling mechanisms, to the intragastric administration of l-leucine, l-valine, l-proline or l-glutamate. Results demonstrated that casr, gprc6a, tas1r1 and tas1r2 mRNAs are modulated by amino acids in the stomach and proximal intestine, with important differences with respect to mammals. Likewise, gut amino acid receptors triggered signaling pathways likely mediated, at least partly, by phospholipase C β3 and β4. Finally, the luminal presence of amino acids led to important changes in ghrelin, cholecystokinin, peptide YY and proglucagon mRNAs and/or protein levels. Present results offer the first set of evidence in favor of the existence of amino acid sensing mechanisms within the fish GIT.

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T1R2+T1R3-independent chemosensory inputs contributing to behavioral discrimination of sugars in mice

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00255.2018 ◽

2019 ◽

Vol 316 (5) ◽

pp. R448-R462 ◽

Cited By ~ 8

Author(s):

Lindsey A. Schier ◽

Chizuko Inui-Yamamoto ◽

Ginger D. Blonde ◽

Alan C. Spector

Keyword(s):

Neural Circuits ◽

Taste Receptor ◽

Sweet Taste ◽

Mouse Strains ◽

Wild Type ◽

Double Knockout ◽

Sweet Taste Receptor ◽

Rat Study ◽

Behavioral Discrimination

Simple sugars are thought to elicit a unitary sensation, principally via the “sweet” taste receptor type 1 taste receptor (T1R)2+T1R3, yet we previously found that rats with experience consuming two metabolically distinct sugars, glucose and fructose, subsequently licked more for glucose than fructose, even when postingestive influences were abated. The results pointed to the existence of an orosensory receptor that binds one sugar but not the other and whose signal is channeled into neural circuits that motivate ingestion. Here we sought to determine the chemosensory nature of this signal. First, we assessed whether T1R2 and/or T1R3 are necessary to acquire this behavioral discrimination, replicating our rat study in T1R2+T1R3 double-knockout (KO) mice and their wild-type counterparts as well as in two common mouse strains that vary in their sensitivity to sweeteners [C57BL/6 (B6) and 129X1/SvJ (129)]. These studies showed that extensive exposure to multiple concentrations of glucose and fructose in daily one-bottle 30-min sessions enhanced lick responses for glucose over fructose in brief-access tests. This was true even for KO mice that lacked the canonical “sweet” taste receptor. Surgical disconnection of olfactory inputs to the forebrain (bulbotomy) in B6 mice severely disrupted the ability to express this experience-dependent sugar discrimination. Importantly, these bulbotomized B6 mice exhibited severely blunted responsiveness to both sugars relative to water in brief-access lick tests, despite the fact that they have intact T1R2+T1R3 receptors. The results highlight the importance of other sources of chemosensory and postingestive inputs in shaping and maintaining “hardwired” responses to sugar.

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An Osteocalcin-deficient mouse strain without endocrine abnormalities

10.1101/732800 ◽

2019 ◽

Author(s):

Cassandra R. Diegel ◽

Steven Hann ◽

Ugur M. Ayturk ◽

Jennifer C.W. Hu ◽

Kyung-eun Lim ◽

...

Keyword(s):

Blood Glucose ◽

Knockout Mice ◽

Male Fertility ◽

Fibroblast Growth Factor 23 ◽

Bone Cell ◽

Wild Type ◽

Double Knockout ◽

Glucose Levels ◽

Blood Glucose Levels

AbstractOsteocalcin (OCN), the most abundant non-collagenous protein in the bone matrix, is reported to be a bone-derived endocrine hormone with wide-ranging effects on many aspects of physiology, including glucose metabolism and male fertility. Many of these observations were made using an OCN-deficient mouse allele (Osc-) in which the 2 OCN-encoding genes in mice, Bglap and Bglap2, were deleted in ES cells by homologous recombination. Here we describe mice with a new Bglap and Bglap2 double knockout (dko) allele (Bglap/2p.Pro25fs17Ter) that was generated by CRISPR/Cas9-mediated gene editing. Mice homozygous for this new allele do not express full length Bglap or Bglap2 mRNA and have no immunodetectable OCN in their plasma. FTIR imaging of cortical and trabecular bone in these homozygous knockout animals finds alterations in the crystal size and maturity of the bone mineral, hydroxyapatite, compared to wild-type littermates; however, μCT and 3-point bending tests do not find differences from wild-type littermates with respect to bone mass and strength. In contrast to the previously reported OCN-deficient mice with the Osc- allele, blood glucose levels and male fertility in the OCN-deficient mice with Bglap/2pPro25fs17Ter allele did not have significant differences from wild-type littermates. We cannot explain the absence of endocrine effects in mice with this new knockout allele. Potential explanations include effects of each mutated allele on the transcription of neighboring genes, and differences in genetic background and environment. So that our findings can be confirmed and extended by other interested investigators, we are donating this new Bglap and Bglap2 double knockout strain to The Jackson Laboratory for academic distribution.Author SummaryCells that make and maintain bone express proteins that function locally or systemically. The former proteins, such as type 1 collagen, affect the material properties of the skeleton while the latter proteins, such as fibroblast growth factor 23, enable the skeleton to communicate with other organ systems. Mutations that affect the functions of most bone cell expressed proteins cause diseases that have similar features in humans and other mammals, such as mice; for example, brittle bone diseases for type 1 collagen mutations and hypophosphatemic rickets for fibroblast growth factor 23 mutations.Our study focuses on another bone cell expressed protein, osteocalcin, which has been suggested to function locally to affect bone strength and systemically as hormone. Studies using osteocalcin knockout mice led other investigators to suggest endocrine roles for osteocalcin in regulating blood glucose levels, male fertility, muscle mass, brain development, behavior and cognition. We therefore decided to generate a new strain of osteocalcin knockout mice that could also be used to investigate these non-skeletal effects.To our surprise the osteocalcin knockout mice we created do not significantly differ from wild-type mice for the 3 phenotypes we examined: bone strength, blood glucose levels, and male fertility. Our data are consistent with findings from osteocalcin knockout rats, but inconsistent with data from the original osteocalcin knockout mice. Because we do not know why our new strain of osteocalcin knockout mice fails to recapitulate phenotypes previously reported for another knockout mouse stain, we have donated our mice to a public repository so that they can be easily obtained and studied in other academic laboratories.

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