The components required for amino acid neurotransmitter signaling are present in adipose tissues

Abstract

The adipocyte does not only serve as fuel storage but produces and secretes compounds with modulating effects on food intake and energy homeostasis. Although there is firm evidence for a centrally mediated regulation of adipocyte function via the autonomous nervous system, little is known about signaling between adipocytes. Amino acid neurotransmitters are candidates for such paracrine signaling. Here, we applied immunohistochemistry to detect components required for amino acid transmitter signaling in rat fat depots. In interscapular brown adipose tissue as well as in interscapular, mesenteric, perirenal, and epididymal white adipose tissues, we demonstrate robust immunosignals for the excitatory neurotransmitter glutamate, the inhibitory neurotransmitter γ-aminobutyric acid (GABA), and the GABA-synthesizing enzyme glutamate decarboxylase (GAD) isoforms GAD65 and GAD67. Moreover, all adipose tissues stained for the vesicular glutamate transporter VGLUT1 and the vesicular GABA transporter VGAT in addition to the vesicle marker synaptophysin. Electron microscopic immunocytochemistry showed that VGLUT1 and VGAT, but not VGLUT2 or VGLUT3, are localized in vesicular organelles in adipocytes. The receptors for glutamate (subunits GluR2/3 and NR1 but not mGluR2) and for GABA (GABAARα2) were present in the adipocytes. The presence of glutamate, GABA, their vesicular transporters, and their receptors indicates a paracrine signaling role for amino acids in adipose tissues. The adipocyte does not only serve as fuel storage but produces and secretes compounds with modulating effects on food intake and energy homeostasis. Although there is firm evidence for a centrally mediated regulation of adipocyte function via the autonomous nervous system, little is known about signaling between adipocytes. Amino acid neurotransmitters are candidates for such paracrine signaling. Here, we applied immunohistochemistry to detect components required for amino acid transmitter signaling in rat fat depots. In interscapular brown adipose tissue as well as in interscapular, mesenteric, perirenal, and epididymal white adipose tissues, we demonstrate robust immunosignals for the excitatory neurotransmitter glutamate, the inhibitory neurotransmitter γ-aminobutyric acid (GABA), and the GABA-synthesizing enzyme glutamate decarboxylase (GAD) isoforms GAD65 and GAD67. Moreover, all adipose tissues stained for the vesicular glutamate transporter VGLUT1 and the vesicular GABA transporter VGAT in addition to the vesicle marker synaptophysin. Electron microscopic immunocytochemistry showed that VGLUT1 and VGAT, but not VGLUT2 or VGLUT3, are localized in vesicular organelles in adipocytes. The receptors for glutamate (subunits GluR2/3 and NR1 but not mGluR2) and for GABA (GABAARα2) were present in the adipocytes. The presence of glutamate, GABA, their vesicular transporters, and their receptors indicates a paracrine signaling role for amino acids in adipose tissues. α-amino-hydroxy-5-methyl-4-isoxazolepropiorate brown adipose tissue central nervous system γ-aminobutyric acid glutamate decarboxylase glial fibrillary acidic protein microtubule-associated protein 2 N-methyl-d-aspartate synaptic vesicle white adipose tissue With the increasing prevalence of overweight and obesity worldwide, a growing interest in the functions of adipocytes has been stirred. The adipose tissue is no longer considered solely as a primary energy storage organ; it has emerged also as an important endocrine organ. Whereas the regulation of hormone synthesis and secretion from the different kinds of adipose cells is still poorly understood, many reports have shown that the release of fuel substrates from adipose tissue is at least partly governed by neuronal inputs. For example, there is extensive neuroanatomical evidence for the sympathetic innervation of white adipose tissue (WAT) (1.Bartness T.J. Bamshad M. Innervation of mammalian white adipose tissue: implications for the regulation of total body fat.Am. J. Physiol. 1998; 275: R1399-R1411Crossref PubMed Google Scholar, 2.Cinti S. The adipose organ.Prostaglandins Leukot. Essent. Fatty Acids. 2005; 73: 9-15Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). In agreement with this, the sympathetic neuronal outflow reportedly modulates glucose and lipid metabolism in WAT of animals and humans (3.Dodt C. Lonnroth P. Wellhoner J.P. Fehm H.L. Elam M. Sympathetic control of white adipose tissue in lean and obese humans.Acta Physiol. Scand. 2003; 177: 351-357Crossref PubMed Scopus (62) Google Scholar). The brown adipose tissue (BAT) also receives a rich sympathetic innervation, and mediastinal BAT reportedly has parasympathetic neurons (4.Giordano A. Frontini A. Castellucci M. Cinti S. Presence and distribution of cholinergic nerves in rat mediastinal brown adipose tissue.J. Histochem. Cytochem. 2004; 52: 923-930Crossref PubMed Scopus (44) Google Scholar). In addition to its function as a heat-producing organ, alterations in BAT metabolism may affect nutritional homeostasis (5.Lowell B.B. Susulic V. Hamann A. Lawitts J.A. Himms-Hagen J. Boyer B.B. Kozak L.P. Flier J.S. Development of obesity in transgenic mice after genetic ablation of brown adipose tissue.Nature. 1993; 366: 740-742Crossref PubMed Scopus (920) Google Scholar). Collectively, these data underlie the hypothesis that a dysregulated sympathetic tone might modify adipose tissues and hence contribute to obesity-related disorders (3.Dodt C. Lonnroth P. Wellhoner J.P. Fehm H.L. Elam M. Sympathetic control of white adipose tissue in lean and obese humans.Acta Physiol. Scand. 2003; 177: 351-357Crossref PubMed Scopus (62) Google Scholar). The adipose depots localized in various parts of the body are seemingly different concerning gene expression profiles and whether they confer increased risk of disease (6.Cousin B. Casteilla L. Dani C. Muzzin P. Revelli J.P. Penicaud L. Adipose tissues from various anatomical sites are characterized by different patterns of gene expression and regulation.Biochem. J. 1993; 292: 873-876Crossref PubMed Scopus (60) Google Scholar, 7.Peiris A.N. Sothmann M.S. Hoffmann R.G. Hennes M.I. Wilson C.R. Gustafson A.B. Kissebah A.H. Adiposity, fat distribution, and cardiovascular risk.Ann. Intern. Med. 1989; 110: 867-872Crossref PubMed Scopus (408) Google Scholar). Moreover, lipectomy in rats followed by autologous transplantation led to increased mass of fat depots not in contact with the grafted fat tissue (8.Lacy E.L. Bartness T.J. Autologous fat transplants influence compensatory white adipose tissue mass increases after lipectomy.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2004; 286: R61-R70Crossref PubMed Scopus (16) Google Scholar, 9.Lacy E.L. Bartness T.J. Effects of white adipose tissue grafts on total body fat and cellularity are dependent on graft type and location.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005; 289: R380-R388Crossref PubMed Scopus (14) Google Scholar). Because of the lack of innervation of the surgically removed adipose tissue, these latter findings indicate important endocrine signaling between adipocytes originating from various, distant organ regions. The nature of this intercellular signaling remains unknown, but the discovery that the excitatory neurotransmitter glutamate and the inhibitory neurotransmitter γ-aminobutyric acid (GABA) can have signaling functions outside of the central nervous system (CNS), such as in the pancreatic islets of Langerhans, opens the intriguing possibility that adipose tissue also could use these signaling substances for paracrine and/or autocrine regulation (10.Anhert-Hilger G. Stadtbaumer A. Strubing C. Scherubl H. Schultz G. Riecken E.O. Wiedenmann B. γ-Aminobutyric acid secretion from pancreatic neuroendocrine cells.Gastroenterology. 1996; 110: 1595-1604Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 11.Reetz A. Solimena M. Matteoli M. Folli F. Takei K. Camilli P.De GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion.EMBO J. 1991; 10: 1275-1284Crossref PubMed Scopus (336) Google Scholar, 12.Thomas-Reetz A. Hell J.W. During M.J. Walch-Solimena C. Jahn R. Camilli P.De A gamma-aminobutyric acid transporter driven by a proton pump is present in synaptic-like microvesicles of pancreatic beta cells.Proc. Natl. Acad. Sci. USA. 1993; 90: 5317-5321Crossref PubMed Scopus (90) Google Scholar, 13.Gammelsaeter R. Froyland M. Aragon C. Danbolt N.C. Fortin D. Storm-Mathisen J. Davanger S. Gundersen V. Glycine, GABA and their transporters in pancreatic islets of Langerhans: evidence for a paracrine transmitter interplay.J. Cell Sci. 2004; 117: 3749-3758Crossref PubMed Scopus (64) Google Scholar). In the CNS, the machinery for synaptic signaling of amino acids includes i) the presence of a transmitter in the presynaptic terminals (e.g., glutamate and GABA); ii) the intraterminal synthesis of the transmitter by specific enzymes [e.g., glutamate decarboxylase (GAD) for GABA]; iii) the packaging of the free transmitter into synaptic vesicles (SVs) in presynaptic terminals by vesicular transporters (e.g., the vesicular glutamate transporters VGLUT1 to VGLUT3 for glutamate and the vesicular GABA transporter VGAT for GABA); iv) the release of transmitter to the synaptic cleft by exocytosis of SVs; and v) transmitter binding to the postsynaptic receptors [e.g., the N-methyl-d-aspartate (NMDA) receptor NMDA-R for glutamate and the GABAA receptor GABAAR for GABA]. Because VGAT carries GABA into SVs, the SVs could be used to mark cells that release GABA by exocytosis, like GABAergic synapses in the CNS (14.Sagne C. Mestikawy S.El Isambert M.F. Hamon M. Henry J.P. Giros B. Gasnier B. Cloning of a functional vesicular GABA and glycine transporter by screening of genome databases.FEBS Lett. 1997; 417: 177-183Crossref PubMed Scopus (239) Google Scholar). VGLUT1 to VGLUT3 are specific for glutamate; hence, their presence in a given tissue compartment suggests the release of glutamate from that particular compartment (15.Fremeau Jr., R.T. Voglmaier S. Seal R.P. Edwards R.H. VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate.Trends Neurosci. 2004; 27: 98-103Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar). Two isoforms of GAD (GAD65 and GAD67) are present in the brain (16.Bu D.F. Erlander M.G. Hitz B.C. Tillakaratne N.J. Kaufman D.L. Wagner-McPherson C.B. Evans G.A. Tobin A.J. Two human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, are each encoded by a single gene.Proc. Natl. Acad. Sci. USA. 1992; 89: 2115-2119Crossref PubMed Scopus (452) Google Scholar, 17.Li L. Jiang J. Hagopian W.A. Karlsen A.E. Skelly M. Baskin D.G. Lernmark A. Differential detection of rat islet and brain glutamic acid decarboxylase (GAD) isoforms with sequence-specific peptide antibodies.J. Histochem. Cytochem. 1995; 43: 53-59Crossref PubMed Scopus (35) Google Scholar). Both enzymes catalyze the formation of GABA from glutamate. Receptors for glutamate and GABA include ionotropic and metabotropic receptors. The former type of receptor mediates fast signals via ligand-gated ion channels, whereas the latter type acts via intracellular second messenger systems. Important ionotropic glutamate receptors are the NMDA-Rs and the α-amino-hydroxy-5-methyl-4-isoxazolepropiorate (AMPA) receptors, whereas GABAA and GABAC receptors constitute the ionotropic GABA receptors. Here, we examined whether different WAT depots and interscapular BAT contain the different elements involved in the handling of transmitter amino acids. We combined the use of immunoperoxidase and immunofluorescence microscopy to localize glutamate and GABA as well as their vesicular transporters and receptors in various rat adipocyte tissues. Glutaraldehyde (25%) and paraformaldehyde were from Merck (Whitehouse Station, NJ). Pentobarbital (9.7 mg/ml) was from Rikshospitalets Apotek (Oslo, Norway). Dextran D-70 was from Amersham (Buckinghamshire, England). All chemicals were of analytical grade. We used two cryostats: 2800 Frigocut-E (Reichert-Jung/Leica) and HM 560 (Microm International GmbH, Walldorf, Germany). The polyclonal antibodies directed against glutamate and GABA were raised in rabbits essentially as originally described by immunizing with the amino acids coupled to BSA by a glutaraldehyde-formaldehyde mixture (18.Storm-Mathisen J. Leknes A.K. Bore A.T. Vaaland J.L. Edminson P. Haug F.M. Ottersen O.P. First visualization of glutamate and GABA in neurones by immunocytochemistry.Nature. 1983; 301: 517-520Crossref PubMed Scopus (777) Google Scholar). The anti-glutamate (No. 607) and anti-GABA (No. 990) sera have been extensively characterized previously (19.Broman J. Anderson S. Ottersen O.P. Enrichment of glutamate-like immunoreactivity in primary afferent terminals throughout the spinal cord dorsal horn.Eur. J. Neurosci. 1993; 5: 1050-1061Crossref PubMed Scopus (95) Google Scholar, 20.Gundersen V. Chaudhry F.A. Bjaalie J.G. Fonnum F. Ottersen O.P. Storm-Mathisen J. Synaptic vesicular localization and exocytosis of L-aspartate in excitatory nerve terminals: a quantitative immunogold analysis in rat hippocampus.J. Neurosci. 1998; 18: 6059-6070Crossref PubMed Google Scholar, 21.Gundersen V. Fonnum F. Ottersen O.P. Storm-Mathisen J. Redistribution of neuroactive amino acids in hippocampus and striatum during hypoglycemia: a quantitative immunogold study.J. Cereb. Blood PubMed Scopus Google Scholar). The antibodies not to amino acid in as they the free amino acids to tissue via the We used an with glutaraldehyde-formaldehyde to as The glutamate was with of and whereas the GABA was with of and a we by the with of the amino acids against the were the glutamate and GABA of the tissue and antibodies and receptor NR1 as well as protein 2 and antibodies were from International The fibrillary acidic protein was from and the was raised in and from Synaptic Germany). The receptor was from whereas the receptor was a from R. The glutamate receptor was from The to the on was raised in and from and antibodies with of and and were from for immunoperoxidase and were from antibodies and were from rats were from The was in with the on the of and was by The animals free to food and an they were with of of the rats were for the with different in by a of in the The animals were with and acid immunoperoxidase or with with or we removed WAT from interscapular, mesenteric, perirenal, and epididymal depots as well as interscapular In the were and with the WAT and BAT they were for at or for at in the used for the of were from animals that were in the was from and the was The tissues for immunoperoxidase were in the at whereas tissues for immunofluorescence were in the in For the tissues were for 2 at with for in a mixture of and and at The WAT and BAT and the brain were on a and on were at they were with the antibodies in and to an immunofluorescence H. localization of receptors in rat J. Neurosci. 1996; PubMed Scopus Google Scholar). The were with a single primary or a mixture of primary antibodies from two different followed by or a mixture of antibodies that were coupled to different For immunoperoxidase the were with the antibodies a L. and in Histochem. Cytochem. PubMed Scopus Google Scholar). The antibodies were used at the to to to to to receptor to and The antibodies and were used at of The and the were used at of In control immunofluorescence in the primary antibodies were or with there was no of the tissue the of the adipose tissue brain were with the antibodies as in all with the adipose tissues. The localization of was with a We data from two to BAT and WAT tissues for microscopy were in to a and immunogold was as described R. Froyland M. Aragon C. Danbolt N.C. Fortin D. Storm-Mathisen J. Davanger S. Gundersen V. Glycine, GABA and their transporters in pancreatic islets of Langerhans: evidence for a paracrine transmitter interplay.J. Cell Sci. 2004; 117: 3749-3758Crossref PubMed Scopus (64) Google Scholar). were with the primary antibodies at the from from and from In of BAT and was with and or VGLUT2 with antibodies In each brain were with the BAT and WAT the of the and were The were with a whether glutamate and GABA, are the excitatory and inhibitory in the may have signaling functions in adipose tissue, we examined the localization of these free amino acids in adipocytes from various anatomical regions. that adipocytes of interscapular BAT and WAT were stained with antibodies glutamate and We could also these two amino acids and there was a glutamate and GABA of the WAT depots in the not In WAT and the amino acid was to the of the adipocyte and from the lipid We whether the presence of GABA in the adipose tissues could be to intracellular synthesis immunofluorescence with antibodies raised against the GABA-synthesizing enzyme We that GAD65 was present in BAT and WAT as well as in the WAT data were with antibodies the not an important role in the energy metabolism of hence, its presence does not a transmitter The amino acid vesicular transporters and VGAT can be used to cells that release glutamate and/or whether the various of adipocytes or of these transporter we of immunofluorescence microscopy and the tissue with antibodies against VGAT and VGLUT1 is the type in the We that VGAT and VGLUT1 were present in BAT as well as in WAT depots. In we that GAD65 and VGAT were partly in these BAT and WAT depots as shown in VGAT and VGLUT1 were present in the adipocytes in BAT and in The intracellular by antibodies against VGAT was to that with the VGLUT1 antibodies a with the with whereas the GAD65 with the localization of the enzyme in were for VGAT and VGLUT1 in the WAT depots not the presence of neurotransmitter components in adipose tissue, we a of and that VGLUT3, and were not present in BAT or these could be in VGLUT3, but not or was present in endocrine pancreatic tissue, as of the protein microtubule-associated protein 2 was in BAT and WAT In brain as in and not In the the primary was we whether VGAT, and VGLUT2 were present in adipose tissue the microscopic immunogold that VGAT and vesicular organelles in BAT and the of VGLUT2 was not immunofluorescence whether the vesicular transporter were to adipocytes or localized in such as cells of we the adipose tissues with antibodies against the and VGAT or was no between and VGAT and VGLUT1 were in the whereas the could be only in cells in BAT and in epididymal of and were also in all WAT depots in the not VGAT and VGLUT1 are an of the of whether such synaptic-like microvesicles in are present in we the tissues with antibodies against the marker synaptophysin. showed that was present in adipocytes of BAT and WAT as well as in adipocytes of the WAT depots not In the was partly with VGAT and the amino acid the GAD65 VGAT, and were all localized throughout the of BAT and WAT the lipid the roles of the amino acids as signaling substances in adipose tissues, we the localization of glutamate and GABA receptors in BAT and the use of immunofluorescence we that the glutamate receptor GluR2/3 and the glutamate receptor NR1 were present in BAT and in all WAT depots GABAA receptor could also be in all adipose tissues examined In to these ionotropic receptors, the metabotropic glutamate receptor was not in of the adipose tissues Amino acid have been in the suggest that glutamate and GABA have a function in the endocrine (10.Anhert-Hilger G. Stadtbaumer A. Strubing C. Scherubl H. Schultz G. Riecken E.O. Wiedenmann B. γ-Aminobutyric acid secretion from pancreatic neuroendocrine cells.Gastroenterology. 1996; 110: 1595-1604Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 11.Reetz A. Solimena M. Matteoli M. Folli F. Takei K. Camilli P.De GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion.EMBO J. 1991; 10: 1275-1284Crossref PubMed Scopus (336) Google Scholar, 12.Thomas-Reetz A. Hell J.W. During M.J. Walch-Solimena C. Jahn R. Camilli P.De A gamma-aminobutyric acid transporter driven by a proton pump is present in synaptic-like microvesicles of pancreatic beta cells.Proc. Natl. Acad. Sci. USA. 1993; 90: 5317-5321Crossref PubMed Scopus (90) Google Scholar, 13.Gammelsaeter R. Froyland M. Aragon C. Danbolt N.C. Fortin D. Storm-Mathisen J. Davanger S. Gundersen V. Glycine, GABA and their transporters in pancreatic islets of Langerhans: evidence for a paracrine transmitter interplay.J. Cell Sci. 2004; 117: 3749-3758Crossref PubMed Scopus (64) Google Scholar). We the evidence for a function of amino novel data that BAT and various WAT depots contain the compounds involved in transmitter amino acid signaling. we could demonstrate that the excitatory amino acid transmitter glutamate and the inhibitory amino acid transmitter GABA were present adipocytes of BAT and WAT depots. In the GABA-synthesizing enzyme isoforms GAD65 and and the vesicular transporters VGAT and as well as the marker could be in the adipose tissue with the localization of the glutamate receptor NR1 and GluR2/3 as well as the GABAA receptor on these BAT and WAT these findings indicate that glutamate and GABA are from intracellular synaptic-like microvesicles to receptor effects on adipocytes paracrine The of and from adipose tissue, brain and tissue for VGLUT2 and VGLUT3, and were not in BAT or whereas they could be in in the has The of the various components of such signaling does not a function of amino acids in adipose tissues. of their at and during of adipose Although considered outside the of this for a role of amino acid in adipose signaling might be by the of amino acids and their vesicular transporters and receptors immunogold and microscopy of adipose tissue in different of Moreover, there are glutamate and GABA receptors in the CNS, and there could be amino acid receptors on adipocytes were examined in this is that intracellular of glutamate and GABA also the synthesis of glutamate and GABA from acid in addition to and/or of a paracrine signaling these amino acids could be of in adipose tissues, metabolism and signaling this to the release or of free acids and substrates to and from the Although the autonomous innervation of BAT and WAT is well a role of the sympathetic system in the of obesity and the has only evidence indicates that from the CNS to adipose tissues might modify the regulation of body and energy homeostasis as well as (3.Dodt C. Lonnroth P. Wellhoner J.P. Fehm H.L. Elam M. Sympathetic control of white adipose tissue in lean and obese humans.Acta Physiol. Scand. 2003; 177: 351-357Crossref PubMed Scopus (62) Google Scholar). Whereas the CNS can affect various fat depots in a signaling might also modify the adipocytes. may be of data from humans have shown important WAT gene expression in the of enzymes involved in lipid and expression of (6.Cousin B. Casteilla L. Dani C. Muzzin P. Revelli J.P. Penicaud L. Adipose tissues from various anatomical sites are characterized by different patterns of gene expression and regulation.Biochem. J. 1993; 292: 873-876Crossref PubMed Scopus (60) Google Scholar, M. G. A. M. J. of human fat expression profiles and gene J. Physiol. 292: PubMed Scopus Google Scholar, A. J. C. of and adipose role in 2005; PubMed Scopus Google Scholar, M. J. P. expression and adipose tissue in lean and obese 2005; PubMed Scopus (35) Google Scholar). in WAT depots might alterations in the risk of For example, WAT with WAT is with increased (e.g., from cardiovascular disease and M. J. P. expression and adipose tissue in lean and obese 2005; PubMed Scopus (35) Google Scholar, S. S. S. L. M.G. P. L. and the risk of in from a 2005; 366: Full Text Full Text PDF PubMed Scopus Google Scholar, A.N. Sothmann M.S. Hoffmann R.G. Hennes M.I. Wilson C.R. Gustafson A.B. Kissebah A.H. Adiposity, fat distribution, and cardiovascular risk.Ann. Intern. Med. 1989; 110: 867-872Crossref PubMed Google Scholar). In this the various components of the amino acid transmitter signaling system could be in interscapular BAT and all WAT depots a role of this signaling the present data not the of amino acid signaling in different of fat depots. In these findings could the for the of to adipose cells the storage and release of fat from their intracellular fat and/or they might and was by from the and the The J. of for the of the