Articles in PresS. Am J Physiol Regul Integr Comp Physiol (January 29, 2014). doi:10.1152/ajpregu.00520.2013
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Supraoptic Oxytocin and Vasopressin Neurons Function as Glucose and
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Metabolic Sensors
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Zhilin Song,1 Barry E. Levin,2,3 Wanida Stevens,1 and Celia D. Sladek1
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Department of Physiology, University of Colorado School of Medicine, Aurora,
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CO;
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Neurology Service, VA Med Ctr, East Orange, NJ, USA
Department of Neurology and Neurosciences, Rutgers, New Jersey Medical
School, Newark, NJ, USA
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Running head: Oxytocin and Vasopressin Neurons: Glucose/Metabolic Sensors
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Corresponding author:
Celia D. Sladek, Ph.D.
Department of Physiology
University of Colorado School of Medicine
12800 E. 19th Ave, Mail Stop 8307
Aurora, CO 80045 USA
303-724-4526
FAX: 303-724-4501
e-mail: celia.sladek@ucdenver.edu
Copyright © 2014 by the American Physiological Society.
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ABSTRACT:
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Neurons in the supraoptic nuclei (SON) produce oxytocin and vasopressin and
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express insulin receptors (InsR) and glucokinase. Since oxytocin is an
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anorexigenic agent and glucokinase and InsR are hallmarks of cells that function
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as glucose and/or metabolic sensors, we evaluated the effect of glucose, insulin,
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and their downstream effector, KATP channels on calcium signaling in SON
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neurons and on oxytocin and vasopressin release from explants of the rat
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hypothalamo-neurohypophyseal system. We also evaluated the effect of
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blocking glucokinase and phosphatidylinositol 3 kinase (PI3K; mediates insulin-
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induced mobilization of glucose transporter, GLUT4) on responses to glucose
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and insulin. Glucose and insulin increased intracellular calcium ([Ca++]i ). The
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responses were glucokinase and PI3K dependent, respectively. Insulin and
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glucose alone increased vasopressin release (p<0.002). Oxytocin release was
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increased by glucose in the presence of insulin. The oxytocin and vasopressin
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responses to insulin+glucose were blocked by the glucokinase inhibitor, alloxan
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(4mM; p≤0.002), and the PI3K inhibitor, wortmannin (50nM; OT: p=0.03; VP:
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p≤0.002). Inactivating KATP channels with 200nM glibenclamide increased
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oxytocin and vasopressin release (OT: p<0.003; VP: p<0.05). These results
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suggest that insulin activation of PI3K increases glucokinase-mediated ATP
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production inducing closure of KATP channels, opening of voltage sensitive
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calcium channels, and stimulation of oxytocin and vasopressin release. The
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findings are consistent with SON oxytocin and vasopressin neurons functioning
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as glucose and ‘metabolic’ sensors to participate in appetite regulation.
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Keywords (3-5): insulin, glucokinase, calcium imaging, hormone release,
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phosphatidylinositol 3 kinase, glucose-sensing
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INTRODUCTION
Obesity has reached epidemic proportions in the US and currently gastric
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bypass is the only effective long term treatment. In these studies, we
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investigated a largely overlooked hypothalamic mechanism for appetite
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regulation that might prove useful for weight intervention. Oxytocin (OT) is an
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anorexigenic agent that inhibits food intake and reduces body weight following
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central or peripheral administration (1, 2, 6, 13, 36, 43, 49, 50, 85), and deficits in
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OT or mutations in the OT receptor (OTR) are associated with obesity in humans
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(11, 22, 81) and mice (8, 46, 70, 71). Studies on the role of OT in appetite
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regulation have largely focused on the paraventricular nucleus (PVN) and its
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hindbrain projections (7, 31, 43, 52, 53, 55, 62). PVN contains both parvocellular
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and magnocellular OT neurons. Parvocellular OT neurons project to
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preganglionic neurons of the autonomic nervous system in the brainstem and
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spinal cord regulating autonomic functions including gastric motility (16). In
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contrast, the magnocellular neurons (MCNs) project to the neural lobe of the
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pituitary and release OT into the peripheral circulation in response to
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anorexigenic and other stimuli (38, 69). However, the OT MNCs in the
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supraoptic nucleus (SON) and PVN are also a major source of OT in the brain.
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They have been shown to project to areas of the brain involved in motivated
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behaviors [e.g. amygdala and nucleus accumbens, (29, 58)]. Since OT neurons
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have not been described in these regions except in the naked mole-rat (57), it is
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likely that the MNCs provide the ligand for OT receptors in these regions, and
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could participate in appetite regulation. This possibility has not received
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attention by investigators studying hypothalamic mechanisms of appetite
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regulation.
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Autoradiographic binding of insulin and prominent insulin receptor (InsR)
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immunohistochemistry was described in SON in the late 1980s (19, 76). Since
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SON contains only OT and vasopressin (VP) MCNs projecting to neural lobe,
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and since insulin serves as an appetite regulating signal in other parts of the
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hypothalamus, expression of InsR in MNCs raises the possibility that, consistent
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with an anorexigenic role for OT, MNCs may have the ability to monitor nutrient
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stores and in turn regulate peptide release to appropriately adjust food intake to
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maintain body nutrient stores. We assessed the ability of SON MNCs to function
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as glucose and metabolic sensors as diagrammed in Fig 1. First, we used
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quantitative real-time PCR (qRT-PCR) to confirm expression of InsR in SON, and
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to evaluate expression of glucokinase (GK), a hallmark of glucose-sensors, in
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order to determine if MNCs expressed molecules characteristic of metabolic
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sensors. We then evaluated the effect of glucose and insulin on intracellular
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calcium signaling in SON MNCs and OT and VP release from explants of the rat
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hypothalamo-neurohypophyseal system (HNS). HNS explants include the SON
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with their projections to neural lobe. Since SON contains only MNCs, HNS
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explants are an excellent preparation for studying responses of MNCs
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independent of parvocellular OT neurons. Finally, we examined the roles of
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ATP-sensitive potassium (KATP) channels, GK, and phosphoinositol 3-kinase
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(PI3K), the kinase that mediates insulin-induced insertion of GLUT4 glucose
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transporters into the membrane in adipocytes and arcuate neurons (9, 47, 48), in
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response to glucose and insulin.
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MATERIALS AND METHODS
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Animals.
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Male Sprague-Dawley rats [CRL:CD(SD)Br; Charles Rivers Laboratories,
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Wilmington, MA], 150-175g, were used in all experiments. All protocols were
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performed in accordance with the National Institutes of Health Guidelines for the
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Care and Use of Laboratory Animals and were approved by the Institutional
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Animal Care and Use Committee of the University of Colorado Denver.
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Detection of mRNA for GK and InsR by qRT-PCR.
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SON was microdissected using the optic chiasm as a landmark. A rectangular
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block of tissue immediately rostral to optic chiasm approximately 2mm wide x
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1mm deep x 3mm long was removed from each side of the brain using
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irridectomy scissors (64). Microdissected samples of SON were collected into
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RNA-Later (QIAGEN) and shipped to B. Levin’s laboratory for processing and
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analysis as described previously (10, 32). Primer sets for cyclophilin, the
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housekeeping reference gene, GK, and insulin receptor mRNA were designed by
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reference to published sequences, and their specificity was verified using
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GenBank and by comparing the sequenced PCR product for cyclophilin, GK and
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InsR to these references. For each mRNA species, a pair of conventional primers
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was used in combination with a sequence-specific 6-carboxyfluoroscein (FAM)-
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labeled probe to allow real-time PCR quantitation using an Applied Biosystems
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7700 Sequence detector set for 40 PCR cycles. The primers for cyclophilin
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[constitutive gene (39)] were GenBank (NM 017101): forward beginning at 253
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bp, CCAATACGTCATTCACAACAACAAG; reverse beginning at 330 bp,
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AAGTTGCTGGAATTCATGGTATAGC; FAM-labeled probe
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TCTACGGAGAGAAATT. The primers for the insulin receptor were Genebank
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(NM_017071): Forward beginning at 1250 bp:
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AACCTGTGAGGATGAGTGTCAGAGT; Reverse beginning at 1321 bp:
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CCTTGCTCTTCATCAGTTTCCA; and FAM-labeled probe
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TGCATCCCCGAGTGC. The primers for GK were Genebank (NM012565):
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Forward primer: CGAGGA GGCCA GTGTAAAGATG; Reverse primer:
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TCTCCGACTTCTGAGCCTTCTG; and probe AACGCACGTAGGTGGG.
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Reference standards were created for cyclophilin and InsR from pooled aliquots
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of arcuate nucleus samples, and these were used to generate standard curves
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from which quantitative data were read. Data were then expressed as the ratio of
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GK and InsR to cyclophilin mRNA.
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Hypothalamo-neurohypophyseal explant preparation.
HNS explants were used for calcium imaging and hormone release
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studies. Explants were prepared as described previously from male rats (67).
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HNS explants include the SON neurons, their axons, and axon terminals in the
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neural lobe as well as organum vasculosum of the lamina terminalis, and
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suprachiasmatic and arcuate nuclei. They do not include the PVN.
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Calcium imaging.
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HNS explants were loaded with the calcium-sensitive dye, Fura-2 AM, as
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described previously (67). They were placed in a recording chamber with the
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ventral surface up allowing easy visualization of SON neurons using the optic
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chiasm as an anatomical landmark (67). SON neurons were identified by the
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size of the cell body (>25 μm in diameter) and their location adjacent to the optic
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chiasm. Explants were perifused at a rate of 3 ml/min with gassed (95% O2/5%
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CO2) specially formulated F12 nutrient mixture modified to contain 0.5 mM
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glucose, 13mM KCl, and 1.7 mM CaCl2. Fura-2-loaded MNCs were alternately
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excited with 340 nm and 380 nm UV light from a Xenon Source (Sutter
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Instruments, Novato, CA). The 380 nm exposure time was between 200-500 ms
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and was tripled for the 340nm exposure. Emitted light was passed through a
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60X fluor water immersion lens attached to an Olympus upright microscope and
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collected at 510 nm by an intensified charge-coupled device camera
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(Hamamatsu, Japan). Paired 340 and 380 nm images were acquired every 3
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sec. using Slide-Book software (Intelligent Imaging, Denver, CO) for a period of
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100 frames. The 340:380 ratio (R) was used as an index of the change in [Ca++]i.
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Rmax was previously determined in ionomycin treated explants and far exceeded
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the highest R obtained with agents studied in these experiment (67). R data are
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presented as percentage of the basal 340:380 R for each cell determined from
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the average R of 10 frames preceding drug exposure. Explants were allowed to
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equilibrate for 1hr.
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Data analysis: Mean±SEM of the percentage values from individual
neurons were calculated and plotted. Parametric one-way ANOVA (F value)
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followed by Student-Newman-Keuls individual mean analysis or Kruskal-Wallis
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one-way ANOVA on ranks (H value) followed with Dunn’s individual mean
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analysis were used to determine significant group differences and paired T-tests
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were used to compare peak responses to the basal [Ca++]i.
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Hormone release from HNS explants.
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Explants were positioned individually in perifusion chambers having a 500
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µl volume, and perifused at 2 ml/hour as described previously (27) with specially
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formulated F12 nutrient mixture containing a final glucose concentration of 1mM.
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Following a 4-5 hour equilibration period to allow hormone release to stabilize at
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basal level, explants were either maintained under control conditions or exposed
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to an increase in glucose (5mM) or insulin (3ng/ml) for 1 hr followed by the
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addition of glucose to achieve 5mM glucose. Since the perifusion medium
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contained 20% fetal bovine serum (FBS), the amount of glucose and insulin
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added was adjusted to account for the glucose and insulin concentration in each
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lot of FBS. Where appropriate drugs were added to inhibit GK or PI3K and
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glibenclamide was used to evaluate the effect of inactivating KATP channels on
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hormone release. Effluent was collected individually at 20 min intervals using a
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refrigerated fraction collector maintained at 4°C. VP and OT concentration in the
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perifusate was determined by radioimmunoassay as described previously (82).
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Changes in VP and OT release from HNS explants reflects altered release from
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nerve terminals in the neural lobe, because although VP and OT are released
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from dendrites in SON and VP from suprachiasmatic nucleus (15), the amount
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from neural lobe far exceeds these other sources (17).
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Data analysis. Basal VP/OT release was determined during the hour
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immediately preceding exposure to insulin, glucose, or drugs. Hormone release
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in response to experimental manipulations is expressed as a % of this initial
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basal release for each explant. Basal release for the explants included in these
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studies was 159±27 pg/ml for VP and 190±10 pg/ml for OT (mean±sem).
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ANOVA with repeated measures followed by post hoc simple main effects
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analysis was performed to evaluate changes in hormone release and to compare
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responses between groups.
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RESULTS
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Detection of mRNA for GK and InsR by qRT-PCR.
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Using primers described previously (10, 32), the transcripts for both InsR and GK
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were found to be abundant in SON. The average number of cycles to detection
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compared to cyclophilin (2-Δct), the reference house-keeping gene, was 4.9 for
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InsR and 6.1 for GK, indicating that InsR mRNA is in the order of 3.4% and GK
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1.5% as abundant as cyclophilin (assuming equal efficiencies in the PCR
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reactions). This is consistent with the prominent expression of InsR observed
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with immunohistochemistry (76). The expression of GK is significant, because it
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raises the possibility that these neurons may function as glucose-sensors (e.g.
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cells that alter their firing pattern proportionally to the extracellular glucose
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concentration). Since the insulin producing β-cells in the pancreas as well as
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glucose-sensing neurons express GK, GK is considered to be the ‘gate-keeper’
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for glucosensing (34).
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hypothesis that these neurons can monitor extracellular glucose.
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ventromedial nucleus (VMN) glucose-sensing neurons also express GK and InsR
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(26), the prominent expression of GK and InsR in SON supports the hypothesis
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that MNCs may function as glucose sensors similar to VMN neurons. Thus,
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these observations provided the crucial evidence to pursue the subsequent
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studies on the effects of glucose and insulin on SON neurons and the role of GK
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and PI3K.
The abundant presence of GK in SON supports the
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Effect of 5mM glucose on [Ca++]i
Since the
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Increasing the glucose concentration to 5mM in F12 containing 3mM K+
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increased [Ca++]i in some, but not all SON neurons (data not shown). Using
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responsiveness to VP or OT as criteria for OT or VP phenotypic identification of
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the neurons (12), 44% of OT neurons and 74% of VP neurons showed
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responses. However, when the K+ concentration in the perifusate was increased
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to 13mM to move neurons closer to their depolarization threshold (80), increasing
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the glucose concentration from 0.5 mM to 5 mM reliably increased [Ca++]i in all of
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the MNCs recorded (Fig 2A and B). The response was sustained in the
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presence of tetrodotoxin (TTX, Fig 2C and D) indicating that the glucose
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response is not synaptically mediated. Since all the MNCs responded to an
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increase in glucose with an increase in [Ca++]i, they would be classified as
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‘glucose-excited’. It should be noted, however, that these experiments were not
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optimally designed to reveal ‘glucose-inhibited’ neurons.
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Effect of 5mM Glucose and Insulin on OT and VP release.
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As seen in Fig 3A, VP release from HNS explants was stimulated by increasing
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glucose alone (Glucose group from hour 4.6 to end; Ftreatment=25.74, p<0.0005;
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Ftime=4.91, p<0.0001), insulin alone (first hour in the Ins+Glucose group;
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Ftreatment=14.47, p=0.0035; Ftime=2.67, p=0.032; Finteraction=2.81; p=0.026), and the
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response to insulin was sustained when glucose was increased in the presence
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of insulin (Ins+Glucose group from hour 4.6 to end; Ftreatment=11.26, p=0.001;
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Ftime=5.13, p<0.0001). OT release was significantly increased during combined
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exposure to insulin and 5mM glucose (Ftime=5.17, p<0.0001; Finteraction=3.57,
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p<0.0001). Thus, glucose and insulin stimulated VP release from the posterior
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pituitary, and OT release was increased by increasing the glucose concentration
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in the presence of insulin.
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Role of KATP Channels in OT and VP neurons
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To assess the effect of inactivating KATP channels on OT and VP release, HNS
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explants were incubated in glibenclamide (200nM), a blocker of KATP channels,
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for 3 hrs. Glibenclamide was solubilized in ethanol and a comparable amount of
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ethanol was added to the control explants. As shown in Figure 4, the addition of
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glibenclamide resulted in a gradual and sustained increase in OT and VP release
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during the duration of exposure exposure to glibenclamide that reached statistical
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significance after 2hrs (OT: Ftreatment=17.24, p=0.002; Ftime=6.32, p<0.0001;
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Finteraction=3.87, p<0.0001; VP: Ftreatment=3.86, p=0.07; Ftime=10.98, p<0.0001;
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Finteraction=2.64, p=0.005). Thus, the presumptive depolarization resulting from
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inactivation of KATP channels is sufficient to increase OT and VP release.
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To assess the role of KATP channels in the glucose-induced increase in [Ca++]i in
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SON neurons, the effect of glucose was evaluated in the absence and presence
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of diazoxide (0.4mM). As shown in Figure 5, increasing the glucose
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concentration from 0.5 to 5mM resulted in a robust and sustained elevation in
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[Ca++]i. After a washout period, the same increase in glucose in the presence of
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diazoxide was ineffective. Thus, the glucose-induced increase in [Ca++]i is
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dependent on closure of KATP channels. This suggests that the glucose-induced
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increase in calcium is most likely due to depolarization induced opening of
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voltage dependent calcium channels, but our studies do not test that directly.
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Role of GK in OT and VP neurons
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To determine if the glucose-induced increase in [Ca++]i and the stimulation of OT
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and VP release by glucose and insulin are GK-dependent, we used alloxan
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(4mM) to block GK. As shown in Figure 6, the glucose-induced increase in
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[Ca++]i was essentially abolished in the presence of alloxan, and alloxan
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prevented the glucose plus insulin-induced increase in OT (Ftreatment= 18.04,
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p=0.0017; Ftime=2.63, p=0.0051; Finteraction=2.667, p=0.0045) and VP release
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(Ftreatment= 17.75, p=0.0001; Ftime=2.97, p=0.0013; Finteraction=4.331, p<0.0001).
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Thus, GK mediates the glucose-induced increase in [Ca++]i and the stimulation of
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OT and VP release induced by the combined increase in glucose and insulin.
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Role of PI3K in OT and VP neurons
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Since insulin activation of PI3K is required for insulin-induced translocation of the
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GLUT4 glucose transporter to the plasma membrane (60), we used wortmannin
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(50nM), a PI3K inhibitor, to determine if the insulin-induced increase in [Ca++]i
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and the stimulation of OT and VP release by insulin and glucose are PI3K-
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dependent. As shown in Figure 7, in the presence of 5mM glucose, the insulin-
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induced increase in [Ca++]i was significantly reduced by 10 or 50nM wortmannin,
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and no increase in OT and VP release was detected in the presence of 50nM
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wortmannin in response to simultaneously increasing the glucose and insulin
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concentrations of the perifusate to 5mM and 3ng/ml respectively (OT:
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Ftreatment=6.289, p=0.031; Ftime=2.887, p=0.0023; Finteraction=2.808, p=0.0029; VP:
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Ftreatment=9.75, p=0.0019; Ftime=5.416, p<0.0001; Finteraction=3.141, p<0.0001).
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Thus, the responses to insulin reflect PI3K-mediated intracellular events that
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could include insulin-induced insertion of GLUT4 transporters into the membrane.
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However, PI3K-dependent insulin actions that are independent of GLUT4
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translocation exist and are not excluded in these experiments (61).
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DISCUSSION
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The ability of glucose and insulin to alter VP and OT release from the
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neurohypophysis is consistent with the possibility that the MNC neurons can
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function as metabolic sensors. Although dendritic release of VP and OT also
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may be affected, the intact HNS explant does not allow us to explore that
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possibility. An important aspect of these findings is that the reported effects of
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glucose and insulin reflect responses to physiologically relevant concentrations of
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glucose and insulin. Glucose concentrations measured in the hypothalamus
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range from 0.5mM in fasting rats to 5mM in severely hyperglycemic rats with
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2.5mM representing post ingestive normoglycemia (63). Thus, the changes in
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glucose utilized in these experiments represented the full physiological range of
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hypothalamic glucose. Insulin is transported into the brain by a saturable
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transport mechanism that is physiologically and regionally regulated (3).
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Hypothalamic insulin levels are among the highest in the brain at 0.4 ng/g (4).
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Thus, it is reasonable, given the diffusion barriers inherent in in vitro
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preparations, that the glucose and insulin concentrations employed in these
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experiments resulted in local fluctuations that SON neurons might encounter in
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vivo.
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Using qRT-PCR, we found that InsR and GK are prominently expressed in
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rat SON. This confirmed the earlier immunohistochemical report showing dense
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InsR expression and the in situ hybridization study showing GK expression in
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SON neurons (45, 76). Since some of the glucose-sensing neurons in VMN also
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express GK and InsR, expression of these molecules in SON neurons is
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consistent with the hypothesis that MNCs function as glucose-sensors. In VMN
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neurons, glucose induces depolarization via GK-mediated inactivation of ATP
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sensitive K+ channels (KATP). Specifically, GK mediated glycolysis is likely to
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result in ATP production, closure of KATP channels, and depolarization (34). We
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found that similar mechanisms regulate OT and VP secretion from the
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neurohypophysis. Specifically, inhibition of KATP channel activity with
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glibenclamide was sufficient to increase OT and VP secretion and the ability of
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glucose to increase [Ca++]i is dependent on closure of KATP channels. This is
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consistent with the reported expression of the KATP channels, Kir6.1 and 6.2 in
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SON (14, 73). Furthermore, the increase in [Ca++]i induced by glucose and the
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increase in OT and VP release induced by combined exposure to increases in
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glucose and insulin are GK-dependent. Finally, since insulin mediated
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membrane insertion of GLUT4 is PI3K dependent (9, 47, 48), the ability of
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wortmannin to significantly reduce the calcium response to insulin and to prevent
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combined glucose and insulin stimulated OT and VP release, is consistent with
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the hypothesis that one role of InsR in SON neurons is to increase glucose
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transport potentially via inducing translocation of glucose transporters to the
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membrane (34, 75). However, since we have not demonstrated that insulin
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increases glucose uptake, it remains possible that the insulin actions, although
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PI3K-dependent, are independent of changes in glucose uptake. Insulin has
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been shown to activate KATP channels in VMN and arcuate neurons via PI3K
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(68). However, this results in membrane hyperpolarization which is not
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consistent with our findings that insulin increases [Ca++]i and hormone release.
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Thus, although future studies should evaluate the effect of insulin on glucose
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uptake, our findings are consistent with the model shown in Fig 1.
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Compartmentalization of InsR with other membrane proteins may allow for
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selective actions in various cells (60).
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Our studies did not demonstrate a differential effect of glucose or insulin
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on OT and VP neurons. How is this compatible with the evidence that OT is the
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neurohypophyseal hormone associated with satiety in rats (49-51)? It is
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consistent with our prior findings that most physiological stimuli and
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neurotransmitters elicit similar responses from MNC OT and VP neurons. In
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previous experiments with HNS explants, the two hormones show similar
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responses to osmolality as well as glutamatergic, adrenergic, purinergic, and
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peptidergic agents (21, 27, 28, 41, 42, 65, 66). Similarly, in vivo, both hormones
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are released by hypertonicity and hypovolemia with only suckling, gastric
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distention, and peripherally administered cholecystokinin (CCK) identified as
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stimuli that selectively activate OT neurons or specifically stimulate OT secretion
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in rats (54, 56). These OT specific stimuli discriminate between OT and VP
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neurons via efferent pathways that selectively activate the OT MNCs not as a
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result of expression of a particular receptor or ion channel being limited to the OT
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neuron (23, 24). The selectivity of the anorexigenic efferents for OT versus VP
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neurons is species dependent: While peripheral administration of CCK induces
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OT release in rats, in monkeys and humans, peripheral CCK stimulates VP
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release but not OT release (39, 78). Species variation in OT and VP receptor
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expression in target brain areas may also contribute to the anorexigenic
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specificity of OT in rats vs VP in primates.
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The hypothesis that, in addition to anorexigenic effects generated by
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peripheral OT (1, 2, 6, 13, 36, 43, 49, 50, 85), OT-induced anorexia partially
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reflects MNC-derived activation of OT receptors in the motivated behavior circuit
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is plausible based on the evidence for differential localization of OT and VP in
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receptors in forebrain regions that participate in appetite regulation (44, 59) as
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well as the recent evidence that MNC OT neurons innervate these regions (29,
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58). OT receptors are present in multiple regions of the motivated behavior
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circuitry including the amygdala, nucleus accumbens, prefrontal cortex, and
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ventral pallidum (caudate/putamen) (59), and all of these areas have been
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implicated in the motivated and hedonic components of appetite regulation (5,
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33). Evidence for innervation of the amygdala and nucleus accumbens by OT
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neurons that also project to the posterior pituitary (e.g. MNCs) has been obtained
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in elegant studies utilizing fluorogold and pseudorabies virus to retrogradely label
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neurons projecting to nucleus accumbens and amygdala (29, 58). Both studies
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identified neurons in SON (which only contains MNCs) that project both to the
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limbic region and the posterior pituitary. Thus, it is likely that axon collaterals
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from OT MNCs provide the substrate for OT receptors in these limbic regions.
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This suggests that glucose and insulin may have similar effects on OT release in
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the motivated behavior circuitry to that reported here on neurohypophyseal
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hormone release.
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While our studies specifically targeted the metabolic sensor capability of
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the MNCs by examining the responses to glucose and insulin, these neurons
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also are activated in response to feeding (25, 35, 40), gastric distention (56),
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activation of vagal afferents (72), and refeeding after an overnight (83) or 48 h
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fast (35). They also respond to other molecules involved in appetite regulation
404
including leptin (20), ghrelin (18, 84), cholecystokinin (23, 24), nesfatin (30, 37),
405
and prolactin releasing peptide (83). This further supports a role for these
406
neurons in appetite regulation.
407
408
Perspectives and Physiological Significance
409
Although involvement of insulin in mediating glucose uptake by the
410
neurohypophyseal system has been recognized since the late 1980’s when Gary
411
Robertson and colleagues reported that in untreated, insulin-dependent diabetics
412
glucose became an osmotic stimulus for VP release (79) and Unger et al (77)
413
reported prominent expression of InsR in the SON, the possibility that the
414
neurohypophyseal system monitors metabolic status and contributes to appetite
415
regulation was not investigated and the anorexigenic actions of OT were
416
attributed primarily to parvocellular OT neurons and hindbrain mechanisms (7).
417
However, the intractability of the recent obesity epidemic has led to the
418
realization that a highly redundant and distributed system is involved in appetite
419
regulation. Thus, while the evidence presented here that neurohypophyseal
420
neurons can monitor nutrient and metabolic status and potentially act on OT
421
receptors in the motivated behavior circuitry may simply represent an additional
422
layer of redundancy, it also might represent a previously under appreciated
423
opportunity to enhance anorexigenic actions, because an integrated peripheral
424
and central release of OT has the potential to influence peripheral metabolism (6)
425
as well as both motivated/hedonic and homeostatic pathways mediating food
426
intake.
427
428
429
430
ACKNOWLEDGEMENTS
431
This work was supported by grants from the National Institutes of Health to C.D.
432
Sladek (R21- HD072428) and B.E. Levin (RO1-DK53181).
433
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696
FIGURE LEGENDS
697
698
Figure 1. Do magnocellular neurons (MNCs) function as glucose and metabolic
699
sensors? Expression of insulin receptors (InsR) and glucokinase (GK) by
700
supraoptic MNCs suggests that MNCs can modulate release of the anorexigenic
701
agent, oxytocin (OT), in response to changes in circulating insulin and the
702
extracellular glucose concentration. We postulate that since glucose transporter
703
3 (GLUT3), the ‘brain glucose transporter’, is saturated at physiological glucose
704
concentrations, glycolysis of glucose by GK, a hexokinase that is not inhibited by
705
its own product (34), provides a glucose concentration dependent source of ATP.
706
GLUT 3 and GK are present in SON (45, 75). In other glucose sensing cells (e.g.
707
pancreatic β-cells and VMN neurons), ATP closes ATP sensitive K+ channels
708
(KATP) channels resulting in membrane depolarization, opening of voltage
709
sensitive calcium channels (vCa), and initiation of exocytosis or generation of
710
actions potentials. Kir6.1 and 6.2 (KATP channels) are present in SON (14, 74).
711
Thus, we postulate that GK-mediated ATP production inactivates (closes) KATP
712
channels inducing depolarization, opening of vCa, and propagation of action
713
potentials to the nerve terminals in posterior pituitary and other CNS regions to
714
initiate exocytosis of OT. We also postulate that insulin mediated membrane
715
insertion of GLUT4 which is PI3K dependent (9, 47, 48) provides additional
716
substrate for GK-mediated glycolysis thus further promoting KATP-mediated
717
depolarization and OT release.
718
719
Figure 2. Effect of increasing glucose on [Ca++]i in SON neurons. A. Time
720
course of change in 340:380 ratio indicative of change in [Ca++]i induced by
721
increasing the glucose concentration from 0.5mM to 5mM. Mean±SEM of 18
722
SON neurons imaged simultaneously in a single HNS explant is shown. B. The
723
peak increase in the 340:380 ratio in response to the addition of glucose in 34
724
neurons imaged in 2 HNS explants. Mean±SEM, * p=<0.001 by signed rank test
725
between basal and peak response. C. Time course of individual neuronal
726
responses to increasing glucose (from 0.5 to 5 mM) in the presence of
727
tetrodotoxin (TTX, 3μM). In this preparation, the responses were not tightly
728
temporally synchronized as in A. This could reflect neurons at different depths
729
within the preparation. D. The peak increase in the 340:380 ratio in response to
730
the addition of glucose in 40 neurons imaged in 2 HNS explants. Mean±sem, *
731
t=17.500, p=<0.001, basal to peak response.
732
733
Figure 3. Effect of glucose, insulin, and insulin plus glucose on VP and OT
734
release from HNS explants. A. and B. Time course of VP and OT responses
735
respectively to increasing the perifusate insulin concentration to 3ng/ml for 1 hour
736
followed by increasing the glucose concentration to 5mM (Ins+Glucose),
737
increasing the glucose concentration alone (Glucose), or maintaining the basal
738
insulin (70pg/ml from fetal bovine serum) and glucose (1mM) concentrations
739
throughout the experiment (Basal). °p<0.05 Ins alone or Ins+Glu vs. basal;
740
*p<0.05 Ins+Glu and Glucose alone vs basal (n=6/group). C. Peak increase in
741
OT or VP release from the same explants in A. and B. during exposure to insulin
742
alone, glucose alone, or insulin+glucose. *p<0.05, **p<0.001.
743
744
Figure 4. Effect of glibenclamide to inhibit KATP channels on OT (A.) and VP (B.)
745
release from HNS explants in 1mM glucose. Glibenclamide increased OT and
746
VP release (OT: *, **, ***p<0.05, 0.01, and 0.001 respectively by Bonferroni post
747
hoc analysis; VP: *p<0.05 by post hoc t-test).
748
749
Figure 5. Effect of activating (opening) KATP channels with diazoxide (0.4mM) on
750
the glucose-induced increase in [Ca++]i in SON neurons. A: Time course of Ca++
751
response to glucose (step rise from 0.5 to 5 mM) in the presence of 13 mM KCl
752
(glucose) and subsequently in the presence of diazoxide (DZX.glucose). Arrows
753
indicate when glucose was administered. B: Comparison of the peak responses
754
under these conditions. Glucose (5 mM) induced an increase in [Ca++]i in the
755
majority of SON neurons in the presence of 13 mM KCl (to increase resting
756
membrane potential). The response was abolished when cells were pretreated
757
with the KATP channel opener, DZX (*H= 47.264, p=< 0.001). Data was combined
758
from neurons imaged in 2 explants. Total # of neurons=32.
759
760
Figure 6. GK-dependence of the effects of glucose on [Ca++]i and
761
Insulin+glucose (Ins+glucose) on OT and VP release. A: Time course of Ca++
762
response to glucose (5 mM) in the presence of 13 mM KCl in the absence
763
(glucose) or presence of alloxan (AOX.glucose; 4 mM AOX). The basal medium
764
contains 0.5 mM glucose. Arrows indicate when glucose was administered. B:
765
Comparison of the peak responses under these conditions. Glucose (5 mM)
766
induced an increase in [Ca++]i in the majority of SON neurons in the presence of
767
13 mM KCl (to increase resting membrane potential). The response was
768
abolished when cells were pretreated with GK inhibitor, AOX (* H= 59.260, p=<
769
0.001). Data was combined from neurons imaged in 2 explants. Total # of
770
neurons=40. C. and D. Effect of alloxan on OT and VP release respectively.
771
Addition of alloxan (4mM) one hour prior to increasing the perifusate glucose and
772
insulin concentrations to 5mM and 3ng respectively did not alter basal release of
773
OT (198±25 pg/ml) or VP (169±46 pg/ml), but it prevented the increase in OT
774
and VP release induced by the addition of glucose and insulin. **p<0.01,
775
***p<0.0001. n=6 explants per group.
776
777
Fig. 7 Effect of the PI3K inhibitor, wortmannin (WMN) on the Ins-induced
778
increase in [Ca++]i and Insulin with glucose (Ins+glucose)-induced OT and VP
779
release. A: Time course of Ca++ response to Ins (3ng/ml) in the presence of 13
780
mM KCl in the absence (Ins) or presence of wortmannin (50nM, WMN.Ins) The
781
medium contained 5 mM glucose. Arrows indicate when Ins was administered.
782
B: Comparison of the peak responses under these conditions. Ins (3 ng/ml)
783
induced an increase in [Ca++]i in the majority of SON neurons in the presence of
784
5 mM glucose and 13 mM KCl (to increase resting membrane potential).
785
(*t=820.0, p=<0.001;). The peak response was greatly reduced in the
786
experiments when cells were pretreated with wortmannin (# H= 42.879, p=<
787
0.001; $ t=-18.299, p=<0.001). Data was combined from neurons imaged in 2
788
explants. Total # of neurons=40 in each experiment. C. and D. Effect of
789
wortmannin on OT and VP release respectively. Addition of wortmannin alone
790
one hour prior to increasing the perifusate glucose and insulin concentrations to
791
5mM and 3ng/ml respectively did not alter basal release of OT (175±22 pg/ml) or
792
VP (231±20 pg/ml), but it prevented the increase in OT and VP release induced
793
by the addition of glucose and insulin. ****p<0.0001. n=6 explants per group.
794
A. B. C. D. A. 300
% of basal
B. VP release
°
200
° *
°
400
* * *
*
C. OT release
°
300
200
100
0
100
0
4
5
Ins
6
Hours
7
5 mM Glucose
4
5
Ins
Control(n=6)
Glucose(n=6)
Insulin + Glucose(n=6)
°ins+gluc vs control
*gluc and ins+gluc vs cont
6
Hours
7
5 mM Glucose
Vehicle (ETOH)
Glybenclamide
OT release
***
***
500
% of basal
400
500
300
200
200
100
100
0
0
4
5
6
Hours
7
* *
* *
400
* **
300
VP release
8
4
5
6
Hours
7
A. B. A. 250
**
**
***
200
D. Control
Ins+Glucose
Alloxan+Ins+Glucose
OT release
Hormone release
% of basal
C. B. VP release
300
****
*
**
200
150
100
100
50
0
4
5
6
7
Hours
Alloxan Ins+5mM Glucose
** P<0.01; ***P<0.0001
0
4
5
6
7
Hours
Alloxan Ins+5mM Glucose
**P<0.01, ****P<0.0001
A. B. C. D. Control
Ins+Glucose
Wortmannin+Ins+Glucose
OT release
Hormone release
% of basal
250
****
200
150
150
100
100
50
50
0
4
5
6
Hours
Wort.
7
Ins+5mM Glucose
****P<0.0001
****
250
200
0
VP release
4
5
Wort
6
Hours
7
Ins+ 5mM Glucose
****P<0.0001