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Noradrenergic signaling in the central nervous system plays an essential role in circuits involving attention, mood, memory, and stress as well as providing pivotal support for autonomic function in the peripheral nervous system. The high affinity norepinephrine (NE) transporter (NET) is the primary mechanism by which noradrenergic synaptic transmission is terminated. Data indicates that NET function is regulated by insulin, a hormone critical for the regulation of metabolism. Given the high co-morbidity of metabolic disorders such as diabetes and obesity with mental disorders such as depression and schizophrenia we sought to determine how insulin signaling regulates NET function and thus noradrenergic homeostasis. Here, we show that acute insulin treatment, through the downstream kinase protein kinase B (Akt), significantly decreases NET surface expression in mouse hippocampal slices and superior cervical ganglion neuron (SCGN) boutons (sites of synaptic NE release). In vivo manipulation of insulin/Akt signaling, with streptozotocin (STZ), a drug that induces a Type 1-like diabetic state in mice, also results in aberrant NET function and NE homeostasis. Notably, we also demonstrate that Akt inhibition or stimulation, independent of insulin, is capable of altering NET surface availability. These data suggest that aberrant states of Akt signaling such as in diabetes and obesity have the potential to alter NET function and noradrenergic tone in the brain. Furthermore, they provide one potential molecular mechanism by which Akt, a candidate gene for mood disorders such as schizophrenia and depression, can impact brain monoamine homeostasis.
Appropriate regulation of critical brain functions such as learning, memory, attention, sleep, mood, and stress depend on the fidelity of noradrenergic signaling in the nervous system. The norepinephrine transporter (NET) is fundamental for maintaining this fidelity by controlling both the duration and strength of NE signaling through its reuptake of synaptic NE (Iversen, 1971; Pacholczyk et al., 1991; Bonisch and Bruss, 2006). Indeed, disruption of NET function has been shown to directly impact both autonomic function and mental health (Ganguly et al., 1986; Klimek et al., 1997; Rumantir et al., 2000; Shannon et al., 2000b; Hahn et al., 2003; Hahn et al., 2005; Kim et al., 2006; Haenisch et al., 2008; Hahn et al., 2008; Hahn et al., 2009).
Prior studies illustrate that NET function is dynamically regulated both by manipulation of transporter turnover rate and by trafficking of the transporter to and from the plasma membrane (Apparsundaram et al., 1998b; Apparsundaram et al., 1998a; Uchida et al., 1998; Apparsundaram et al., 2001; Miner et al., 2003; Sung et al., 2003; Jayanthi et al., 2004; Dipace et al., 2007). Importantly, previous studies implicate a clear role for insulin, a metabolic hormone, in the regulation of NET function. Indeed, insulin inhibits NE uptake in whole brain neuronal cultures, dissociated brain cells, and whole brain synaptosomes (Boyd et al., 1985; Boyd et al., 1986; Masters et al., 1987; Raizada et al., 1988). Furthermore, Figlewicz et al. demonstrated the ability of nanomolar concentrations of acute insulin to decrease NE uptake from both hypothalamic and hippocampal slices (Figlewicz et al., 1993). These studies were elegantly extended to demonstrate that insulin also inhibits NE uptake in PC12 cells which endogenously synthesize NE and express NET (Figlewicz et al., 1993). Conversely, more recent studies have shown in different preparations that insulin increases NE uptake (Apparsundaram et al., 2001). Our studies, both in vitro and in vivo, indicate that insulin plays an inhibitory role in the regulation of NET function by controlling its surface availability. Moreover, we reveal that protein kinase B (Akt), a multifunctional kinase downstream of numerous signaling pathways including insulin, is required for this insulin regulation. Importantly, we also show that Akt activity potently regulates NET surface levels independently of changes in insulin status.
Diseases characterized by aberrant insulin and Akt signaling such as diabetes and obesity have a high co-morbidity with monoamine related mental disorders such as schizophrenia and depression (Mukherjee et al., 1989; Mukherjee et al., 1996; Lustman and Clouse, 2005; Zhao et al., 2006). Our data reveal for the first time, Akt function as a potent regulator of NET activity/trafficking and thus provide an interesting and plausible link between metabolic dysfunction and mood disorders. Considering the identification of Akt as a candidate susceptibility gene in schizophrenia and perhaps depression (Hsiung et al., 2003; Emamian et al., 2004; Karege et al., 2007; Arguello and Gogos, 2008), these data also provide a plausible molecular mechanism linking anomalous Akt function to altered monoamine homeostasis which is characteristic of these disorders.
We gratefully acknowledge Dr. Randy Blakely for the gift of CHO cells stably transfected with HA tagged human NET (hNET cells). The cells were maintained in Ham’s F12 Media/10% FBS/L-Glu/pen/strep. Cells were plated on poly-D-lysine (Chemicon/Millipore, Billerica, MA)-coated plates for each experiment and incubated for 24 to 48 h before each experiment. Transient transfections with Akt-KD, a kinase dead (catalytically inactive) construct of Akt with the lysine at residue 179 replaced with an arginine (AKT-K179R) (Garcia et al., 2005), was graciously provided by Dr. R. Roth Stanford University (Stanford, CA). Transfections were performed with Fugene6 according to manufacturer’s instructions (Roche), and experiments on transfected cultures were performed 48 hours following transfection. Mouse superior cervical ganglion neurons (SCGNs) were cultured according to Savchenko et al. (Savchenko et al., 2003; Matthies et al., 2009). Briefly, superior cervical ganglions were dissected from 1–3 day old C57BL/6J mice and dissociated for 30 minutes in 3 mg/mL collagenase/0.5 mg/mL trypsin, followed by 10% FBS in DMEM. Cells were plated and incubated with 3% fetal bovine serum in UltraCulture medium containing NGF for 2 hrs at 37° C to allow fibroblasts to adhere. Supernatant medium (containing SCG cells) was centrifuged, resuspended in medium supplemented with FBS, and cultured on poly-D-lysine- and collagen-coated glass-bottom plates for 14 days before experiments (treating with 1 μM 5-fluoro-5-deoxyuridine after 24 hrs).
Monoclonal anti-mNET (NET-05) and monoclonal anti-hNET (NET 17-1) from MAb Technologies, Stone Mountain, GA (Matthies et al., 2009)), monoclonal anti- Na+/K+ ATPase α subunit (Developmental Studies Hybridoma Bank, Iowa City, IA), anti-Tyrosine Hydroxylase (MAB5280, Millipore/Chemicon, Billerica, MA), anti-ERK ½ (V114A, Promega, Madison, WI), anti-Akt (9272, Cell Signaling, Danvers, MA), anti-phospho-Akt (ser473) (4060, Cell Signaling) were used at dilutions of 1:200, 1:1000, 1:50, 1:1000, 1:5000, 1:1000 and 1:1000, respectively, for immunoblotting. Detection was obtained with secondary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) and by enhanced chemiluminescence reaction (ECL Plus Western Blotting Detection System, RPN2133, GE Healthcare). Monoclonal anti-mNET (NET-05 MAb Technologies, Stone Mountain, GA (Matthies et al., 2009)), anti-Rab11a (Lapierre et al., 2001), anti-Akt1 (2938, Cell Signaling), anti-Akt2 (D-17, sc-7127, Santa Cruz Biotechnology), anti-insulin Rβ (C-19, sc-711, Santa Cruz Biotechnology) were used at dilutions of 1:500, 1:110, 1:400, 1:100, and 1:50 for immunocytochemistry. Fluorescent secondary antibodies included highly cross-absorbed anti-mouse, goat, or rabbit IgG (Molecular Probes/invitrogen, Carlsbad, CA). Insulin from bovine pancreas (I6634), clozapine (C6305), NE, and STZ (S0130) were obtained from Sigma (I6634 St. Louis, MO). Akt1/2, Akt1, and Akt2 inhibitors (Lindsley et al., 2005) were generously provided by Dr. C. Lindsley (Vanderbilt University, Nashville, TN).
Biotinylation experiments were performed on intact cells as described previously (Sung et al., 2003; Garcia et al., 2005; Dipace et al., 2007). Briefly, hNET cells were plated at a density of 1 × 106 per well in a six-well poly-(D-lysine) coated plate. Cells were serum starved for 30 minutes to 1 hour prior to treatment, and washed with cold PBS containing Ca2+/Mg2+. Then, cells were incubated with 1.0 mg/mL sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate [NHS-SS-biotin] (Peirce/ThermoScientific, Rockford, IL) for 30 minutes, washed, quenched with 100 mM glycine, and extracted in lysis buffer (PBS Ca2+/Mg2+, 1% Triton 100-X, and 0.5 mM PMSF at 4°C). Lysates were centrifuged, total fractions reserved, and supernatants incubated with immobilized streptavidin beads (Pierce/ThermoScientific) for 1 hr at room temperature. Beads were washed three times in lysis buffer, and bound proteins eluted with 2X sample buffer containing 2-mercaptoethanol. Proteins were separated by SDS-PAGE and immunoblotted. For estimation of relative amounts of proteins, the exposed films of the immunoblots were scanned, and band intensities were measured with Scion Image (Scion Corporation, Frederick, MD). For brain slice preparation and biotinylation all procedures were performed according to Vanderbilt University Institutional Animal Care and Use Committee approved procedures. Brain slices were prepared from C57BL/6J 15 to 20-week-old male mice from Jackson Laboratories that were anesthetized with isoflurane and rapidly decapitated. Following, brain removal the brain was chilled in oxygenated 4°C sucrose solution (sucrose 210 mM; NaCl 20 mM; KCl 2.5 mM; MgCl2 1 mM; NaH2PO4•H2O 1.2 mM), and then while in sucrose solution 300 μm coronal slices were made using a vibratome. Slices were then collected in oxygenated artificial cerebral spinal fluid (ACSF) (NaCl 125 mM, KCl 2.5 mM, NaH2PO4•H2O 1.2 mM, MgCl2 1 mM, CaCl2•2H2O 2 mM). For in vitro drug treatments slices were then allowed to recover for 1 hour at 37°C in oxygenated ACSF and were then followed by drug treatment (i.e. insulin) and subsequent biotinylation. For in vivo treated animals (STZ or Clozapine experiments), slices were immediately washed twice with oxygenated 4°C ACSF following collection, and then incubated with 4°C ACSF solution containing 1 mg/mL of EZ-Link Sulfo-NHS-SS-Biotin (Pierce/ThermoScientific; Rockford, IL.) for 45 min. After biotin incubation, the slices were rinsed twice quickly and for two 10min washes in oxygenated 4°C ACSF. The reaction was quenched by washing twice for 20min each with oxygenated 4°C ACSF containing glycine. Following quenching, slices were frozen on dry ice and the cortex was cut out and frozen at −80°C until used. For each experiment, a minimum of 4 animals were used for the collection of slices per treatment group, and a single slice was utilized for each sample (represented in the text as “N”). Single slices were lysed in 1% Triton buffer (25 mM Hepes, 150 mM NaCl, 2 mM Sodium orthovanadate, 2 mM NaF, plus a cocktail of protease inhibitors). Lysates were then centrifuged at 17,000g for 30 min at 4°C. After isolation of supernatant 0.1% Triton pulldown buffer (25 mM HEPES, 150 mM NaCl, 2 mM Sodium orthovanadate, 2 mM NaF, plus a cocktail of protease inhibitors) was added. Total protein was taken and the samples were processed for protein concentration determination using Bio-Rad’s protein assay and spectrometry at 595 nm Biotinylated proteins were then isolated using ImmunoPure immobilized streptavidin beads (Pierce/ThermoScientific) overnight at 4°C with agitation. Beads were washed three times with 0.1% Triton pulldown buffer and biotinylated proteins were then eluted in 50 μL of 2X SDS-PAGE sample loading buffer at 95°C and then room temperature. Total slice lysates and the biotinylated (slice surface) fraction underwent immunodetection for NET, pAkt473, total Akt, Na+/K+ ATPase, and TH as described previously.
SCG neurons were either serum starved for one hour (insulin treatment) or non-starved (Akt inhibitor treatment) in DMEM:F12 and treated with vehicle, insulin, Akt1, or Akt2 inhibitor for 20 or 60 minutes respectively. Slices were obtained as previously described (cell and slice biotinylation methods). Slices and neurons were subsequently fixed with PBS Ca2+/Mg2+/4% paraformaldehyde, washed three times with PBS Ca2+/Mg2+, permeabilized and blocked with PBS/4% bovine serum albumin (BSA)/0.15% Tween-20, and immunostained with the appropriate antibody dissolved in PBS plus 4% BSA and 0.05% Tween-20. Primary antibodies were visualized with the appropriate covalently Alexa-labeled secondary antibody from Molecular Probes. Immunofluorescence was imaged using a Perkin Elmer UltraView confocal with a Nikon Eclipse 2000-U microscope equipped with a 60X lens with N.A.=1.49, or an Olympus FV 1000 using a 60X lens of N.A.=1.45 (VUMC Cell Imaging Shared Resource). Image processing was performed using Image J and Adobe Photoshop.
The quantitation of NET intracellular accumulation was achieved using pixel intensity plots of a single confocal section along a line through the center of each bouton using ImageJ. The line intersects the brightest spot on one side of the widest portion of the bouton perimeter. The line was extended beyond the limits of the bouton to generate a background value, and then divided into 20 bins (bin # 10 is approximately at the center of the bouton) for the pixels spanning the bouton. NET fluorescence intensity of each bin was normalized to the fluorescence intensity of the brightest spot on the bouton perimeter (100%) and the mean ± SEM pixel intensity of each bin plotted.
Only images in which there was no pixel saturation were analyzed. Background fluorescence was first subtracted in ImageJ by selecting an unstained area of each image and running the background subtraction plugin available at UHN research facilities (CA). The intensity correlation quotient (ICQ) was then determined by running the intensity correlation analysis (ICA) plugin for ImageJ developed by Tony Collins and Elise Stanley (Toronto Western Research Institute), also available at the above link. The ICQ indicates whether the intensity of staining for two proteins varies in synchrony over space. An ICQ value of +0.5 means that in any pixel with a certain intensity of staining for one protein, the intensity of staining for the other protein studied will be exactly the same, while an ICQ value of 0 signifies no relation between the two staining patterns. Consistently, an ICQ value of −0.5 indicates an inverse relationship for colocalization. This method is based on the synchrony around which two signals vary in space (Li et al., 2004).
hNET cells were seeded into 6-well plates 24–48 h prior to the experiment and grown to confluency. After 30 minutes of serum starvation, the cells were incubated with 10 nM insulin for 1 min (Sigma, St Louis, MO, USA) in KRH/glucose buffer at 37°C. The cells were then placed into 37°C KRH/glucose uptake buffer for 10 min (130 mM NaCl, 1.3 mM KCl, 10 mM Hepes, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.2 mM CaCl2, 10mM glucose, pH 7.4) containing, 100 μM ascorbic acid, 100 μm pargyline, and [3H]DA (50 nM) for uptake and depending on the treatment group the uptake buffer also had vehicle, 10 nM insulin, or cocaine to measure non-specific uptake. Immediately following uptake, cells were washed in cold 4°C KRH/glucose buffer 3 times. All solution was removed and plates were allowed to dry on the 37°C plate. Once dry, cells were lysed with 1 mL of 0.01% SDS. Radioactivity was measured in a Beckman scintillation counter with UniverSol cocktail. Specific uptake was defined as total uptake minus non-specific uptake in the presence of 10 μM cocaine.
All procedures were approved by the Vanderbilt University Medical Center and the University of Texas Health Science Center at San Antonio Institutional Animal Care and Use Committees and were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For all experiments, male C57BL/6J 15 to 20-week-old mice from Jackson Laboratories served as experimental subjects. STZ is an antibiotic that destroys the insulin-secreting beta cells of the pancreas and has previously been used to induce chronic hypoinsulinemia in rats by our laboratories (Galici et al., 2003; Owens et al., 2005). STZ (Sigma-Aldrich;) was freshly dissolved in ice-cold 100 mM citrate saline (pH 4.5) for all studies. Mice received STZ (200 mg/kg, i.p. for biochemical and high-speed chronoamperometry (HSCA) studies) and were returned to their home cages for 7–10 days. Weight and blood glucose were measured prior to injection and post injection. Blood glucose was measured with a glucometer (Advantage Accu-Chek, Roche Diagnostics;) before STZ, at 48 hours after injection and at animal sacrifice. Animals were considered hypoinsulinemic when their blood glucose levels exceeded 300 mg/dl.
The brain sections were homogenized in 100–750 ul of 0.1M TCA, containing 10-2 M sodium acetate, 10-4 M EDTA, 5ng/ml isoproterenol (as internal standard) and 10.5 % methanol (pH 3.8). Samples were spun in a microcentrifuge at 10000 g for 20 minutes. The supernatant was removed and stored at −80 degrees (3). The pellet was saved for protein analysis. Supernatant was then thawed and spun for 20 minutes. Samples of the supernatant were then analyzed for biogenic monoamines and/or amino acids. Biogenic amines were determined by a specific HPLC assay utilizing an Antec Decade II (oxidation: 0.5) electrochemical detector operated at 33° C. Samples of the supernatant were injected using a Water 717+ autosampler, twenty onto a Phenomenex Nucleosil (5u, 100A) C18 HPLC column (150 × 4.60 mm). Biogenic amines were eluted with a mobile phase consisting of 89.5% 0.1M TCA, 10-2 M sodium acetate, 10-4 M EDTA and 10.5 % methanol (pH 3.8). Solvent was delivered at 0.6 ml/min using a Waters 515 HPLC pump. Using this HPLC solvent the following biogenic amines elute in the following order: noradrenaline, MHPG, adrenaline, DOPAC, dopamine, 5-HIAA, HVA, 5-HT, and 3-MT (2). HPLC control and data acquisition are managed by Millennium 32 software.
HSCA is an electrochemical recording technique, which affords the kinetics of NE clearance to be measured in vivo. Detailed methods are published elsewhere (Daws & Toney, 2007; Daws et al. 2005). Mice were anaesthetized by intraperitoneal injection (2 ml/kg body weight) of a mixture of chloralose (25 mg/kg) and urethane (250 mg/kg) followed by tracheal intubation and placement into a stereotaxic frame. Body temperature was maintained at 36–37°C and blood oxygen levels monitored (MouseOximeter, StarrLifeSciences, USA) and maintained above 90%. A Nafion-coated carbon fiber electrode was attached to a glass micropipette containing NE. The assembly was lowered into the brain region of interest (stereotaxic coordinates in mm: DG region of hippocampus AP −1.6 to −1.7; ML 0.5; DV −1.8 to −2.0) and NE pressure ejected to achieve concentrations at the recording electrode ranging from about 0.2–4.0 μM. High-speed chronoamperometric recordings were made using the FAST-12 and FAST-16 systems (Quanteon, USA). Oxidation potentials consisted of 100-ms pulses of +0.55 V. Each pulse was separated by a 900-ms interval during which the electrode potential was maintained at 0.0 V. Voltage at the active electrode was applied with respect to a Ag/AgCl reference electrode positioned in the extracellular fluid of the ipsilateral superficial cortex. Electrode placement was confirmed by making an electrolytic lesion at the recording site at the conclusion of the experiment.
All data are expressed as the mean ± s.e.m. Mean differences between groups were determined using t-tests or one and two-way ANOVAs followed by post hoc tests when the main effect or interaction was significant at P < 0.05. Statistical analyses were conducted using software from Graph-Pad Prism. The number of animals and specific statistical analyses used in each experiment are indicated in the figure legends and/or text.
NET plays a pivotal role in controlling global noradrenergic tone and its aberrant regulation has the capacity to impact mental health (Ganguly et al., 1986; Klimek et al., 1997; Rumantir et al., 2000; Shannon et al., 2000a; Hahn et al., 2003; Hahn et al., 2005; Kim et al., 2006; Haenisch et al., 2008; Hahn et al., 2008; Hahn et al., 2009). Evidence indicates that insulin signaling regulates NE homeostasis (Shimizu, 1991; Figlewicz et al., 1993; Figlewicz et al., 1996; Barber et al., 2003). Thus, understanding how insulin/Akt fine tunes NET trafficking and/or function may provide an interesting and plausible link between metabolic dysfunction and mental disorders such as depression and schizophrenia (Mukherjee et al., 1989; Mukherjee et al., 1996; Lustman and Clouse, 2005; Zhao et al., 2006). Our goal is to determine the nature of insulin’s regulation of NET in the central and peripheral nervous systems. Here, we provide evidence for the role of insulin in the down regulation of the transporter from the surface. Utilizing a new assay, slice biotinylation, we show for the first time that acute in vitro treatment of mouse hippocampal slices with 1 nM insulin significantly diminishes surface levels of NET (Fig. 1A). Tyrosine hydroxylase (TH), a cytosolic protein, is detected primarily in the total fraction and comprises less than 1% of the surface fraction. Therefore, these data demonstrate that the biotinylated fraction represents cell surface proteins and speaks to the health of noradrenergic TH positive neurons in the assay. These results provide a molecular mechanism to describe the decreased NE uptake described by Figlewicz and collaborators (Figlewicz et al., 1993). In addition to revealing a role for insulin in the regulation of NET trafficking in slices from the central nervous system, we also sought to demonstrate the ability of insulin to regulate NET in the peripheral nervous system.
Superior cervical ganglion neurons (SCGN) enable us to investigate the role of insulin in the peripheral nervous system. Importantly, due to the large diameter (2–4 micron) of SCGN boutons, these cultures also provide us with the unique opportunity to study NET regulation at presynaptic sites of NE release (Matthies et al., 2009). Before exploring the role of insulin in the regulation of these noradrenergic neuronal cultures, we sought to demonstrate that these cells express the insulin receptor (IR). Not only is IRβ staining abundant throughout the SCGN (data not shown), the receptor is also specifically localized to single boutons (Fig. S1). After demonstrating the presence of IRs on noradrenergic SCGNs, we serum-starved the SCGN cultures for 1 hour and then treated them with vehicle, 1 nM, or 100 nM insulin for 20 minutes and discovered a dramatic increase in intra-bouton NET immunoreactivity (Fig. 1B). We quantified these observations using pixel intensity plots of a single confocal section (see Materials and Methods). Pixel intensity plots were obtained from a straight line drawn across the widest region of the approximately spherical bouton, starting at the brightest spot of fluorescence on one side of the bouton. The subsequent line spanned the entire diameter of the bouton, and was extended beyond the limits of the bouton to provide a background value. This line was then divided arbitrarily into 20 bins, and the NET fluorescence intensity of each bin was normalized to the fluorescence intensity of the brightest spot on the perimeter of the bouton (100%). The mean ± s.e.m. pixel intensity (normalized NET intensity) is plotted against each bin (normalized distance). Figure 1B shows that in insulin-treated neurons, the NET intra-bouton fluorescence signal is significantly higher than in vehicle-treated neurons as shown by a shift in the curve upward. This demonstrates that NET is accumulated intracellularly upon 100 nM insulin treatment. Our result that 100 nM insulin treatment significantly increases intracellular NET accumulation is further substantiated by co-staining for the recycling endosome marker Rab11a. Rab11a is a GTPase that plays an important role in the trafficking of numerous proteins including NET via “slow” recycling endosomes (Matthies et al. in press in the Journal of Neuroscience). Importantly, we show that 100 nM insulin exposure significantly enhances NET colocalization with the strictly intracellular protein Rab11a (Fig. 1C). This increase in colocalization was quantified by utilizing the intensity correlation analysis/intensity correlation quotient (ICA/ICQ) method (Li et al., 2004). We calculated the ICQ of NET and Rab11a in boutons and found a significant increase upon 100 nM insulin treatment (Fig. 1C).
Our results, for the first time, demonstrate the ability of insulin to induce NET trafficking, in both the central and peripheral nervous systems. Furthermore, this regulation is seen not only at the level of hippocampal slices but also at the level of a single bouton. In order to dissect this insulin-induced regulation further, we chose to study it in a more malleable system, Chinese hamster ovary (CHO) cells with stably transfected HA-tagged human NET (hNET cells). Here we validate this model by demonstrating that insulin-induced trafficking of NET away from the cell surface is both time and dose-dependent in hNET cells. Incubation of hNET cells for 5 minutes with 1 μM insulin results in a significant reduction of NET in the biotinylated fraction (Fig. 1D). Additionally, 5 minutes of insulin exposure at varying doses results in significant decreases in surface NET (Fig. 1E). In addition, to insulin’s ability to modulate NET surface availability in these cells, uptake assays also support an insulin mediated decrease in NET function. Consistent with previous results, our data reveal a significant decrease in [3H] DA uptake after exposure to 10 nM insulin (Vehicle 1.162 ± 0.020 and Insulin 0.986 ± 0.020 pmol*well−1*min−1; mean ± s.e.m., **P<0.01 by Student’s t-test; N=4-3). The time and dose dependent changes in NET surface levels and function in response to insulin in hNET cells illustrates the utility of this system for studying the intricacies of regulation of this transporter by insulin. In particular, we sought to uncover specifically which components of the insulin signaling pathway are essential for insulin-induced NET regulation.
Insulin receptors are tyrosine kinase receptors which upon activation result in the recruitment of both phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) to the membrane for subsequent activation. Akt, in particular, is a multifunctional kinase that is involved with diverse pathways in cell growth, survival, metabolism etc. Indeed aberrant Akt function has been implicated in a vast array of disorders such as diabetes, obesity, cancer, autoimmune disease, as well as mental disorders such as depression and schizophrenia (Hsiung et al., 2003; Emamian et al., 2004; Dummler and Hemmings, 2007; Karege et al., 2007; Manning and Cantley, 2007; Krishnan et al., 2008). While genetic evidence for the involvement of Akt in mental disorders grows, how Akt dysregulation impacts these diseases at the molecular level is still unclear. Importantly, it has been shown that Akt is critical for the regulation of other transporters such as the glucose and dopamine transporter by insulin (Carvelli et al., 2002; Garcia et al., 2005; He et al., 2007; Zaid et al., 2008). Here, we investigate whether Akt plays a critical role in the insulin regulation of NET by utilizing the isoform specific Akt1, Akt2, or Akt1/2 inhibitors (DeFeo-Jones et al., 2005; Lindsley et al., 2005; She et al., 2008). These inhibitors are allosteric inhibitors that require the pleckstrin homology (PH) domain of Akt to inhibit phosphorylation and activation of the kinase. While they require this domain for inhibition, binding of the inhibitors to Akt requires the whole protein since in vitro assays show the PH domain alone is insufficient for binding. Furthermore, the inhibitors reversibly inhibit both the activation and activity of Akt and are highly specific for Akt compared to other similar kinases such as PKA, PKC, and SGK. In our studies, 5 μM application of the dual Akt1/2 inhibitor to hNET cells 30 minutes prior to and during insulin exposure prevents insulin stimulated trafficking of NET away from the surface (Fig. 2A). In addition to pharmacological blockade, we also utilize a “kinase-dead” dominant negative mutant (K179R) construct of Akt (Akt-KD) to show that Akt activity is required for insulin induced regulation of the transporter (Garcia et al., 2005). Consistent with our previous results, transient transfection of hNET cells with the Akt-KD construct 48 hours prior to insulin application abolishes insulin stimulated trafficking of NET away from the plasma membrane (Fig. 2B). While both genetic and pharmacological inhibition of Akt prevents insulin induced regulation of the transporter in hNET cells, we sought to extend these observations to a more physiologically system.
Given the abundance of NET expression in mouse hippocampal slices, we chose this experimental system to investigate the role of Akt in the insulin regulation of NET. While all three isoforms of Akt are expressed within the brain, the distribution and differences in their expression across various regions of the brain is relatively uncharacterized (Easton et al., 2005). We anticipate that for Akt to regulate the transporter it should be localized in the same neuronal domain. Indeed, NET, Akt1, and Akt2 co-staining of slices reveals that Akt1 and Akt2 are heavily expressed in NET positive hippocampal terminals (Fig. 3A). Thus, Akt is poised for regulating the transporter in hippocampal slices. Consistent with these findings, insulin-stimulated decreases in NET are abolished by pre-treatment with either the Akt1 or Akt2 inhibitor for 1 hour prior to and during insulin exposure (Fig. 3B). These data demonstrate that insulin-induced down-regulation of NET requires Akt activity. Interestingly, changes in the phosphorylation status of Akt at serine residue 473, which relates to Akt activation, correlate well with changes in NET surface levels (Fig. 3C). This correlation is seen consistently throughout our studies and led us to hypothesize that Akt alone, which can be stimulated by numerous pathways, is a pivotal regulator of the transporter. Before investigating the effects of altering Akt activity independent of insulin signaling, however, we sought to determine if in vivo manipulation of insulin and Akt signaling results in significant changes in NET regulation and consequential monoamine homeostasis.
To induce a hypoinsulinemic state, mice were injected with streptozotocin (STZ), a selectively toxic compound targeting the insulin producing pancreatic beta cells (Lenzen, 2008). Original studies on STZ injected mice demonstrate that these mice display hallmarks of hypoinsulinemia such as aberrant glucose regulation, hyperphagia, polyuria, etc (Hernandez and Briese, 1972; Bell and Hye, 1983). In our studies, mice received one 200 mg/kg i.p. injection of STZ, and 48 hours later blood glucose levels were measured to confirm drug efficacy. Control mice, which received vehicle injections, maintained normal glucose levels while STZ injected mice showed aberrantly elevated levels of blood glucose (Fig. S2). After measurement of blood glucose levels, mice were left untreated in the STZ-induced diabetic state for 7 to 10 days and were then sacrificed for biochemical studies. STZ mice display deficits in Akt phosphorylation at ser473 in the hippocampus, which is accompanied by an increase in NE tissue content in the region (Fig. 4A, B). Importantly, studies show that appropriate NET function is critical for regulating NE tissue content (Xu et al., 2000). Thus, given the deficits in Akt phosphorylation and the significant elevation of NE tissue content, we hypothesize that these mice will display significantly enhanced NET levels. Moreover, evidence from previous studies show that STZ rats have elevated steady state levels of NET mRNA in the locus coeruleus (Figlewicz et al., 1996). Importantly, hippocampal slice biotinylation reveals a significant increase in surface NET in mice with STZ-induced hypoinsulinemia (Fig. 4C). To determine if enhanced surface expression of NET in hippocampal slice preparations from STZ-treated mice results in increased NE clearance in hippocampus in vivo, we utilized high speed chronoamperometry (HSCA). Clearance rate of NE locally injected into the dentate gyrus was significantly increased in STZ treated mice, as indicated by the decreased time required for the NE signal to diminish in STZ mice as compared with saline treated mice (Fig. 4D, left panel). Consistently, the average clearance rate of NE over a range of concentrations, measured as described in Material and Methods, was significantly enhanced in STZ mice as compared to control animals, such that the apparent maximal velocity for NE clearance was increased approximately 2-fold compared to control mice (Fig. 4D, right panel). Similarly, the average time required for NE clearance (NE clearance time) over the same range of concentrations was significantly reduced in STZ treated mice (Fig. 4D, bottom panel). Thus, STZ mice display aberrant Akt regulation and increased NE tissue content in the hippocampus which corresponds with increased NET surface expression and function. Importantly, the STZ-induced diabetic mice display normal protein levels of other noradrenergic markers in the hippocampus such as TH (CTR N=9 100.0 ± 5.5; STZ N=11 94.5 ± 7.2; unpaired T-Test p=0.57) and dopamine beta hydroxylase (CTR N=7 100.0 ± 8.5; STZ N=9 101.7 ± 8.2; unpaired T-Test p=0.89). These data indicate that changes in NE tissue content correlates with altered NET surface expression and function and are not due to changes in NE synthesis. In addition, to support our hypothesis that hypoinsulinemia induced by STZ underlies the alterations observed in NET expression and function, we sought to reverse these deficits in vitro and in vivo with acute insulin treatment. Again, STZ treated mice had significantly reduced levels of Akt ser473 phosphorylation (Fig. 5A) and enhanced surface expression of NET (Fig. 5B) in biotinylated hippocampal slices relative to control mice. However, 1nM insulin treatment for 20 minutes restores Akt phosphorylation levels and surface expression of the transporter to control levels (Fig. 5A, B). Thus, acute in vitro insulin treatment is sufficient to rectify STZ-induced alterations in hippocampal preparations. To determine if a similar rescue is possible in vivo we again utilized HSCA. As before, the average time required for NE clearance was significantly reduced, indicating enhanced NET function, in STZ treated, hypoinsulinemic mice, relative to saline treated control mice (Fig. 5C, left panel). Similar to our in vitro paradigm, local infusion of insulin (10 μM/100 nL to deliver 1 pmol) was then utilized in the dentate gyrus of STZ mice in an attempt to rectify the changes observed in NET function. First, exogenous NE was locally applied (200 μM/10 nl to deliver 2 pmol) to achieve reproducible signals with amplitudes recorded at the carbon fiber electrode in the range of 1 μM. Insulin was then infused and two minutes later NE was applied again. Importantly, insulin itself did not produce any electrochemical signal. Exogenous insulin application 5 minutes before NE infusion, however, significantly increased the time required for NE to clear from the extracellular fluid in the dentate gyrus thus implying a significant reduction in NET function (Fig. 5C, center and right panel). The ability of in vitro and in vivo insulin application to restore STZ-induced deficits further supports the notion that peripheral hypoinsulinemia underlies NET dysfunction in these mice. Thus, our data from biotinylated hippocampal slices and in vivo HSCA in STZ-induced diabetic mice show for the first time, that in vivo manipulation of insulin levels in the periphery impacts both Akt signaling/phosphorylation and NET function in the brain with consequences for monoamine homeostasis.
We hypothesize that manipulation of Akt signaling either by pharmacological means or receptor stimulation, independently of insulin, alters the surface availability of NET. To investigate if Akt signaling is capable of regulating NET trafficking, we exposed hNET cells to the Akt1/2 inhibitor (5 μM) for varying time periods (Fig. 6A). Inhibition of Akt significantly increased NET surface expression in a time dependent manner. Importantly, this pharmacological manipulation of Akt is independent of insulin status. To determine if Akt regulates NET at sites of NE release, we extended these studies to boutons of SCGN cultures. Co-staining of SCGN cultures for NET, Akt1, and Akt2 indicates that Akt is indeed present in the bouton and poised for regulation of the transporter (Fig. S3). Given the close proximity of both Akt isoforms to NET in the SCGN boutons, we exposed SCGN cultures to vehicle, Akt1 inhibitor, or Akt2 inhibitor for 1 hour to determine if both isoforms are capable of driving NET to the surface. In order to allow for Akt signaling inhibition by the Akt inhibitors the neurons were not serum starved. Here we show, at sites of synaptic NE release, that incubation of cultures with either the Akt1 or Akt2 isoform specific inhibitors results in a significant increase in NET immunoreactivity on the perimeter of the bouton (Fig. 6B). Thus, both Akt1 and Akt2 are capable of regulating NET surface availability in SCGN. Again, we quantified these observations using pixel intensity plots of a single confocal section (see Materials and Methods). Upon Akt inhibition, the Pixel intensity plots reveal a significant downward shift, indicating an increase in NET surface levels in response to Akt inhibition. Furthermore, the average NET intensity across inner bins (bins 3–17) reveals a significant decrease in NET immunoreactivity within the bouton in the presence of either Akt inhibitor (Fig. 6B, inset). These data demonstrate, for the first time, that Akt activity is a potent regulator of NET.
Next, we determined if activation of Akt by signaling pathways distinct from insulin are capable of altering NET surface expression. Numerous studies have shown that antipsychotics, such as clozapine, influence the phosphorylation status and activity of Akt in the cortex (Emamian et al., 2004; Kang et al., 2004; Roh et al., 2007). Our evidence suggests that alterations in the phosphorylation status of Akt correlate well with changes in NET surface expression. Thus, we determined if in vivo i.p. injections of clozapine are capable of altering both the phosphorylation state of Akt at ser473 as well as surface levels of NET. Mice received 30 mg/kg i.p. single injections of clozapine and one hour after injection the mice were sacrificed and cortical slices were taken and biotinylated to examine both Akt phosphorylation and surface levels of NET. As expected, clozapine treatment significantly enhanced phosphorylation of Akt at ser473 in the cortex while total levels remained unaltered (Fig. 7A). Importantly, this increase in Akt phosphorylation is accompanied by a significant decrease in the levels of surface NET (Fig. 7B). Thus, activation of Akt through receptor signaling distinct from that of insulin alters the dynamics of NET surface expression.
Through its re-uptake of synaptic NE and other monoamines such as DA, NET is pivotal for maintaining the integrity of monoaminergic signaling in the brain and periphery (Iversen, 1971; Pacholczyk et al., 1991; Xu et al., 2000; Moron et al., 2002). NET function is intricately controlled not only through regulation of transporter activity but also through the dynamic trafficking of the transporter to and from the plasma membrane (Apparsundaram et al., 1998b) (Apparsundaram et al., 2001; Sung et al., 2003; Wersinger et al., 2006; Dipace et al., 2007). Importantly, single nucleotide polymorphisms in NET that impact surface availability have been shown to be directly linked to both central and peripheral nervous system disorders, such as depression, ADHD, orthostatic intolerance, and blood pressure abnormalities (Halushka et al., 1999; Hahn et al., 2003; Hahn et al., 2005; Haenisch et al., 2008; Hahn et al., 2009). Thus, the ability of abnormal NET surface expression to impact mental health and autonomic function makes it imperative that we understand how dysregulation of signaling pathways linked to these disorders impacts NET function. Notably, aberrant Akt signaling has been implicated in diseases such as diabetes and obesity, which have a high co-morbidity with monoamine related mental disorders, as well as with the pathology of schizophrenia and depression (Mukherjee et al., 1989; Mukherjee et al., 1996; Hsiung et al., 2003; Emamian et al., 2004; Lustman and Clouse, 2005; Zhao et al., 2006; Karege et al., 2007; Krishnan et al., 2008). Here, our goal was to characterize how changes in Akt signaling, regulates NET function in the nervous system.
We demonstrate for the first time, that insulin and Akt signaling are capable of fine tuning NET surface levels and function in both the central and peripheral nervous systems. We show that acute insulin treatment significantly decreases NET surface availability in mouse hippocampal slices, at sites of synaptic NE release (SCGN boutons), and in heterologous cells. Moreover, our studies demonstrate that Akt is required for insulin regulation of NET. Consistently, in vivo manipulation of insulin signaling in the periphery via STZ treatment, results in aberrant Akt signaling in the hippocampus, and this deficit correlates with enhanced NE levels, NET surface expression, and NE clearance. The aberrant NE levels and NET regulation in STZ-induced diabetic mice provides a strong proof of principle, that metabolic dysfunction in the periphery has the potential to impact NET function and monoamine homeostasis in the brain. These data provide one plausible avenue to explain the depressive-like behavioral deficits observed in diabetic rodents (Hilakivi-Clarke et al., 1990). STZ-induced diabetic mice undergo physiological changes beyond hypoinsulinemia, and therefore we cannot exclude the possibility that these changes in NET expression are exclusively due to insulin deficits. Still, our in vitro data strongly indicate a direct role for insulin in regulating NET surface levels. Furthermore, the ability of acute insulin treatment in STZ hippocampal slices to restore NET surface levels and phosphorylation of Akt to control levels, as well as its ability to restore NET function in STZ animals in vivo supports our notion that hypoinsulinemia underlies these alterations. Altogether, these data provide convincing support for an inhibitory role of insulin/Akt signaling in the regulation of NET function, and importantly, this regulation of NET in the brain can be altered by abnormal insulin signaling in the periphery.
Interestingly, this down regulation of NET in response to insulin is contrary to the role of insulin signaling in the regulation of the dopamine transporter (DAT). For example, early studies showed that in vivo manipulation of insulin levels via STZ treatment and food deprivation results in decreased DAT mRNA and DAT-mediated DA uptake respectively (Figlewicz et al., 1996; Patterson et al., 1998). More recent studies confirm these original observations and substantial evidence supports the notion that insulin/PI3K/Akt signaling is critical for maintaining basal levels of DAT at the surface (Carvelli et al., 2002; Galici et al., 2003; Garcia et al., 2005; Owens et al., 2005; Zhen et al., 2006; Wei et al., 2007; Williams et al., 2007; Lute et al., 2008; South and Huang, 2008). This ability of insulin to differentially regulate DAT and NET surface expression is intriguing given the high homology of the two transporters, and thus speaks to the specificity of insulin signaling in the regulation of monoamine homeostasis.
In addition, to describing the nature of insulin’s regulation of NET we also establish a novel role for Akt in controlling NET surface availability. We show that Akt inhibition, independent of insulin, in heterologous cells and SCGN boutons results in enhanced NET surface expression. Indeed, overall, our studies show that NET surface expression correlates well with the phosphorylation status of Akt at ser473. Therefore, Akt is not only required for insulin regulation of the transporter, but manipulation of Akt alone impacts NET surface availability. Distinct signaling pathways such as receptor tyrosine kinase (RTK), integrin, and G-protein coupled receptor (GPCR) signaling converge on Akt. Thus, divergent signaling pathways have the potential to similarly impact NET function via Akt signaling as a final common pathway. Indeed, here we demonstrate that similar changes in NET surface availability can be induced by either stimulation of insulin signaling or manipulation of GPCR signaling with an antipsychotic. The ability of the atypical antipsychotic clozapine, which can target α2-adrenergic receptors present on presynaptic noradrenergic terminals, to enhance Akt phosphorylation at ser473 is well documented in the cortex (Ashby and Wang, 1996; Emamian et al., 2004; Kang et al., 2004; Karkoulias et al., 2006; Roh et al., 2007). We show that i.p. injection of clozapine not only increases Akt ser473 phosphorylation, but also, similar to acute insulin treatment, it stimulates a subsequent decrease in NET surface expression. These data substantiate the hypothesis that completely distinct signaling pathways which converge on Akt have the potential to similarly influence NET availability and function. In addition, the ability of clozapine to manipulate the surface availability of NET could be important for its enhanced efficacy in the treatment of cognitive symptoms associated with schizophrenia (Woodward et al., 2005). Indeed, a number of clinical trials utilizing NET specific inhibitors are ongoing and promise to reveal if NET specific inhibition will prove effective in treating schizophrenia cognitive symptoms such as poor concentration and memory deficits (clinicaltrials.gov).
The importance of the discovery of Akt as a potent regulator of NET is further emphasized when one considers the wealth of information supporting a role for Akt dysfunction in the development of mental disorders such as schizophrenia and depression. A link between Akt and schizophrenia was first described in (Emamian et al., 2004). The authors revealed decreased levels of Akt protein in lymphocytes and post-mortem brain tissue from schizophrenic patients along with evidence for a genetic association between schizophrenia and an Akt haplotype. Since these original studies numerous reports substantiate an association between Akt dysfunction and schizophrenia (Norton et al., 2007; Arguello and Gogos, 2008; Dawn et al., 2008). In particular, seminal human genetic and imaging studies corroborate a relationship between an Akt1 variant associated with schizophrenia and disruptions in DA-associated behaviors linked to the disorder (Tan et al., 2008). These studies indicate a possible link between Akt dysregulation, monoamine function, and predisposition to schizophrenia. Correspondingly, our studies provide one molecular mechanism by which Akt dysfunction has the potential to impact monoamine homeostasis, via its regulation of NET, a protein critical for controlling both global NE homeostasis and cortical DA homeostasis (Yamamoto and Novotney, 1998; Moron et al., 2002). Thus, future studies investigating the impact of schizophrenia-linked Akt1 variants on the noradrenergic system (i.e. NET expression, NE homeostasis) and ultimately NE-related behaviors will strengthen the growing link between aberrant Akt function, monoamine homeostasis, and schizophrenia. Interestingly, related studies from our laboratory in mice show that ablation of phosphorylation of Akt at Ser473 in neurons results in dramatically enhanced NET surface expression as well as behavioral and biochemical phenotypes that are hallmark characteristics of schizophrenia (Siuta and Robertson et al. in press in PLoS Biology).
While a role for abnormal Akt signaling in schizophrenia is well supported, studies supporting a role for Akt in depression are just beginning to appear. For example recent studies reveal deficits in Akt activity in the PFC post-mortem tissue of depressed individuals (Hsiung et al., 2003; Karege et al., 2007). Interestingly, social defeat induced depression in mice has also been shown to depend on a decrease in Akt activity (Krishnan et al., 2008). Thus impairments in Akt activity may sustain depression. In addition, chronic antidepressant treatment in both mice and humans results in enhanced levels of phosphorylated Akt (Krishnan et al., 2008) which our studies correlate with decreased surface NET. Interestingly, chronic antidepressant treatment has also been shown to result in a down regulation of NET (Kitayama et al., 2006; Song et al., 2008; Jeannotte et al., 2009b; Jeannotte et al., 2009a). Whether this antidepressant-stimulated decrease in surface NET is Akt dependent, however remains to be determined. Thus, increases in NET cell surface expression could underlie the etiology of depression. Indeed, one NET SNP F528C that is associated with major depression has enhanced membrane expression of the transporter along with deficits in trafficking of the transporter away from the plasma membrane (Hahn et al., 2005; Haenisch et al., 2008).
Given the widespread influence of NET on monoamine homeostasis in both the CNS and PNS, and the evidence supporting a link between NET dysfunction and disease, substantial effort must be invested into enhancing our understanding of NET trafficking and regulation. Here, we show that Akt signaling is key for fine tuning NET surface availability. As our picture of NET regulation becomes more complete, so too will our ability to create unique approaches for treating disorders such as, depression, schizophrenia, drug addiction, and ADHD.
Sponsorship: Work supported by NIH Grants MH058921 and DA13975 (A.G.), DA14684 (A.G. & L.C.D), MH084755 (S.D.R.)
We would like to thank members of the Galli, Blakely, Colbran, and Kenworthy labs for useful discussion. We would also like to acknowledge Dr. J. R. Goldenring for use of the anti-Rab11a antibody, Dr. R.D. Blakely for the use of the stably transfected hNET cell line, and the Vanderbilt Neurochemistry Core.