Leptin and insulin stimulation of signalling pathways in arcuate nucleus neurones: PI3K dependent actin reorganization and KATPchannel activation
© Mirshamsi et al; licensee BioMed Central Ltd. 2004
Received: 11 May 2004
Accepted: 06 December 2004
Published: 06 December 2004
Leptin and insulin are long-term regulators of body weight. They act in hypothalamic centres to modulate the function of specific neuronal subtypes, by altering transcriptional control of releasable peptides and by modifying neuronal electrical activity. A key cellular signalling intermediate, implicated in control of food intake by these hormones, is the enzyme phosphoinositide 3-kinase. In this study we have explored further the linkage between this enzyme and other cellular mediators of leptin and insulin action on rat arcuate nucleus neurones and the mouse hypothalamic cell line, GT1-7.
Leptin and insulin increased the levels of various phosphorylated signalling intermediates, associated with the JAK2-STAT3, MAPK and PI3K cascades in the arcuate nucleus. Inhibitors of PI3K were shown to reduce the hormone driven phosphorylation through the PI3K and MAPK pathways. Using isolated arcuate neurones, leptin and insulin were demonstrated to increase the activity of KATP channels in a PI3K dependent manner, and to increase levels of PtdIns(3,4,5)P3. KATP activation by these hormones in arcuate neurones was also sensitive to the presence of the actin filament stabilising toxin, jasplakinolide. Using confocal imaging of fluorescently labelled actin and direct analysis of G- and F-actin concentration in GT1-7 cells, leptin was demonstrated directly to induce a re-organization of cellular actin, by increasing levels of globular actin at the expense of filamentous actin in a PI3-kinase dependent manner. Leptin stimulated PI3-kinase activity in GT1-7 cells and an increase in PtdIns(3,4,5)P3 could be detected, which was prevented by PI3K inhibitors.
Leptin and insulin mediated phosphorylation of cellular signalling intermediates and of KATP channel activation in arcuate neurones is sensitive to PI3K inhibition, thus strengthening further the likely importance of this enzyme in leptin and insulin mediated energy homeostasis control. The sensitivity of leptin and insulin stimulation of KATP channel opening in arcuate neurones to jasplakinolide indicates that cytoskeletal remodelling may be an important contributor to the cellular signalling mechanisms of these hormones in hypothalamic neurones. This hypothesis is reinforced by the finding that leptin induces actin filament depolymerization, in a PI3K dependent manner in a mouse hypothalamic cell line.
Leptin and insulin function as peripherally-derived hormone signals involved in the long-term regulation of energy balance [1–4]. Their circulating levels are directly proportional to adipose mass and CNS access occurs via saturable receptor-mediated processes. The primary CNS target for these adipostats is the ARC, where leptin and insulin receptors are highly expressed, and where direct administration of either hormone has a potent effect on food intake and body weight. Two specific ARC neurone populations have been strongly implicated in sensing changes in levels of circulating leptin and insulin and transducing these signals into neuronal outputs [1, 3]. These "first-order" neurones encompass the melanocortin precursor, POMC containing neurones and NPY and AgRP co-containing neurones, the former associated with catabolic, the latter anabolic, outputs. Leptin and insulin increase POMC mRNA levels and decrease NPY & AgRP mRNA levels respectively.
However, transcriptional control is not the only effector mechanism elicited by these hormones on ARC neurones. Electrophysiological studies have shown that leptin depolarizes and increases the firing rate of ARC POMC neurones and inhibits the tone of NPY/AgRP neurones . Although the electrophysiological actions of insulin have not been reported for identified POMC and NPY/AgRP neurones, both leptin and insulin have been demonstrated to inhibit, by hyperpolarization, the firing of a sub-population of ARC neurones, identified by their sensitivity to changes in extracellular glucose concentration [6, 7]. For these latter neurones, termed glucose-responsive (GR), KATP channels have been identified as an effector mechanism through which leptin and insulin elicit neuronal inhibition. Consequently, leptin and insulin signal the status of body energy stores by activating their receptors on ARC neurones, eliciting changes in the electrical activity and amounts of releasable peptides in specific neuronal populations, leading to compensatory effector outputs, such as changes in food intake, energy balance and glucose homeostasis .
Obese humans have elevated leptin and insulin levels, indicative of central resistance to these hormones . The mechanisms underlying this resistance are unclear, with defective hormone passage through the BBB and flawed receptor-signal transduction in ARC neurones being the prime candidates [10, 11]. Consequently, it is important to understand the molecular mechanisms underlying leptin and insulin receptor modulation of ARC first-order neurones. Leptin and insulin, by stimulation of their respective receptors, have been demonstrated to activate various signalling pathways in peripheral tissues [10–13]. However, as these hormones induce seemingly identical actions on ARC neurones, both in terms of behavioural output and effects on ARC neurone excitability, some parallelism or convergence of signalling is likely [12, 13]. Leptin, by binding to the long form of the leptin receptor (ObRb) has been demonstrated to activate three main signalling cascades, JAK2 – STAT3, MAPK and PI3K, the latter two of which are also intermediates in insulin receptor activation [14, 15]. However, recent studies have strongly implicated PI3K as the key signalling intermediate in leptin and insulin actions on hypothalamic neurones influencing food intake and body weight [16, 17].
Thus, to elucidate further the pathways that contribute to convergent actions of leptin and insulin on ARC neurones, we have examined the phosphorylation status of key leptin and insulin signalling intermediates in the ARC and have explored the linkage, with a focus on PI3K mediated signal transduction pathways, between these hormones and ARC neurone KATP channel activation.
Leptin and insulin stimulate phosphorylation of signalling proteins in ARC
Leptin and insulin activate KATPchannels in acutely isolated ARC neurones
Leptin and insulin activation of ARC neurone KATPis PI3K dependent
Leptin and insulin activation of GR neurone KATPrequires actin filament re-organisation
PI3K mediates leptin-induced actin filament reorganisation in GT1-7 cells
PI3K – a pivotal enzyme in ARC signalling
Previous studies have demonstrated that leptin applied in vivo stimulates hypothalamic ObRb to increase phosphorylation of the signalling protein intermediates STAT3 and MAPK and that both leptin and insulin increase hypothalamic PI3K activity [12, 29]. Here we have applied hormones directly to ARC wedges isolated from hypothalamic slices to enable improved signal detection (with respect to amplitude and temporal resolution), localisation of signalling to the arcuate nucleus and to fix external conditions so that potential compensatory changes associated with in vivo studies are obviated. Exposure of ARC wedges to leptin or insulin induced rapid (≤1 minute) phosphorylation of MAPK (ERK1 & 2 subfamilies), STAT3 and the PI3K activity indicators, PKB and its downstream target GSK3. These hormone-induced increases in phosphorylation were transient in the majority of experiments, usually lasting 1–5 minutes at ~34°C with return to control levels of phosphorylation within 30 minutes. Such rapid recovery has also been noted in other studies [13, 30] and may be due to activation of endogenous phosphatases such as PTP1B curtailing this acute signalling process [14, 31]. The phosphorylation of MAPK is quite modest and at present there are few data which link this pathway directly with the actions of either insulin  or leptin  on energy homeostasis, although recently it has been shown that centrally driven insulin-mediated sympathoactivation of brown adipose tissue is MAPK-dependent .
As expected, exposure of ARC wedges to leptin induced an increase in tyrosine phosphorylated STAT3 [11, 13, 29, 34]. However, unexpectedly insulin also induced an increase in tyrosine phosphorylation of STAT3 in ARC neurones. In a previous study  in vivo application of insulin (icv) demonstrated no such change, unless leptin was co-applied. The data reported here indicate that insulin per se is capable of increasing STAT3 phosphorylation, as no exogenous leptin was present or endogenous leptin likely to remain in the ARC sections following the extensive washes and incubations prior to stimulation. This difference may be due to an increased signal to noise delivered using ARC tissue over whole hypothalamus and that rapid transient signals are more readily detectable by this method. Both leptin and insulin rapidly increased the phosphorylation of PKB and its downstream effector GSK3 in a wortmannin and LY294002 sensitive manner, indicative of increased PI3K activity in ARC neurones, in agreement with previous in vivo studies [17, 35].
However, our results did not demonstrate that either leptin or insulin induced a significant increase in IRS-2-associated PI3K activity measured directly in ARC tissue. This may be due to a low signal to noise ratio, as only a (unknown) proportion of cells would be expected to respond to the hormones in the ARC tissue block, and/or that hormone mediated increases in PI3K activity are limited to plasma membrane microdomains. This is supported by the very modest increase in PI3K activity detected in GT1-7 cells when stimulated by leptin. Although hypothalamic activation of PKB by insulin has been reported previously , these are the first reports that leptin increases PKB activity and that both hormones increase the phosphorylation of GSK3 in the ARC. The presence of the PI3K inhibitors, wortmannin or LY294002, also reduced the leptin and insulin driven increase in MAPK phosphorylation. The mechanism by which leptin and insulin cause phosphorylation of this protein is most likely through the Ras pathway, as this protein has been demonstrated to interact directly with the catalytic subunit of PI3K  and inhibitors of PI3K have been reported to inhibit insulin induced increased MAPK activity, for example in rat adipocytes . The insulin mediated enhanced STAT3 tyrosine phosphorylation in an interesting observation that requires further examination. Although phosphorylation of tyrosine-705 on STAT3 is a prerequisite for dimerisation and translocation of STAT3 to the nucleus , phosphorylation of serine-727 may also be required for maximal activation of STAT3 DNA binding .
Interestingly one pathway candidate for phosphorylating serine-727 is the Ras/Raf/MEK signalling cascade, and indeed a recent study has demonstrated that leptin can induce S727 phosphorylation of STAT3 in a PD98059 dependent manner in macrophages, and this is required to produce full stimulation of STAT3 . Insulin mediated serine phosphorylation of STAT3 has also been reported, using transfected Chinese hamster ovary cells, to be mediated by a MEK-dependent pathway . A similar mechanism in hypothalamic neurones would indicate an inter-connection between the three identified signalling pathways activated by these hormones and an important effector molecule, STAT3. Studies are underway to examine this proposal.
The importance of STAT3 signalling to the central mechanisms that control energy homeostasis has recently been directly demonstrated by transgenic mouse studies. Using a 'knock-in' strategy to induce defective STAT3 binding to ObRb  or a 'knock-out' strategy to ablate STAT3 from some hypothalamic neurones , loss or reduction in hypothalamic STAT3 signalling initiates hyperphagia, increased body weight and adiposity with alterations in glucose homeostasis. Indeed, the JAK2-STAT3 and IRS2-PI3K signalling pathways are purported to underpin the genomic and acute or membrane functions of these signalling pathways respectively . Clearly, further work is required to determine the exact signalling mechanisms controlling insulin stimulated STAT3 phosphorylation in hypothalamic neurones.
Leptin and insulin signalling to KATPchannels
Leptin and insulin cause inhibition, by hyperpolarization through activation of a sulphonylurea-sensitive K+ conductance, of a subset of hypothalamic neurones, defined by their acute sensitivity to changes in external glucose concentration, termed GR neurones [6, 7, 19]. Single channel recordings from acutely isolated ARC neurones demonstrate that both hormones activate the same K+ channel, the sulphonylurea-sensitive large conductance KATP channel. This action is rapid and independent of transcriptional events, so most likely is mediated by MAPK or PI3K signalling. Pharmacological inhibition of the MAPK pathway with PD98059 did not reverse leptin (as shown above) or insulin  stimulated KATP activity, abrogating this pathway from causing the hyperpolarising response. In contrast, inhibition of PI3K with either wortmannin or LY294002, reversed both leptin (as shown above) and insulin  raised KATP activity. Furthermore, use of the fusion protein PH-GRP1-GFP as a specific detector of PtdIns(3,4,5)P3 in isolated neurones also demonstrated that both hormones rapidly increase the cellular content of this PI3K lipid product in a sub-population of neurones. These results are consistent with class 1 PI3K  acting as a point of convergence for leptin and insulin signal transduction pathways to KATP channels in GR neurones. The functional significance of PI3K in the control of energy balance has been demonstrated by in vivo studies, which show that leptin  and insulin  stimulate IRS2-associated PI3K activity in the hypothalamus and pharmacological inhibition, using wortmannin and LY294002, of hypothalamic PI3K activity prevents the anorectic actions of icv leptin or insulin, whereas the MAPK inhibitor PD98059 had no effect on leptin driven attenuation of food intake .
Remodelling of cortical actin filaments as a leptin and insulin signalling event
Leptin and insulin stimulated KATP activity in isolated ARC neurones was also reversed, within 5–10 minutes, by the marine sponge toxin, jasplakinolide. This toxin binds to F-actin with high affinity, resulting in its stabilization and prevention of depolymerization to its monomer G-actin . These data indicate that the adiposity hormones require actin filament depolymerization for KATP activation to occur. Such a mechanism is supported by reports that agents, which promote actin depolymerization, activate KATP channels in cardiac myocytes [45, 46] and the insulin-secreting cell line, CRI-G1 . Furthermore, in this latter study leptin stimulated KATP channel activity was also shown to depend on actin filament depolymerization. Insulin is also well documented to cause actin filament re-organization in peripheral cells associated with various functional outputs, which depend on PI3K activity, including metabolic and mitogenic effects . The reversal of hormone-stimulated KATP activity by jasplakinolide was faster (5–10 minutes) than for the PI3K inhibitors (15–20 minutes). This temporal difference suggests that the site of jasplakinolide action is downstream from the PI3K signal transduction pathway to KATP channels.
However, alteration of the cellular cortical actin structure is inferred through the use of natural agents like jasplakinolide. In order to verify directly that hormone-driven structural re-arrangements did occur we decided to use the hypothalamic cell line, GT1-7, as preliminary experiments using freshly isolated neurones did not produce reliable and reproducible data due to the presence of dead and dying cells showing as false positives for hormone induced actin depolymerization. Use of this cell line also obviated any problems with identification of ObRb containing neurones and neuronal subtypes in slices. RT-PCR analysis indicates that this cell line does express the main signalling form of the leptin receptor and analysis of PI3K activity shows functional coupling of this receptor to this signalling pathway. We have shown, by cell staining of fixed cells and, independently by analysis of cellular G- and F-actin concentration from live cells, that leptin disrupts cortical actin structure by disturbing the processes that maintain the equilibrium between F-actin and G-actin, in the direction of depolymerization to G-actin. This effect of leptin was completely inhibited by the presence of either jasplakinolide or the PI3K inhibitors. In addition, there is a good temporal and spatial association between PtdIns(3,4,5)P3 production, as determined by PH-GRP1-GFP binding, and actin filament depolymerization. Thus, leptin and insulin signalling in, at least some sub-groups of hypothalamic neurones maintains a close parallel with leptin signalling in insulin-secreting cells, where it has been reported that leptin increases KATP activity by a PI3K-dependent cortical actin re-arrangement .
The effect of leptin and insulin on the phosphorylation status of various cellular signalling intermediates and on KATP channel activation in arcuate neurones indicates that both hormones activate the same signalling cascades, and can produce common outputs. The sensitivity of both KATP opening and the phosphorylation of certain intermediates to PI3K inhibition is significant as this enzyme has been previously demonstrated to play an important role in leptin and insulin mediated energy homeostasis control. Furthermore it is interesting that leptin and insulin induce rapid phosphorylation of MAPK and STAT3 as these data support the view that these hormones may influence genomic and membrane neuronal outputs by common mechanisms. The inhibition of leptin and insulin stimulation of KATP channel opening of arcuate neurones by jasplakinolide suggests a role for cytoskeletal dynamics in modulation of membrane events such as neuronal hyperpolarization. This hypothesis is further strengthened by the finding that leptin induces actin filament depolymerization in a mouse hypothalamic cell line, which is PI3K dependent, demonstrating that this cell line may be a useful model for further analysis of leptin signalling mechanisms in hypothalamic neurones.
Preparation of hypothalamic lysates and immunoblots
Male Sprague-Dawley rats (50–100 g) were killed by cervical dislocation in accordance with Schedule 1 of the UK Government Animals (Scientific Procedures) Act (1986). The brain was rapidly transferred to ice-cold aCSF solution, containing (in mM): 128 NaCl, 5 KCl, 1.2 NaH2PO4, 26 NaHCO3, 1.2 CaCl2, 2.4 MgSO4, and 10 glucose, equilibrated with 95% O2, 5% CO2 to give a pH of 7.4. The tissue was maintained in ice-cold aCSF whilst horizontal 400 μm coronal brain slices were prepared using a Vibratome (Intracel, Royston, Herts. UK). Slices containing the ARC were incubated in aCSF at room temperature for 20 minutes, and then at 33–35°C for 1 hour. Hypothalamic wedges, predominantly containing the ARC were cut, and these were incubated in aCSF ± hormones and/or kinase inhibitors (10 mls) for the required time. The reaction was stopped by the addition of 2 ml of cold lysis buffer containing (in mM) 100 NaCl, 10 NaF, 25 Tris HCl, 10 NaPPi, 5 EGTA, 1 EDTA, 1 Na3VO4, 1 Benzamidine, 0.1 PMSF, 0.1% (v/v) mercaptoethanol, 1% Tritron X-100 (v/v) and 92 mg ml-1 sucrose. The tissue was homogenised on ice, the lysate sonicated for two 10 s periods and then centrifuged for 10 minutes at 12000 rpm at 4°C. The supernatant was retained and the pellet discarded. The protein content of the clarified lysate was determined by the method of Bradford . Proteins (10 μg) were separated by SDS-PAGE, and subsequently transferred to nitrocellulose membranes. Membranes were incubated in blocking buffer (10% non-fat dried milk in TBST (20 mM Tris HCl, 150 mM NaCl, 0.5% Tween, pH 7.4)) for 1 hour at room temperature following which phospho-specific p44/p42 MAPK (Thr202/Tyr204), phospho-specific STAT3 (Tyr705), phospho-specific GSK-3α/β(Ser21/9), phospho-specific PKB (Thr308) and PKB (all polyclonal and used at 1:1000) antibodies were applied overnight at 4°C with gentle shaking. All antibodies were obtained from Cell Signalling Technology Inc. The membranes were washed four times with TBST and incubated for 1 hour at room temperature with horseradish peroxidase conjugated Goat anti-Rabbit IgG (1:5000). After further washing with TBST, total amount of specific protein was visualised by enhanced chemiluminescence detection as described by the manufacturer (NEN Life Science Products). Immunoreactive bands were scanned and quantified using AIDA software. As an internal control, the membranes were immunoblotted with a monoclonal anti β-actin antibody (Sigma: used at 1:5000) or with the PKB antibody. The values for proteins were normalized with respect to the internal control to account for variations in gel loading.
Determination of PI 3-kinase activity
Cell and tissue lysates were made as described. The immunoprecipitation and PI3K activity assay were carried out as previously described . Briefly, frozen samples were thawed before centrifugation to remove precipitated material. 10 μl Protein-G-Sepharose beads pre-coupled to 5 μg anti-IRS2 antibody (Upstate Biotechnology) was used to immunoprecipitate PI3K activity from ~0.5 mg cell lysate. The immunoprecipitated material was washed once with ice cold lysis buffer and three times with ice cold assay buffer, both of which were freshly supplemented with protease inhibitors, reducing agent and sodium vanadate as described . Washed beads were re-suspended in 40 μl assay buffer supplemented with 1 μM unlabelled ATP, 25 μCi/assay radiolabelled ATP and phosphatidylinositol/phosphatidylethanolamine vesicles (final concentration of each lipid 100 μM). Samples were incubated at 37°C for 30 mins and the reaction was stopped by addition of 0.6 ml methanol/chloroform/12 M HCl (80:40:1, v/v), 0.2 ml chloroform and 0.32 ml 0.1 M HCl. Samples were processed and PtdIns(3)P separated from contaminating materials by thin layer chromatography (TLC) as previously described . Bands corresponding to [32P]PtdIns(3)P were located using a phosphorimager (Fuji FLA 5000) and analyzed with AIDA software.
Preparation of acutely isolated ARC neurones and electrophysiology
Coronal slices containing the medial hypothalamus were obtained (as described above) and sections containing the ARC were removed. The sections were transferred to 5 ml aCSF containing 1 mg ml-1 protease XIV (Sigma-Aldrich, Dorset, U.K) and incubated for 1 hour at room temperature. The aCSF was continuously gassed with 95% O2: 5% CO2 for the entire incubation period. Sections were removed and washed in 50 ml aCSF five times prior to re-suspension in 5 ml normal saline containing (in mM): 135 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, 3 glucose, pH 7.4. Sections were sequentially triturated with fire polished Pasteur pipettes with decreasing tip size. The cell suspension was evenly distributed onto concanavalin A (Sigma-Aldrich) pre-treated 35 mm diameter culture dishes. The culture dishes were left for 15–20 minutes allowing cell adhesion prior to use.
Cell-attached single channel currents were recorded from single neurones at room temperature, using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA USA). Patch pipettes were prepared from thick walled borosilicate glass and had open tip resistances of 8 – 15 MΩ when filled with high K+ solution containing (in mM) 140 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, pH 7.2. This solution was used in order to allow easy identification of K+ currents in the cell-attached configuration . All cell-attached recordings were made in the presence of normal saline, with no applied pipette potential, thus utilizing the cell membrane potential to drive current flow (with inward current shown as downward deflections). Single channel recordings from inside-out patches isolated from ARC neurones were made either under asymmetrical conditions, in the presence of normal saline, or under symmetrical K+ conditions with the intracellular aspect of the membrane exposed to a bathing solution containing (in mM): 140 KCl, 1 MgCl2, 2.7 CaCl2, EGTA 10 (free Ca2+ of 100 nM), HEPES 10, pH 7.2.
Data were recorded onto digital audio-tape using a Biologic DTR 1200 recorder and analysed off-line. Pre-recorded data were transferred via a Digidata 1200 interface into a PC, digitised at 10 kHz and measured using the PCLAMP6 software, Fetchan 6. The mean current (I) and single channel amplitude (i) were determined for recordings ranging in duration from 30 s to 120 s and channel activity (Nf.Po) determined as described previously , where Nf is the number of functional channels and Po is the open probability. Drug effects were measured by comparison of Nf.Po from individual patches in the presence and absence of the drug. Data for a given set of experiments were normalised and statistical significance determined by employing the Students t-test for unpaired data. Results are presented as mean ± SEM and the number of experiments denoted by 'n.'
Leptin receptor mRNA expression
Reverse transcription was performed in a 20 μl reaction containing 1 × First Strand Buffer, 1 mM DTT, 0.5 mM of dNTP, 0.5 μg anchored oligo(dT)18, 4 μg RNA and 1 μl (200 U) M-MLV Reverse Transcriptase (Gibco), at 25°C for 5 minutes, 42°C for 60 minutes, 70°C for 15 minutes and stored at -20°C. After RT, a 2 μl aliquot of the reaction was added to 48 μl of PCR mix. The mix containing 1 × PCR buffer, 2.5 mM MgCl2, 0.5 mM PCR nucleotide mix, 1 μM each of the gene specific primers (mObRcom F:ggaatgagcaaggtcaaaa; mObRcom R:gtgacttccatatgcaaacc; mObRb F:tcttctggagcctgaacccatttc; mObRb R:ttctcaccagaggtccctaaact; ref ) and 5 units of Taq DNA polymerase (Promega). PCR was performed using the following profile: 94°C for 5 minutes, 25 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, with a final extension at 72°C for 7 minutes.
GT1-7 cell culture, staining and actin analysis
The mouse hypothalamic cell line GT1-7  was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Sigma), 1 mM L-glutamine and 1% penicillin-streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were passaged every 3–4 days, plated on poly-L-lysine (Sigma) coated glass coverslips in 3.5 cm Petri dishes and used 1–2 days after plating. Cells were treated with 10 μM LY294002 or 10 nM wortmannin or 100 nM jasplakinolide (all Sigma) in normal saline for 10 minutes, prior to a challenge with 10 nM leptin (or saline), in the continuous presence of inhibitor, for 20 or 60 minutes before fixing. GT1-7 cells were fixed in 4% methanol-free formaldehyde in cytoskeletal buffer (10 mM MES, 3 mM MgCl2, 138 mM KCl, 2 mM EGTA, pH 6.1) with 0.32 M sucrose for 30 minutes . They were then washed in phosphate-buffered saline (PBS), permeabilised in PBS/0.5% Triton X-100 for 10 minutes, rinsed in PBS, blocked with 20% goat serum (Sigma) for 30 minutes, rinsed in PBS and incubated with rhodamine conjugated phalloidin, or 2 μg ml-1 Alexa 594-DNase I and 2 U ml-1 Alexa 488-phalloidin (all Molecular Probes) for 90–120 minutes, rinsed in PBS, and mounted on coverslips. Cells were observed with a 63X oil objective and images acquired using a laser-scanning confocal microscope (Zeiss LSM 510), under identical conditions with randomly selected regions of each coverslip. For quantitative analysis of G- and F-actin cellular pools, we used a direct method to partition the actin pools from live cells . In brief, equal cell numbers were added to 3.5 cm culture dishes and cells grown to 80% confluence. The Triton-X-100 soluble (G-actin) pool was isolated first, by incubating cells for 5 minutes at room temperature with 1 ml PBS containing 1% Triton-X-100, protease inhibitors and 1 μg ml-1 phalloidin (to prevent filament dissociation). Cells were then washed with PBS, and the Triton-X-100 insoluble pool (F-actin) prepared by addition of 1 ml of PBS lysis buffer, containing 1% Triton-X-100, protease inhibitors, 2% SDS and 1 μg ml-1 phalloidin for 5 minutes prior to harvesting cells from dishes. For determination of total actin, cells were exposed to the second step only. Each cellular pool was passed through a 25 gauge needle and total protein concentration determined, before equal amounts of protein were loaded onto SDS-PAGE gels, and actin detected using an actin monoclonal antibody (Chemicon). Quantitative measurements of G- and F-actin in fixed cells were made using Velocity software (Improvision), where individual cell total fluorescence, normalized to cell area, was determined and background fluorescence subtracted. Average fluorescence intensity was calculated for 8 cells in each experiment, and expressed relative to control (non-drug exposed cells). Actin bands on gels were quantified by densitometry, where total density was determined with respect to constant area, background subtracted and average relative band density calculated.
PH-GRP1-GFP fusion protein overlays
Following stimulation with hormone for 10–20 minutes, acutely isolated neurones (room temperature) or GT1-7 cells (37°C) were fixed at room temperature with 2–4% paraformaldehyde for 15 and 30 mins, respectively. Cells were permeabilized by washing with 0.05% PBS-Tween 20 (PBS-T; x2 for 10 mins). Non-specific binding was minimised by blocking with 3% BSA for 1 hour at room temperature. Cells were subsequently washed with 0.05% PBS-T prior to incubation with wild type PH-GRP1-GFP or K273A mutant PH-GRP1-GFP (50 μg ml-1) fusion protein for 1 hour at room temperature, and images acquired by confocal microscopy.
List of abbreviations
artificial cerebrospinal fluid
ATP-sensitive potassium channel
- DNase I:
green fluorescent protein
- GR neurone:
general receptor for phosphoinositides-1
glycogen synthase kinase 3
insulin receptor substrate 2
janus kinase 2
mitogen-activated protein kinase
- PH domain:
pleckstrin homology domain
protein kinase B
4,5)P3, phosphatidylinositol 3,4,5-trisphosphate
signal transducer and activator of transcription 3
Supported by the Wellcome Trust (068692), Tenovus Scotland and Biovitrum. CS is a Diabetes (UK) Senior Fellow.
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