© 2000 Nature America Inc. • http://neurosci.nature.com
brief communications
Insulin activates ATPsensitive K+ channels in
hypothalamic neurons of
lean, but not obese rats
© 2000 Nature America Inc. • http://neurosci.nature.com
D. Spanswick1, M. A. Smith1, S. Mirshamsi1, V. H. Routh1,2
and M. L. J. Ashford1
1
Department of Biomedical Sciences, University of Aberdeen, Aberdeen Centre
for Energy Regulation and Obesity, Institute of Medical Sciences, Foresterhill,
Aberdeen AB25 2ZD, UK
2
Present address: Department of Pharmacology and Physiology, New Jersey
Medical School, 185 South Orange Avenue, University Heights, Newark, New
Jersey 07103-2714, USA
Correspondence should be addressed to M.L.J.A. (mike.ashford@abdn.ac.uk)
Insulin-sensitive ARC and VMN neurons also responded to
decreased extracellular glucose (10 to 0 mM), which induced hyperpolarization and reduced firing and input resistance, actions
reversible on re-application of 10 mM glucose (Fig. 1b). The corresponding current–voltage relationships show the conductance
increase reversed at –89 ± 2 mV (n = 3), consistent with potassium
channel opening3. Another characteristic of GR neurons is sensitivity to the sulfonylurea tolbutamide4. Tolbutamide (50–200 µM),
applied to GR neurons hyperpolarized by glucose removal (n = 4) or
insulin (n = 10), reversed these effects (Fig. 1a and b). Removal of
tolbutamide restored the insulin (n = 5/7; Fig. 1a) or glucose-free
(n = 3/4) response in most neurons. Current–voltage relationships
before and during tolbutamide application revealed a reversal potential of –89 ± 2 mV, indicating decreased potassium conductance (data
not shown). The effects of insulin were also examined on non-GR
neurons, defined by insensitivity to glucose removal and tolbutamide.
In 17 non-GR neurons (11 ARC, 6 VMN), insulin (10–100 nM) had
no effect on membrane properties. In 3 non-GR neurons, insulin
induced a 2–4 mV excitation (data not shown).
Tolbutamide effects on insulin responses implicated activation
of KATP channels. Insulin (0.1–10 nM) applied during cell-attached
recordings from freshly dissociated hypothalamic neurons increased
channel activity (Fig. 1c and d) in 50% of patches (19/39 neurons).
Average channel activity 1–2 minutes after forming the cell-attached
Insulin and leptin receptors are present in hypothalamic regions that
control energy homeostasis, and these hormones reduce food intake
and body weight in lean, but not obese, Zucker rats. Here we demonstrate that insulin, like leptin, hyperpolarizes lean rat hypothalamic
glucose-responsive (GR) neurons by opening KATP channels. These
findings suggest hypothalamic KATP channel
a
b
function is crucial to physiological regulation
of food intake and body weight.
Insulin regulates intermediary metabolism in peripheral tissues by stimulating glucose uptake and metabolism, and in the CNS
links peripheral events with central control
of food intake and body weight1. The mechanism(s) by which insulin acts on hypothalamic neurons is unclear. Leptin, another
systemically produced anorectic hormone,
hyperpolarizes certain hypothalamic neurons2, termed glucose-responsive neurons3.
As insulin and leptin have similar actions on
energy homeostasis, we examined the effects
c
d
of insulin on hypothalamic GR neurons and
on KATP channel activity in lean and obese
Zucker rats.
Insulin (5–300 nM) inhibited a subpopulation of neurons, causing decreased firing,
hyperpolarization and increased conductance
(Fig. 1a). These effects, in 17 arcuate (ARC)
and 15 ventromedial (VMN) neurons, were
fully or partly reversible in 12/32 neurons and
persisted (up to 80 min) in 20/32 neurons. Fig. 1. Insulin or glucose removal inhibits hypothal- e
amic neurons via KATP channels. (a) Insulin (50 nM)
The mean resting membrane potential was hyperpolarization of
an arcuate neuron, reversed
–49.4 ± 4.7 mV before, and reached by tolbutamide (200 µM). Note time breaks
–58.7 ± 6.8 mV 15–20 minutes after insulin (arrows). Bottom, current–voltage relationships
exposure (n = 22). Hyperpolarization was from neuron shown. (b) Glucose-free medium
associated with a decreased input resistance induces hyperpolarization, reversed by tolbutamide
of 37.6 ± 16.0% (1027 ± 380 MΩ to 632 ± 268 (200 µM), actions recovered with 10 mM glucose.
MΩ; n = 22), which persisted when the mem- (c) Insulin (0.1 nM) increases K+ channel activity
brane potential was clamped to resting levels (openings downward), cell attached. Biphasic
and in tetrodotoxin (500 nM, n = 3), consis- deflections denote action potentials. (d) Cellattached recordings. In control, few channels are
tent with a direct effect. Current–voltage relaopen, whereas 10 nM insulin activates K+ channel activity, blocked reversibly by tolbutamide (200 µM).
tionships before and during insulin-induced Relationship between N P (n.o. of functional channels × open probability of channel) and time for
f o
membrane hyperpolarization (Fig. 1a) recording shown. (e) Inside-out recording shows channel activity reversibly inhibited by 2 mM
showed a reversal potential of –90 ± 3 mV, Mg-ATP. C, closed state. Experiments used slices of 100–250 g rat (Sprague–Dawley) hypothalamus,
indicating a potassium conductance increase. acutely dissociated hypothalamic neurons and recording conditions as described2.
nature neuroscience • volume 3 no 8 • august 2000
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© 2000 Nature America Inc. • http://neurosci.nature.com
brief communications
© 2000 Nature America Inc. • http://neurosci.nature.com
a
b
Phosphoinositide 3-kinase (PI3 kinase) is pivotal in signal transduction between insulin receptor activation and
cellular responses in peripheral tissues5, and links leptin
activation of KATP channels in an insulin-secreting cell line6.
Thus, we determined the actions of PI3-kinase inhibitors
on insulin-induced KATP channel activation in cell-attached
recordings from acutely dissociated GR neurons. Insulin
(0.1–10 nM) increased KATP activity from 0.18 ± 0.02 to
0.55 ± 0.13, after which 10 µM LY 294002 (Fig. 2b) reduced
activity to 0.20 ± 0.03 (n = 4, p < 0.05, Student’s paired
t-test). In another series, insulin increased activity from
0.08 ± 0.04 to 0.42 ± 0.11, which was returned to
0.17 ± 0.04 (n = 3, p < 0.05, Student’s paired t-test) by
10 nM wortmannin. This PI3 kinase inhibitor (100 nM)
did not affect KATP activity in inside-out patches (n = 4).
Identical cell-attached experiments using the MAPK kinase
inhibitor PD 98059 (50 µM) demonstrated no effect on
insulin-induced channel activity (n = 3).
Our main finding is that physiological levels (< 1 nM)
Fig. 2. Insulin and leptin act on the same neurons via PI3 kinase. (a) Pressure ejection of insulin activate GR neuron KATP channels of control and
of insulin over the slice induced hyperpolarization, reduced firing and decreased input lean, but not obese Zucker rats, consistent with findings
resistance, effects partly reversible over time. Subsequent bath application of leptin that intracerebroventricular insulin reduces food intake
induced similar effects and stopped firing, actions reversed by tolbutamide. (b) Cell- and body weight of lean, but not obese Zucker rats7. These
attached recording in control and after 20 min insulin (10 nM). LY294002 (10 µM) data indicate disconnection between central insulin monreversed the increase in channel activity. Relationship between Nf Po and time.
itoring and output, driven by this genetic defect. The obese
Zucker lesion (fa) is a single missense mutation of the leptin receptor gene (ObRb)8 causing hypothalamic insensitivity to leptin with respect to food intake9 and GR neuron KATP
configuration was 0.07 ± 0.04, which increased to 0.47 ± 0.16
(n = 14) at the peak of the insulin response (12.7 ± 1.3 minutes after
channel activation2. Clearly the ability of insulin to activate GR neuapplication). No change in channel activity occurred in control
ron KATP channels and decrease food intake and body weight of these
experiments (30 minutes; n = 5). Insulin-induced channel activity
animals depends on normal functioning of the brain leptin pathway.
was inhibited by 200 µM tolbutamide (Fig. 1d) by 81.7 ± 11.9%
The ObRb(fa) receptor has impaired signal transduction capabili(n = 4, p < 0.01), indicating the channel was KATP. Inside-out recordty10,11 and displays constitutive activity without ligand12,13. Thus lep+
ings, following cell-attached insulin-induced activation of K chantin-induced refractoriness of intracellular signaling linked to KATP
nels (Fig. 1e) confirmed this. Bath application of 2 mM Mg-ATP
channels may explain the observed insulin insensitivity, particularly
reversibly reduced channel activity by 65.1 ± 4.5% (n = 5). This
as both hormones act on the same population of GR neurons. The
channel, characterized in symmetrical 140 mM KCl, had a linear
finding that insulin action on GR neurons involves PI 3-kinase illuscurrent–voltage relationship, a conductance of 137 ± 23 pS (n = 3)
trates its importance to both leptin and insulin signaling5,6. The
and was voltage independent (data not shown). This is consistent
ObRb(fa) mutation is linked to peripheral dysfunctional PI 3-kinase
with insulin activating large-conductance KATP channels, reported
activity14,15. A similar lesion centrally may explain the inability of lepin GR neurons3,4 and activated by leptin2.
tin and insulin to open KATP channels of hypothalamic GR neurons,
Recordings were made in hypothalamic slices from lean and obese
and could underlie the functional deficit in regulation of food intake
Zucker rats. Insulin (10–100 nM) inhibited the activity of 12 GR (10
and energy homeostasis associated with the ObRb(fa) mutation.
ARC, 2 VMN) neurons in lean Zucker rats. Insulin induced hyperACKNOWLEDGEMENTS
polarization from –50.4 ± 1.7 to –61.8 ± 5.0 mV (n = 12), and
The Wellcome Trust (grant no. 042726) and Pharmacia & Upjohn supported this work.
decreased input resistance from 1534 ± 242 to 856 ± 235 MΩ
S.M. is a Pharmacia & Upjohn student, and M.A.S. is a Wellcome Trust Prize student.
(n = 12) with reversal at –86.8 ± 2.6 mV. These effects of insulin were
reversed (n = 12) by 200 µM tolbutamide. Glucose-free medium
RECEIVED 16 FEBRUARY; ACCEPTED 30 MAY 2000
induced hyperpolarisation in Zucker lean rats from –50.6 ± 1.9 to
–63.2 ± 3.9mV and decreased input resistance from 1509 ± 205 to
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660 ± 334MΩ (n=5). Conversely, insulin (10–100 nM) had no effect
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