Article

Brain Glucose Sensors Play a Significant Role in the Regulation of Pancreatic Glucose-Stimulated Insulin Secretion

Department of Medicine, University of Cambridge Metabolic Research Laboratories, and National Institute for Health Research, Cambridge Biomedical Research Centre, Cambridge, UK
Diabetes (Impact Factor: 8.1). 12/2011; 61(2):321-8. DOI: 10.2337/db11-1050
Source: PubMed

ABSTRACT

As patients decline from health to type 2 diabetes, glucose-stimulated insulin secretion (GSIS) typically becomes impaired. Although GSIS is driven predominantly by direct sensing of a rise in blood glucose by pancreatic β-cells, there is growing evidence that hypothalamic neurons control other aspects of peripheral glucose metabolism. Here we investigated the role of the brain in the modulation of GSIS. To examine the effects of increasing or decreasing hypothalamic glucose sensing on glucose tolerance and insulin secretion, glucose or inhibitors of glucokinase, respectively, were infused into the third ventricle during intravenous glucose tolerance tests (IVGTTs). Glucose-infused rats displayed improved glucose handling, particularly within the first few minutes of the IVGTT, with a significantly lower area under the excursion curve within the first 10 min (AUC0-10). This was explained by increased insulin secretion. In contrast, infusion of the glucokinase inhibitors glucosamine or mannoheptulose worsened glucose tolerance and decreased GSIS in the first few minutes of IVGTT. Our data suggest a role for brain glucose sensors in the regulation of GSIS, particularly during the early phase. We propose that pharmacological agents targeting hypothalamic glucose-sensing pathways may represent novel therapeutic strategies for enhancing early phase insulin secretion in type 2 diabetes.

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Brain Glucose Sensors Play a Signicant Role in the
Regulation of Pancreatic Glucose-Stimulated
Insulin Secretion
Mayowa A. Osundiji,
1
Daniel D. Lam,
2
Jill Shaw,
1,2
Chen-Yu Yueh,
1,3
S. Pauliina Markkula,
1
Paul Hurst,
1
Carolina Colliva,
4
Aldo Roda,
4
Lora K. Heisler,
2
and Mark L. Evans
1
As patients decline from health to type 2 diabetes, glucose-stimulated
insulin secretion (GSIS) typically becomes impaired. Although
GSIS is driven predominantly by direct sensing of a rise in blood
glucose by pancreatic b-cells, there is growing evidence that
hypothalamic neurons control other aspects of peripheral glu-
cose metabolism. Here we investigated the role of the brain in
the modulation of GSIS. To examine the effects of increasing or
decreasing hypothalamic glu cose sensing on glucose tolerance
and insulin secr etion, glucose or inhibitors of glucoki nase, re-
spectively, were infused into the third ventricl e during intrave-
nous glucose tolerance tests (IVGTTs). Glucose-infused rats
displayed improved glucose handling, particularly within t he
rst few minutes of the IVGTT, with a signicantly lower area
under the excursion curve within the rst 10 min (AUC
0-10
). This
was explained by increased insulin secretion. In contrast, infusion
of the glucokinase inh ibitors glucosamine or m annoheptulose
worsened glucose tolerance and d ecreased GSIS in the rst
few minutes of IVGTT. Our data suggest a role for brain glucose
sensors in the regulation of GSIS, particularly during the early
phase. We pr opose that pharmaco logica l agents targeting hypo-
thalamic glucose-sensing pathways may represent novel thera-
peutic strategies for enhancing early phase insulin secretion in
type2diabetes.Diabetes 61:3213 28, 2012
T
he rise in prevalence of type 2 diabetes reects
a primary medical challenge of the 21st century.
The mechanisms underlying glucose homeos ta-
sis in gener al, and g lucose-stimulated insulin
secretion (GSIS) in particular, are not fully understood. In
health, a rise in blood glucose triggers a biphasic pattern of
insulin response, consisting of a rapid (,10 min) rst
phase and a less prominent but sustaine d second phase
(1). The precise mechanisms mediating the early phase of
the insulin response are unclear. Nevertheless, impaired
rst-phase GSIS is a major pathological hallmark of the
early stages of type 2 diabetes (2), suggesting that this may
be an important determinant of the transition to diabetes
in at-risk subjects.
Pancreatic b-cells can directly sense changes in blood
glucose and alter insulin release as appropriate. In addition,
the pancreas has rich autonomic innervations, and a number
of experimental approaches have demonstrated that neural
inputs may modulate insulin release (3,4) via muscarinic
receptors or a-adrenergic signaling (5,6). A preponderance
of the neural inputs that inuence pancreatic b-cell activity
emanate from the hypothalamus (5); however, the specic
nature of hypothalamic pathways regulating insulin secre-
tion is less clear.
In particular, hypothalamic melanocortin signaling (79)
and inammation (10) have been implicated in the control
of insulin release. Additionally, there is increasing evidence
of a role for hypothalamic nutrient-sensing pathways in
the control of other facets of peripheral glucose metabo-
lism, in particular for hepatic glucose production (11,12). It
is therefore plausible that similar mechanisms might allow
central facilitation of insulin secretion in response to a rise
in blood glucose.
In order to control peripheral glucose metabolism, the
brain must rst rapidly and accurately detect changes in
glucose availability. The hypothalamus contains glucose-
sensing neurons, although the mechanisms used to sense
glucose are not fully dened. However, some reports sug-
gest that these hypothalamic neurons may sense products
of glucose metabolism such as cellular A TP levels (11,13).
To be metabolized, glucose must rst be phosphorylated
by hexokinases (HKs). The specialized low afnity HK
isozyme glucokinase (GK), thought to be central in pan-
creatic glucose sensing, may also play a key role in hy-
pothalamic glucose sensing (14). Accordingly, here we
investigated the effects of acute activation of hypotha-
lamic glucose sensing (by brain glucose infusion) or in-
hibition (using the competitive GK inhibitors glucosamine
[GSN] or mannoheptulose [MH]) on insulin secretion and
glucose handling during intravenous glucose tolerance
tests (IVGTT) in rats.
RESEARCH DESIGN AND METHODS
Animals. Healthy adult male Sprague Dawley rats ;250350 g were used
throughout. For each study, the cohorts were matched for weight and ran-
domized into treatment groups. Procedures were approved in advance by both
a local university and a national ethical review process (U.K. Home Ofce
License held under the Animals [Scientic Procedures] Act). Chemicals were
from Sigma-Aldrich (Gillingham, U.K.) unless otherwise stated.
Surgical preparation. Under inhaled anaesthetic, rats underwent stereotaxic
insertion of a guide cannula into the base of the third ventricle (coordinates
from bregma: 2.2 mm posterior, 0.9 mm lateral, 8.4 mm below skull surface
angled at to vertical toward the midline) and placement of jugular vein
catheter as previously described (15). Peri- and postoperative injectable an-
algesia and antibiotic were provided routinely, and only animals that had
regained preoperative body weight with no signs of infection or illness were
studied 1 week later.
From the
1
Department of Medicine, University of Cambridge Metabolic Re-
search Laboratories, and National Institute for Health Research, Cambridge
Biomedical Research Centre, Cambridge, U.K.; the
2
Department of Pharma-
cology, University of Cambridge, Cambridge, U.K.; the
3
Department of Family
Medicine, Chang Gung Memorial Hospital at Chiayi, Chang Gung Institute of
Technology, Chiayi, Taiwan; and the
4
Department of Pharmaceutical Scien-
ces, University of Bologna, Bologna, Italy.
Corresponding authors: Mark L. Evans, mle24@cam.ac.uk, and Lora K. Heisler,
lkh30@medschl.cam.ac.uk.
Received 28 July 2011 and accepted 10 November 2011.
DOI: 10.2337/db11-1050
Ó 2012 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for prot,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
diabetes.diabetesjournals.org DIABETES, VOL. 61, FEBRUARY 2012 321
ORIGINAL ARTICLE
Page 1
Effect of intracerebroventricular infusion of D-glucose versus urea
on glucose handling during IVGTT. Ch ronically catheterized intra-
cer ebrove ntricu la r (ICV) and intravenous (IV) rats prepared as above were
brought to the study room on 2 subsequent days. Day 1 was for acclimatization,
with animals left undisturbed. On day 2, overnight-fasted rats were acclimatized
for 2 h before vascular catheters were opened and ushed and an ICV infusion
initiated. Nine milligrams of
D-glucose (or equimolar urea) wa s delivered over
30 min (0.5 ml/min, primed for 3 min at 1.5 ml/min). Next, r ats underwent
IVGTT with 0.35 g/kg delivered over 1 m in (glucose dose was reduced by
9 mg in ICV glucose rats so that total g lucose dose delivered was similar).
The gluco se dose in these stu dies was se lecte d to be toward the lower end of
the range used in rodent studies, given that we were anticipating an en -
hanced response with ICV glucose. Blood samples were collected over the
ensuing 60 min for plasma glucose and insulin ass ays. After 60 min, rats were
ane sthetized deeply and brains perfused, removed, and examined to verify
correct cannula positioning.
Effect of ICV infusion of GSN or MH versus vehicle on glucose handling
during IVGTT. GSN and MH (CMS Chemicals, Abingdon, U.K.) were dissolved
in articial extracellular uid (aECF; CMA Microdialysis AB, distributed by
Linton Instrumentation, Diss, U.K.). Chronically catheterized rats were pre-
pared, acclimatized, and fasted overnight as above. Rats received ICV infusion
(0.3 ml/min, primed for 3 min at 0.9 ml/min) of GSN (75 nmol/min or 150 nmol/
min), MH (300 nmol/min), or vehicle (aECF). 90 min after the start of ICV
infusions, all animals received an IVGTT (0.5 g/kg glucose over 1 min) with
blood sampling as above. By design, the glucose dose in these studies was
higher than in the ICV glucose studies described above, given that we were
anticipating a reduced response with GK inhibition. At the end of the studies,
rats were anesthetized deeply and brains perfused with sali ne then xative
and removed to verify correct cannula positioning.
Hormonal assays. Glucose was measured using a benchtop glucose analyzer
(Analox GM9 [glucose oxidase method]; Analox Instruments, London, U.K.)
and insulin by RIA (Linco).
Insulinogenic index. Insulinogenic index was calculated as the ratio of areas
under insulin and glucose excursion curves.
Assay of GK activity. Sections of hypothalami were dissected from brain
samples using the stereotaxic atlas of Paxinos and Watson as a reference guide
(16). Thereafter, hypothalamic and liver protein samples were prepared as
previously described with minor modication (17). In brief, tissues were ho-
mogenized in ice-cold lysis buffer (50 mM HEPES, 150 mM KCl, 5 mM MgCl
2
,
and 1 mM EDTA [pH 7.4], supplemented with 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl uoride, and 10 mM leupeptin HCl) and centrifuged at
13,000 rpm for 20 min at 4°C. Supernatants were collected and stored at 280°C
for further analysis. HK and GK activity was assayed spectrophotometrically
(340 nm, room temperature, Beckmann DU-64 spectrophotometer) by cou-
pling glucose phosphorylation to a reporter assay, which oxidizes glucose-6-
phosphate to 6-phosphogluco-d-lactone with simultaneous reduction of NAD+
to NADH, as described previously (18). The reaction mixture in 1 mL nal
volume contained 20 mM HEPES (pH 7.1), 25 mM KCl, 2 mM MgCl
2
,1mM
dithiothreitol, 1 mM NAD+, 1mM ATP, 10 units glucose-6-phosphate de-
hydrogenase from Leuconostoc mesenteroides, 1 mM 3-O-methyl N-acetyl GSN
(Axxora UK Ltd., Bingham, U.K.), 100 mL liver/hypothalamus protein extract,
and glucose in concentrations of 0.5 mM, 1 mM and 20 mM. In all assays for
GK activity, 3-O-methyl N-acetyl GSN was incorporated to inhibit N-acetyl
GSN kinase (19), except inhibition studies with MH and GSN. GK activity was
calculated by subtracting glucose phosphorylation at 0.5 mM (hypothalamus)
or 1 m M (liver) glucose from that measured at 20 mM glucose. Data were
analyzed by tting sigmoidal curves to the dose-response studies by nonlinear
least square minimizat ion method.
Determination of extent of GSN distribution following ICV infusion.
Nonfasted catheterized rats underwent 90 min ICV infusion of 150 nmol/
min GSN or aECF. Plasma samples were collected before and after ICV
infusion. After 90 min, rats were rapidly euthanized and their brains removed;
the hypothalamus, brain stem, and cortex were dissected rapidly and frozen
in liquid nitrogen. Samples were stored at 80°C before being analyzed.
Brain samples were homogenized in ice-cold lysis buffer, centrifuged at
13,000 rpm for 20 min at 4°C, and supernatants were collected for further
analysis.
GSN concentration in plasma and brain homogenate was assayed by high-
performance liquid chromatographyelectrospray ionizationtandem mass
spectometry (HPLC-ESI-MS/MS). Analysis was performed using an Alliance
2695 chromatograph (Waters, Milford, MA) coupled with a triple-quadrupole
MS (Quattro LC; Micromass UK Ltd., Manchester, U.K.). Sample preparation
was optimized by a slight modi cation of a previously published method for
direct determination of GSN in plasma (20). Briey, 45 mLofsamplewas
mixedwith5mLof
D-[1-
13
C] GSN as t he internal standard; then, 25 m Lof
trichloroacetic acid (200 g/L) were add ed to achieve protein prec ipitation.
3 mL of the supernatant was injected into the HPLC-ESI-MS/MS system.
A polymer-based amino column (5 mm, 2.0 mm i.d. 3 150 mm), supplied by
Showa Denko K.K. (Kanagawa, Japan), and mobile phase of 80:20 (volume for
volume) acetonitrile:10 mmol/L ammonium acetate (pH = 7.5) at 0.3 mL/min
ow rate were used. MS/MS detection, using ESI source in positive ionization,
was performed in multiple reaction monitoring mode, selecting the charge/
mass ratio transition 18072 for GSN and charge/mass ratio18173 for in-
ternal standard. The limit of quantication of the method was 0.28 mmol/L for
both rat plasma and brain homogenate. Matrix-matched standards were used
for calibration, obtaining good linearity up to 56 mmol/L (R
2
$0.9937). For
calculating absolute concentrations, the density of brain tissue was assumed
to be 1.04 g/mL (21).
RESULTS
ICV infusion of D-glucose impro ved glucose handling
during IVGTT. We hypothesized that if the brain plays
a role in controlling GSIS, then increasing glucose levels in
the hypothalamus would lead to increased insulin secre-
tion. To investigate this, glucose was infused into the third
ventricle (ICV) of adult male rats for 30 min followed by an
IVGTT (0.35 g/kg) (Fig. 1A and B). ICV infusion of glucose
did not change systemic plasma glucose during the 30 min
infusion period (230 to 0 min). However, ICV glucose-
infused rats displayed improved glucose handling during the
rst10minoftheIVGTT,withasignicantly lower (P ,0.05)
area under the excursion curve (AUC
0-10
) relative to control
urea-infused animals (Fig. 1C and D).
Despite being exposed to equivalent or lower circulating
plasma glucose levels prior to and during IVGTT respec-
tively, plasma insulin levels in ICV glucose-infused rats were
signicantly higher than controls, seemingly starting to rise
even before the delivery of an external glucose load (Fig.
1E). Insulin secretion between the start of the brain infusion
and the 10 min time point of the IVGTT (40 min total),
which was used as the cutoff between early and late-phase
insulin secretion, was signicantly higher in ICV glucose-
infused rats (Fig. 1F). These data suggest that elevation of
hypothalamic glucose leads to a centrally driven insulin
secretory response.
ICV infusion of GSN impaired glucose handling and
insulin secretion during IVGTT. As a possible mecha-
nism underlying this observation, we examined the role
of hypothalamic GK-dependent sensing pathways in in-
sulin secretion in response to a systemic glucose load.
The competitive inhibitor of glucose phosphorylation,
GSN, has previously been shown to inhibit hepatic GK
(22). To conrm that GSN inhibits hypothalamic GK, we
rst examined the effect of GSN on GK activity in hepatic
and hypothalamic protein preparations ex vivo. GK ac-
tivity was detectable in hepatic and hypothalamic protein
preparations from healthy Sprague Dawley rats ex vivo
(Fig. 2A and B). GSN dose-dependently inhibited rat liver
GK activity, with a half-maximal inhibitory concentration
(IC
50
)of4.2mMinthepresenceof20mMglucose
(Fig. 2C). GSN also dose-dependently inhibited hypotha-
lamic GK activity with an IC
50
of 5.0 mM in the presence
of 20 mM glucose (Fig. 2D).
To determine the appropriate infusion regimen to be
used in subsequent studies and the sites and conc en-
t rations of GSN achieved, the extent of GSN distribution
following ICV infusion (150 nmol/min) was measured using
HPLC-ESI-MS/MS method. ICV GSN resulted in a marked
rise in hypothalamic GSN to levels of approximately 1 mM,
a modest rise in brain stem GSN levels (;15% of hypo-
thalamic levels), but did not alter cerebral cortex levels
(Table 1). Importantly, although our sensitive assay was
able to detect a small statistically signicant change in
plasma values, levels of GSN in the blood stream remained
BRAIN CONTROL OF INSULIN SECRETION
322 DIABETES, VOL. 61, FEBRUARY 2012 diabetes.diabetesjournals.org
Page 2
at low micromolar levels, three orders of magnitude below
the levels required to inhibit GK.
Next, we examined whether hypothalamic levels of GSN
attained by 90 min ICV infusion (1 mM) would be sufcient
to inhibit hypothalamic GK activity. We aimed to measure
hypothalamic GK activity in the presence of 1 mM GSN in
vitro at a glucose concentration that is likely to simulate
ambient hypothalamic glucose levels during the IVGTT.
A concentration of 3 mM glucose was selected for in vitro
assays based on peak plasma glucose levels observed
during IVGTT (17 mM, Fig. 1) and on the fact that accepted
approximations of brain extracellular glucose concentrations
are roughly 20% of that of plasma glucose (23,24). GK rep-
resented ;5% of total hypothalamic glucose phosphorylation
activity at 3 mM glucose and was selectively inhibited by 1
mM GSN (reduced to 48.1 6 7.3% and total glucose phos-
phorylating activities was 97
+ 1% of control, n =3).These
kinetic studies suggested that ICV GSN in vivo, even at the
highest dose used, is unlikely to result in major spillage of
GSN outside the brain and that levels achieved might be
sufcient to inhibit, at least in part, hypothalamic GK activity.
We next investigated the effects of 90 min ICV infusion
of GSN at 75 and 150 nmol/min on plasma glucose and
insulin responses during IVGTT (0.5 g/kga higher dose
than that used in ICV-glucose studies above) (Fig. 3A). ICV
GSN signicantly and dose-dependently impaired glucose
handling during IVGTT (Fig. 3B an d C). Despite the higher
plasma glucose in the ICV GSN rats, insulin responses
FIG. 1. ICV infusion of glucose improved glucose handling during IVGTT. A and B: Schematic representation of experimental design. Vascular and
third ventricle (ICV) catheters were implanted on day 1. Rats were acclimatized to the study room on day 6 and fasted overnight from 1500 hrs on
day 6. On day 7, chronically catheterized rats underwent IVGTT (0.35 g/kg) preceded by ICV infusion of either 9-mg glucose or equimolar urea (n =
67) delivered over 30 min. C: ICV glucose rats showed reduced plasma glucose levels particularly during the rst few minutes of IVGTT. D:In
particular, the AUC
0-10
(plasma glucose) was signicantly (P < 0.05) lower in ICV glucose-treated rats relative to control urea-treated rats. E and
F: Despite exposure to lower glycemia, plasma insulin responses were signicantly greater in ICV glucose animals. Data are mean 6 SEM. *P <
0.05. (A high-quality color representation of this gure is available in the online issue.)
M.A. OSUNDIJI AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 61, FEBRUARY 2012 323
Page 3
were reduced, particularly during the rst few minutes of
the glucose challenge, with peak and AUC
0-10
insulin being
signicantly lower in ICV 150 nmol/min GSN rats compared
with controls. As might be expected, ICV 75 nmol/min GSN
showed a pattern intermediate between the other two
groups. Insulinogenic index was signicantly lower in both
ICV 150 nmol/min and 75 nmol/min GSN rats relative to
controls (Fig. 3DF). Taken together, these data show that
third ventricle infusion of a low dose of a GK inhibitor, GSN,
predominantly distributed into the surrounding hypothala-
mus, impaired insulin secretion and glucose tolerance dur-
ing the rst few minutes of an IVGTT in rats. Moreover,
these ndings suggest a role for hypothalamic GK mediated
glucose sensing in the central regulation of GSIS.
FIG. 2. Ex vivo GK activity assays in protein preparations. A: Sigmoidal dependence of hepatic GK (n = 2) on glucose concentration. B: Glucose
increases hypothalamic GK activity. C and D: GSN dose dependently inhibits hepatic (n =35) and hypothalamic (n = 3) GK ex vivo. E: MH dose
dependently inhibits hypothalamic GK ex vivo (n = 3), although with reduced potency as compared with GSN (IC
50
=12 mM vs. 5 mM, MH vs. GSN,
respectively). **P < 0.01.
BRAIN CONTROL OF INSULIN SECRETION
324 DIABETES, VOL. 61, FEBRUARY 2012 diabetes.diabetesjournals.org
Page 4
ICV infusion of MH impaired glucose handling and
insulin secretion during IVGTT. A limitation of the
study above is that GSN has other biological actions in
addition to inhibiting glucose metabolism via GK, for in-
stance acting through the hexosamine pathway (25). The
study was repeated using MH, an alternative GK inhibitor
that is structurally unrelated to GSN and does not act
through the hexosamine pathway. Again, we rst con-
rmed ex vivo that MH dose-dependently inhibited hypo-
thalamic GK (Fig. 2E). Given the lower potency of MH for
GK inhibition, a higher dose of MH was used for in vivo
studies. In keeping with an effect media ted by GK in-
hibition, ICV 300 nmol/min MH-treated rats displayed im-
paired glucose handling with a signicantly higher AUC
0-10
glucose relative to control rats (Fig. 4A and B). Consis-
tently, insulinogenic index was signicantly lower in ICV
300 nmol/min MH rats relative to controls (Fig. 4C and D).
These ndings further support a role for hypothalamic GK-
mediated glucose sensing in the regulation of GSIS in re-
sponse to an IV glucose challenge.
DISCUSSION
Although there is increasing evidence that hypothalamic
glucose sensing may contribute to the integr ated control of
aspects of whole body glucose homeostasis (26) such as
hepatic glucose output (11,12) and hypoglycemia counter-
regulation (13), its role in the regulation of insulin secretion
has been less clear. Here, we prov ide the rst direct
evidence that hypothalamic glucose sensors play a signi-
cant role in the control of insulin secret ion, one of the
most important systems in the maintenance of whole-body
glucose homeostasis.
Specically, we observed that activation of hypotha-
lamic glucose sensing by ICV infusion of glucose improved
glucose handling and insulin secretion during IVGTT.
Furthermore, we demonstrated that pharmacological in-
hibition of hypothalamic glucose sensing by ICV infusion
of GK and HK inhibitors, GSN and MH, signicantly im-
paired glucose handling and rst-phase insulin secretion
during IVGTT. These data suggest a critical role for brain
glucose sensing in the regulation of the rst phase of
pancreatic GSIS and in turn whole-body glucose tolerance.
Such a role is consistent with emerging evidence that hy-
pothalamic glucose sensors contribute to the integr ated
control of peripheral glucose homeostasis (26).
As in humans, glucose infusion in rats elicits a biphasic
insulin response (27). In this study, we elected to use an
IVGTT rather than oral challenge because it allowed in-
vestigation of GSIS without a confounding effect from
incretins. Furthermore, IVGTT as a measure of insulin
secretion has been used successfully in rats. Similar to the
pattern seen in humans, both an early peak response
during the rst few minutes after a glucose load and a later
sustained insulin release can be identied (28). Analogous
to humans, the early phase of insulin release is suppressed
in some rodent models of diabetes (29).
Although GSN and MH inhibit other HKs in addition to
inhibiting GK, the conditions of our studies suggest that
these effects are likely mediated via GK. Brain extracellular
glucose concentration is ;20% of that of plasma glucose
(23,24). Plasma glucose levels during I VGTT studies
attained peak values of approximately 1720 mM (Figs. 13)
and 20% of these values are roughly 34 mM. However, it is
possible that glucose levels sensed by hypothalamic arcuate
nucleus (ARC) neurons may be higher than this because of
the proximity of the ARC to the median eminence, where
the blood-brain barrier is thought to be leaky (30). Since GK
is active in the hypothalamus at glucose concentrations
ranging from about 3 to 20 mM (31), it is better suited for
high capacity glucose phosphorylation necessary for glu-
cose sensing in this range of glucose concentrations than
other high afnity HKs (which are easily saturated at glu-
cose concentrations less than 500 mM). Our in vitro simu-
lation studies demonstrating that 1 mM GSN selectively
inhibits hypothalamic GK without interfering with other
HKs at 3 mM glucose further suggest that ICV GSNs effects
during IVGTT are mediated via inhibition of hypothalamic
GK activity.
It is important to note that our studies do not permit us to
exclude a potential contribution from extrahypothalamic
brain regions inuenced by third ventricle infusion in me-
diating centrally driven GSIS responses. However, given
that the relative levels of GSN achieved by our third
ventricle infusion in hypothalamus were approximately
5-fold higher than in the brain stem, the data suggest that
the hypothalamus received the highest concentration of GSN.
Furthermore, even though the data clearly demonstrate
a direct effect of brain glucose sensors on pancreatic GSIS,
the involvement of additional synergistic effects to im-
prove glucose handling by hypothalamic efferents, for
example, altering hepatic glucose output directly, cannot
be excluded. To delineate further the specic role of the
hypothalamus and specic hypothalamic subnuclei in the
effects of GSIS, localized injections of glucose and inhib-
itors of GK should be used. In addition, many elegant ge-
netic and pharmacogenetic tools are now available which will
enable further rened probing of the discrete role of spe-
cic chemically dened neurons, such as those expressing
the melanocortin neuropeptides in the ARC, in GSIS.
TABLE 1
GSN concentrations in brain and plasma
GSN levels in brain areas following 90-min
ICV infusion (mM)
GSN levels in
plasma (mM)
Hypothalamus Brain stem Cortex
Before ICV
infusion
After 90-min
ICV infusion
ICV 150 mol/min GSN 922 6 632* 142 6 119* 5 6 226 136 1*
ICV aECF 1 6 026 126 1ND ND
Data are mean 6 SEM. GSN concentrations in brain areas (n =35) and plasma (n = 11) following 90-min ICV 150 nmol/min GSN or aECF
(vehicle). Chronically catheterized (jugular vein and third ventricle) adult male Sprague Dawley rats underwent 90-min ICV infusion of 150
nmol/min GSN or aECF, with blood sampling from ICV GSN rats at start and end of infusion. Following 90 min ICV infusions, animals were
killed and brains collected for ex vivo GSN assays. ND, no data. *P , 0.05 Mann-Whitney test for GSN vs. aECF and after vs. before ICV
infusion.
M.A. OSUNDIJI AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 61, FEBRUARY 2012 325
Page 5
Overall, our data support a role for hypothalamic glu-
cose sensing in the integrated control of peripheral glu-
cose homeostasis. In particular, our ndings suggest a
novel model for the regulation of G SIS. We identied for
the rst time that GK-dependent glucose phosphorylation in
the hypothalamus may play a facilitatory role in the regu-
lation of the rst phase of insulin secretion in response to a
systemic glucose load. GK activators have been highlighted
as potential therapeutic candidates for type 2 diabetes (32),
in particular focusing on effects mediated by activation of
FIG. 3. ICV infusion of a GK inhibitor, GSN-impaired glucose handling, and insulin secretion during IVGTT. A: Experimental design. On day 7,
chronically catheterized (jugular vein and third ventricle) rats, which had been fasted overnight, underwent IVGTT (0.5 g/kg) preceded by 90-min
ICV infusion of either GSN at 75 nmol/min or 150 nmol/min, or vehicle (aECF) (n =913). B: ICV GSN rats showed impaired glucose handling,
particularly during the rst few minutes of IVGTT. C: The AUC
0-10
(plasma glucose) increased signicantly (P < 0.05) and dose-dependently in ICV
GSN-treated rats as compared with ICV vehicle-treated rats. D and E: In spite of higher plasma glucose levels in ICV GSN-infused rats, plasma
insulin levels were reduced signicantly and dose-dependently relative to vehicle-treated rats. Insulinogenic index was also signicantly reduced
by both ICV 150 and 75 nmol/min treatment as compared with ICV aECF. Data are mean 6 SEM. *P < 0.05; **P < 0.01 (A high-quality color
representation of this gure is available in the online issue.)
BRAIN CONTROL OF INSULIN SECRETION
326 DIABETES, VOL. 61, FEBRUARY 2012 diabetes.diabetesjournals.org
Page 6
hepatic and pancreatic GK. Our data suggest that a further
benecial action might be targeting GK-mediated glucose
sensing in the hypothalamus.
In conclusion, our ndings delineate a novel central
mechanism i n the control of glucose-stimulated insul in
release and suggest that this may o ffer a future thera-
peutic target for improving glycemic control in type 2
diabetes.
ACKNOWLEDGMENTS
This study was supported by Juvenile Diabetes Research
Foundation regular grants (1-2003-78 and 1-2006-29) and
Diabetes UK (BDA: RD05/003059) to M.L.E. and National
Institute of Diabetes and Digestive and Kidney Diseases
(DK065171), the Wellcome Trust (WT081713), and the
American Diabetes Association to L.K.H. and by the
Medical Research Council Centre Grant (MRC-CORD) to
all authors. M.A.O. was supported by a Diabetes Research
and Wellness Foundation Ph.D. studentship, D.D.L. by a
Gates Cambridge Trust studentship, and S.P.M. by MRC
Studentship.
No potential conicts of interest relevant to this article
were reported.
M.A.O., D.D.L., P.H., S.P.M., and M.L.E. conceived and
designed the experiments; performed the experiments;
analyzed the data; contributed reagents, materials, and
analysis tools; and wrote the manuscript. J.S. conceived
and designed the experiments; performed the experi-
ments; contributed reagents, materials, and analysis tools;
and wrote the manuscript. C.-Y.Y. conceived and designed
the experiments, performed the experiments, and analyzed
the data. L.K.H. conceived and designed the experiments;
analyzed the data; contributed reagents, materials, and
analysis tools; and wrote the manuscript. C.C. and A.R.
performed the experiments, analyzed the data, and contrib-
uted reagents, materials, and analysis tools. M.L.E. is the
guarantor of this work and, as such, had full access to all the
data in the study and takes responsibility for the integrity of
the data and the accuracy of the data analysis.
Parts of this study were presented in abstract form at the
42nd European Association for the Study of Diabetes annual
meeting, Copenhagen, Denmark, 14 1 7 Sept emb er 2006
FIG. 4. ICV infusion of a GK inhibitor, MH, impairs insulin secretion during IVGTT. A: ICV MH rats showed impaired glucose handling, particularly
during the rst few minutes of IVGTT. B: The AUC
0-10
(plasma glucose) was signicantly (P < 0.05) higher in ICV MH-treated rats as compared
with ICV vehicle-treated rats. C and D: In spite of higher plasma glucose levels in ICV GSN-infused rats, plasma insulin levels were reduced relative
to vehicle-treated rats. Insulinogenic index was signicantly reduced by both ICV 300 nmol/min MH treatment as compared with ICV aECF. Data
are mean 6 SEM (n =913). *P < 0.05. (A high-quality color representation of this gure is available in the online issue.)
M.A. OSUNDIJI AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 61, FEBRUARY 2012 327
Page 7
and at the 67th Scientic Sessions of the American Diabe-
tes Association, Chicago, Illinois, 2226 June 2007.
The authors are grateful to Keith Burling, Department of
Clinical Biochemistry, Addenbrookes Hospital, Cambridge,
for performing hormonal assays.
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BRAIN CONTROL OF INSULIN SECRETION
328 DIABETES, VOL. 61, FEBRUARY 2012 diabetes.diabetesjournals.org
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    • "Disrupting brain sensing of macronutrients such as glucose leads to metabolic disturbances, including defects in pancreatic glucose-stimulated insulin secretion (e.g. [18] [19] [20]). For hormones, the effects of deletion of receptors for insulin and leptin from specific populations of hypothalamic neurons demonstrate that hormone sensing by brain circuits is essential for peripheral glucose homeostasis and normal body weight [21] [22] [23]. "
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    Full-text · Article · Apr 2013 · Physiology & Behavior
  • Source
    • "Central control of insulin secretion could be viewed as an indirect pathway, e.g., leptin and NPY modulates insulin secretion mainly through receptors in the hypothalamus [72] and NTS [26], respectively. Sensing of glucose by brain regions [99] including the hypothalamus, has recently been shown to trigger insulin release [14,80] establishing a brain-endocrine pancreatic axis. There is strong evidence for vagal stimulation of beta cell secretion [98]. "
    [Show abstract] [Hide abstract] ABSTRACT: Secretin (Sct), traditionally a gastrointestinal hormone backed by a century long research, is now beginning to be recognized also as a neuroactive peptide. Substantiation by recent evidence on the functional role of Sct in various regions of the brain, especially on its potential neurosecretion from the posterior pituitary, has revealed Sct's physiological actions in regulating water homeostasis. Recent advances in understanding the functional roles of central and peripheral Sct has been made possible by the development of Sct and Sct receptor (SctR) knockout animal models which have led to novel approaches in research on the physiology of this brain-gut peptide. While research on the role of Sct in appetite regulation and fatty acid metabolism has been initiated recently, its role in glucose homeostasis is unclear. This review focuses mainly on the metabolic role of Sct by discussing data from the last century and recent discoveries, with emphasis on the need for revisiting and elucidating the role of Sct in metabolism and energy homeostasis.
    Full-text · Article · Dec 2012 · General and Comparative Endocrinology
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