Am J Physiol Regul Integr Comp Physiol 301: R668–R673, 2011.
First published June 15, 2011; doi:10.1152/ajpregu.00058.2011.
TRANSLATIONAL PHYSIOLOGY
Insulin and glucagon share the same mechanism of neuroprotection
in diabetic rats: role of glutamate
Rami Abu Fanne,1* Taher Nassar,1* Samuel N. Heyman,2 Nuha Hijazi,1 and Abd Al-Roof Higazi1,3
1
Department of Clinical Biochemistry, 2Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem,
Israel, and 3Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia Pennsylvania
Submitted 28 January 2011; accepted in final form 6 June 2011
stroke; brain damage; glutamate
acute ischemic stroke, diabetes is
found at presentation (5, 24), ⬃70% have elevated blood
glucose levels (27), and about 25% develop persistent hyperglycemia (21). Hyperglycemia is associated with increased
infarct size (5, 9, 12, 21, 36, 44), as well as with a ⬃3-fold
higher risk of death (15), and survivors have more profound
neurologic deficits and disability (26). Hyperglycemia is also
associated with aggravated postischemic brain damage in animal models. In cats, acute hyperglycemia is associated with a
⬃3-fold increase in the volume of hemispheric infarcts induced
by cerebrovascular occlusion (19), and in dogs, even moderate
hyperglycemia has been shown to increase brain damage and
mortality induced by ischemia (33). Consistent with these
IN ⬃30 – 40% OF PATIENTS WITH
* R. Abu Fanne and T. Nassar contributed equally to this work.
Address for reprint requests and other correspondence: Abd Al-Roof
Higazi, Dept. of Pathology and Laboratory Medicine, Univ. of Pennsylvania, 513A Stellar-Chance, 422 Curie Blvd., Philadelphia, PA 19104
(e-mail: [email protected]).
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findings, treatment of hyperglycemia with insulin in these
models, improves the outcome (10, 29, 45). Notwithstanding
extensive clinical and experimental data indicating that hyperglycemia exacerbates poststroke brain damage and evidence
from animal models that reversal of hyperglycemia with insulin attenuates injury (10, 29, 45), in clinical practice, this
approach is controversial (8, 23, 25, 31, 35, 37). This disconcerting lack of clinical success is not entirely surprising, since
insulin has pleiotropic effects on cell metabolism that extend
beyond the lowering of blood glucose.
We hypothesize that in the acute setting of stroke, hyperglycemia, as such, is not neurotoxic and that the deleterious
effects are mediated through the associated increase in circulating amino acids, including the neurotoxic amino acid glutamate. In all stages of diabetes, including early prediabetic
insulin resistance (IR), increased levels of amino acids, such as
alanine, proline, valine, leucine/isoleucine, phenylalanine, tyrosine, glutamate/glutamine, and ornithine, have been observed
(41); furthermore, in addition to its effect on glucose levels,
insulin reduces the concentrations of these amino acids in the
circulation (11, 30).
Two important corollaries of this concept are 1) following
brain injury or ischemia, neurotoxic amino acids are elevated
for only ⬃30 min in rodents (42) and ⬃6 h in humans (13), and
thus, it can be predicted that insulin given after that time would
be ineffective; and 2) reduction of CNS glutamate alone may
improve the outcome without necessarily affecting blood glucose levels. On the basis of these postulates, we suggest that
the predominant effect of insulin in poststroke management is
off-target, i.e., insulin primarily lowers blood glucose rather
than glutamate levels, and in the clinical setting, its brief
therapeutic window has not been taken into consideration.
Recently, we reported that glucagon improves the outcome
of traumatic brain injury (TBI) in mice, although it increases
the concentration of glucose (1). In the present communication
in a rat model, we report that insulin, glucagon, and the
glutamate scavenger, oxaloacetate, improve poststroke outcome in animal models by decreasing glutamate in the circulation and in the cerebrospinal fluid (CSF) (14, 32) The
neuroprotective effect of glucagon, insulin, and oxaloacetate
did not correlate with glucose levels, which were affected in
opposite directions, if at all.
MATERIALS AND METHODS
Animals. All experimental protocols involving the use of vertebrate
animals were approved by the Israeli Board for Animal Experiments.
Adult male, 8- to 10-wk-old Sprague-Dawley rats (average weight
0363-6119/11 Copyright © 2011 the American Physiological Society
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Abu Fanne R, Nassar T, Heyman SN, Hijazi N, Higazi AA.
Insulin and glucagon share the same mechanism of neuroprotection
in diabetic rats: role of glutamate. Am J Physiol Regul Integr Comp
Physiol 301: R668 –R673, 2011. First published June 15, 2011;
doi:10.1152/ajpregu.00058.2011.—In patients with acute ischemic
stroke, diabetes and hyperglycemia are associated with increased
infarct size, more profound neurologic deficits and higher mortality.
Notwithstanding extensive clinical and experimental data, treatment
of stroke-associated hyperglycemia with insulin is controversial. In
addition to hyperglycemia, diabetes and even early prediabetic insulin
resistance are associated with increased levels of amino acids, including the neurotoxic glutamate, in the circulation. The pleiotropic
metabolic effects of insulin include a reduction in the concentration of
amino acids in the circulation. In this article, we show that in diabetic
rats exposed to transient middle cerebral artery occlusion, a decrease
of plasma glutamate by insulin or glucagon reduces CSF glutamate,
improves brain histology, and preserves neurologic function. The
neuroprotective effect of insulin and glucagon was similar, notwithstanding their opposite effects on blood glucose. The therapeutic
window of both hormones overlapped with the short duration (⬃30
min) of elevated brain glutamate following brain trauma in rodents.
Similar neuroprotective effects were found after administration of the
glutamate scavenger oxaloacetate, which does not affect glucose
metabolism. These data indicate that insulin and glucagon exert a
neuroprotective effect within a very brief therapeutic window that
correlates with their capacity to reduce glutamate, rather than by
modifying glucose levels.
NEUROPROTECTION BY INSULIN AND GLUCAGON IN DIABETES
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When jugular blood was available, blood glucose levels were reconfirmed by measuring serum glucose on a Kodak analyzer, as described
previously (1).
CSF glutamate. Several groups of rats were used for CSF sampling
only. CSF samples were taken from the cisterna magna of sham or
post MCAO anesthetized rats, treated or not treated with insulin or
glucagon (as indicated) (7). About 70 ␮l of CSF were obtained from
each rat. After CSF draining, the animals were killed. Pooled CSF
from two or three animals was frozen at ⫺70°C. Amino acid concentrations were determined on a Bio-Chrom 20-amino acid analyzer, as
above.
Diabetic rats. Adult male Sprague-Dawley rats were randomly
placed for 14 wk on a normal fat (NF) or high fat (HF) diet (Harlan
Teklad, Madison, WI) corresponding to 10% or 55% of the calories
from fat and provided with water ad libitum (n ⫽ 30 in each group).
Animals were maintained in a temperature-controlled barrier facility,
with a 12-h alternating light-dark cycle. Food intake monitored
throughout the study was almost identical in the two groups. In
addition to body weight, fasting blood glucose was monitored weekly,
using an Elite Glucometer (Bayer, Mishawaka, IN). At the end of 14
wk, the group fed the HF diet had significantly higher fasting blood
glucose levels than the controls (4.88 ⫾ 2.31 vs. 8.9 ⫾ 3.11 mmol/l,
respectively, P ⫽ 0.02). Glucose tolerance tests were performed by
tail vein injection of glucose (1 g/kg) after an overnight fast, as
described previously (38). HF rats displayed the expected glucose
intolerance (data not shown), compared with the control NF group.
Statistical analysis. All data are presented as means ⫾ SE. Differences were analyzed using the Student’s t-test or one-way ANOVA
with the Newman-Keuls post hoc test, as indicated in RESULTS. In the
case of neuroscoring data, the “between-group” comparisons were
performed by one-way ANOVA with the Newman-Keuls test and by
the Kruskal-Wallis rank test; in addition to the means ⫾ SE, the
median values are also shown in Figs. 2– 4. Statistical significance
was set at P ⬍ 0.05.
RESULTS
To examine the role of insulin in the development of
poststroke brain damage, normal rats were treated with 2 IU/kg
of insulin or normal saline (NS) at 10 min prior to the induction
of transient middle cerebral artery occlusion (tMCAO). Injection of insulin decreased the infarct size by about 65% (P ⬍
0.002, one-way ANOVA with Newman-Keuls post hoc test)
(Fig. 1) and improved the neuroscoring (Fig. 2A) significantly
Fig. 1. Time-dependent neuroprotective effect of insulin on postischemic brain
damage The middle cerebral artery (MCA) of rats was occluded with an
intraluminal filament. The thread was withdrawn 2 h later. Ten minutes before
(⫺10=) or 10, 30, or 60 min after (⫹10= to ⫹60=) the occlusion of the MCA,
normal saline (NS) or saline containing insulin (Insulin) (2 U/kg ip) was
injected. In two of the groups, 10 min after occlusion of the MCA, the
administration of insulin was followed by intravenous injection of glutamate
(Glutam) or glucose (Glucose) (20% in 0.75 ml). The mean ⫾ SE of data from
8 –14 animals/group at each time point is shown. *Significant difference from
controls receiving only NS (*P ⬍ 0.002).
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250 –280 g) (Harlan Laboratories, Jerusalem, Israel) were anesthetized with an intraperitoneal injection of ketamine (75 mg/ml) and
xylazine (5 mg/ml; Kepro Barneveld, Holland) before the experiments.
Transient occlusion of the middle cerebral artery. Transient occlusion of the middle cerebral artery (MCAO) was performed exactly as
described previously (2, 4). Briefly, the left common carotid artery
(CCA) was exposed and permanently ligated. An incision was made
in the CCA to insert a 4 – 0 monofilament nylon suture. The different
treatments were given before or after the induction of ischemia, as
described below. The monofilament was inserted through the CCA
into the lumen of the internal carotid artery and was advanced into the
circle of Willis, effectively occluding the middle carotid artery. Two
hours after MCA occlusion, the monofilament was removed, the
surgical wound was closed, and the animals were returned to their
cages to recover. The mortality rate was about 15% and was similar
in the different groups. Almost all of the deaths happened during or
immediately after the induction of MCAO, and all of these rats were
excluded.
Injection of insulin and glucagon. Glucagon and insulin were given
intraperitoneally 10 min before or 10, 30 or 60 min after the induction
of MCAO. Insulin doses of 2 U/kg to wild-type (WT) and 3 U/kg
given to diabetic rats were based on the literature (20, 40), as well as
on a preliminary test that we performed to decrease plasma glutamate
with insulin to the same levels achieved with glucagon. The higher
dose of insulin in diabetic rats was due to the insulin resistance typical
of type II diabetes.
Injection of oxaloacetate, glutamate, and glucose. Immediately
after insulin or glucagon injection and as indicated, rats were given
saline alone (30 ␮l·min⫺1·100 g⫺1), or saline containing oxaloacetate
(30 ␮mol·min⫺1·100 g⫺1) or oxaloacetate and sodium glutamate
(Sigma, St. Louis, MO) (30 ␮mol·min⫺1·100 g⫺1 each) (47) into the
tail vein over 30 min. Glucose was injected intraperitoneally (20% in
0.75 ml of water for injection) immediately after the injection of
insulin.
Neuroscoring and exclusion criteria. The neurological scoring was
performed at 24 h after induction of stroke. The neuroscores were
determined twice by independent observers who were blinded to the
experimental protocol. The scoring of the neurological deficits was a
composite of motor, sensory, reflex, and balance tests, as described in
detail by Chen et al. (18) and others (6, 17). The severity of injury was
graded as follows: Points were awarded for the inability to perform
the required test or for the absence of a reflex tested. Thus, the higher
the score, the more severe was the injury. Neurological function was
graded on a scale of 0 to 18 (normal score, 0; maximal deficit, 18
points). Scores of 13–18 were considered to reflect severe injury;
scores of 7–12 reflected moderate injury and scores of 1– 6 indicated
mild injury.
Measurement of infarct size. Infarct size was determined as
previously reported (2, 4). The infarcted area in each section was
traced manually and measured using an image analysis system. The
total infarct volume was determined by integrating the areas from
all sections, and the results were expressed as a percentage of the
total volume of the ipsilateral hemisphere, as described previously
(2, 4, 43).
Blood tests. For the measurement of blood glucose, blood samples
were taken during the experiments from the tail of anesthetized rats to
measure basal glucose levels, using a manual glucometer. For the
measurement of plasma glutamate, rats were anesthetized as above.
Blood samples were taken from the jugular vein acceded by venesection. In some rats, blood was obtained by cardiac puncture just prior
to euthanizing the animals. The blood was collected into commercially available tubes (Vacutainer, Plymouth, UK) containing EDTA
as anticoagulant. Plasma was separated immediately in a refrigerated
centrifuge and deproteinized with sulfosalicylic acid. Amino acid
concentrations were determined using HPLC (1, 6) on a Bio-Chrom
20 amino acid analyzer (Pharmacia Biotech, Heidelberg, Germany).
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Fig. 2. Time-dependent neuroprotective effect of insulin on postischemic
neurological recovery. The experiments were performed as in Fig. 1. Twentyfour hours after middle cerebral artery occlusion (MCAO), the neurological
scores were determined. A: scores for each rat besides the mean ⫾ SE for each
group. The median value for each group is presented at the bottom of the
graph. B: asterisks above the bars denote a significant difference from controls
receiving only NS. Blood and cerebrospinal fluid (CSF) samples taken 15 min
after insulin injection were used to measure glucose (B) and glutamate (C) in
the plasma and CSF, respectively. The mean ⫾ SE is shown. C: significant
difference from controls receiving only NS (#P ⬎ 0.004). Significant difference compared with sham operated animals (*P ⬎ 0.001).
(P ⬎ 0.0002, one-way ANOVA with Newman-Keuls post hoc
test and by Kruskal-Wallis rank test). Fifteen minutes after
insulin injection, the concentrations of glucose in the blood
were 63 ⫾ 9.3 mg/dl in animals injected with insulin compared
with 117 ⫾ 12.19 mg/dl in animals given saline alone (P ⫽
0.001, Student’s t-test) (Fig. 2B). The decrease of glucose
levels was accompanied by decreased glutamate levels in the
circulation and the CSF of the treated animals. Fifteen minutes
after insulin injection, the concentrations of glutamate in the
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Fig. 3. Time-dependent neuroprotective effect of glucagon on postischemic
neurological recovery. The experiments were performed as in Fig. 1. NS or
NS-containing glucagon (Glucagon) (5 ␮g ip) was injected 10 min before
(⫺10=) or 10, 30, or 60 min after (⫹10= to ⫹60=) the occlusion of the MCA.
As in Fig. 1, one group received intraperitoneal glucagon followed by intravenous glutamate (Glutam). Twenty-four hours later, the brain infarct size was
measured (A) after determining the neurological score (B). The individual data
and the mean ⫾ SE from 8 –14 animals/group at each time point is shown.
*Significant difference from controls receiving only NS: A: *P ⫽ 0.001;
B: *P ⫽ 0.01.
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CSF were 1.89 ⫾ 0.21 ␮mol/l in animals injected with insulin
compared with 11 ⫾ 1.6 ␮mol/l in animals given saline alone
(P ⫽ 0.017, Student’s t-test) (Fig. 2C); insulin also significantly decreased the plasma concentrations of glutamate from
201.3 ⫾ 29 to 87.3 ⫾ 19 ␮mol/l (P ⫽ 0.004, Student’s t-test).
To examine the therapeutic window of the insulin treatment,
animals were treated at 10 min before or 10, 30, or 60 min after
the induction of tMCAO. Figure 1 shows that the beneficial
effect of insulin on brain damage, as reflected by decreased
infarct size and improved neuroscoring (Fig. 2A), was observed
in pretreated rats or rats treated up to 30 min after initiation of
tMCAO; animals treated with insulin 60 min after the initiation
of tMCAO did not differ significantly from controls (Figs. 1
and 2A). Neuroprotection afforded by lower doses of insulin
(0.5–1.5 U/kg) induced dose-dependent neuroprotection that
correlated with its effects on blood and CSF levels of glutamate
(data not shown).
We next examined the effect of the administration of glucose
or glutamate together with insulin. Intravenous injection of
glutamate (47) prevented the decrease of glutamate in the
circulation by insulin [187.7 ⫾ 37 ␮mol/l in rats given glutamate and insulin compared with 71 ⫾ 14 ␮mol/l in animals
given insulin alone (P ⫽ 0.001, Student’s t-test)] and prevented
its neuroprotective effect (Figs. 1 and 2A). We next examined
the effect of preventing the reduction of glucose levels following administration of insulin. Injection of glucose (20% in 0.75
NEUROPROTECTION BY INSULIN AND GLUCAGON IN DIABETES
ml of water for injection) intraperitoneally immediately after
insulin prevented the hypoglycemic effect of insulin; 15 min
after injection of insulin, plasma glucose concentrations were
132 ⫾ 27 mg/dl in rats given insulin and glucose compared
with 59 ⫾ 12 mg/dl (P ⫽ 0.005, Student’s t-test) in animals
given insulin alone. However, prevention of the decrease in
glucose levels did not affect insulin-induced neuroprotection
(Figs. 1 and 2A). To evaluate the potential long-term neuroprotection by insulin, we examined neurobehavioral functions
at 1, 7, 14, and 28 days after tMCAO. Similar to the prolonged
neuroprotective effect of glucagon (1), we found that tMCAO
mice treated with insulin showed significantly improved neuAJP-Regul Integr Comp Physiol • VOL
roscoring at all time points studied, compared with the controls
(data not shown).
To further substantiate our findings on the irrelevance of
glucose concentration to the outcome of brain injury, we
injected glucagon instead of insulin. Glucagon is known to
induce gluconeogenesis, leading to increased blood glucose
levels and a decrease of glucogenic amino acid concentrations,
such as glutamate (11, 34, 46). Injection of glucagon 10 min
before or 10 min after induction of stroke decreased infarct size
by ⬃70% (P ⫽ 0.001, one-way ANOVA with Newman-Keuls
post hoc test) (Fig. 3A) and led to an improvement in the
neuroscore (P ⫽ 0.01) (Fig. 2B); injection of glucagon 1 h after
induction of stroke had no protective effect (Fig. 3, A and B).
The protective effect of glucagon was accompanied by a
decrease in blood and CSF glutamate and increased glucose
concentrations. Fifteen minutes after the injection of NS or
glucagon (5 ␮g IP), blood glucose in animals treated with
glucagon was 176 ⫾ 6.4 compared with 121 ⫾ 5.18 mg/dl
(P ⬍ 0.001, Student’s t-test) after NS. When the concentration of glutamate was maintained by giving intravenous
glutamate along with glucagon (47) [glutamate concentrations were 173.7 ⫾ 28 ␮mol/l in rats treated with glucagon
and glutamate, compared with 65 ⫾ 22 ␮mol/l (P ⫽ 0.03,
Student’s t-test) in animals treated with glucagon alone],
and neuroprotection was abolished (Fig. 3, A and B).
In contrast to the widely accepted concept that diabetes (26)
and hyperglycemia (12, 15, 36, 44) are risk factors for exacerbation of ischemic brain injury, our data suggest that neurotoxicity is mediated through increased glutamate concentrations, rather than by poststroke hyperglycemia. To directly
examine this hypothesis in the setting of diabetes, we looked at
the effect of insulin and glucagon on the outcome of stroke in
animals with basal hyperglycemia. Intraperitoneal injection of
saline containing 5 ␮g of glucagon or 3 U/kg of insulin 10 min
before or after tMCAO improved the outcome of ischemic
stroke in diabetic rats, compared with those given saline alone
(Fig. 4, A–C). Both hormones provided comparable neuroprotection (Fig. 4, A–C) with similar therapeutic windows (Fig. 4,
A and B). The improvement in neurological recovery did not
correlate with glucose levels, which were affected by insulin
and glucagon in opposite directions, i.e., injection of insulin
Fig. 5. The neuroprotective effect of oxaloacetate in diabetic rats. NS or NS
containing 1 mmol oxaloacetate/100 g rat weight (Oxal) was given intravenously to diabetic rats 10, 30, or 60 min after (⫹10= to ⫹60=) the occlusion of
the MCA, as indicated. Twenty-four hours later, the brain infarct sizes were
measured. As indicated, in some groups, 10 min after occlusion of the MCA,
the administration of oxaloacetate was followed by intravenous injection of
glutamate (Glutam). The mean ⫾ SE of data from 7 to 12 animals/group is
shown. *Significant difference from controls receiving only NS (P ⬎ 0.004).
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Fig. 4. The neuroprotective effect of insulin and glucagon in diabetic rats. NS
or NS-containing insulin (3 U/kg) (A) or glucagon (5 ␮g) (B) was given
intraperitoneally to diabetic rats 10 min before (⫺10=) or 10 or 60 min after
(⫹10= or ⫹60=) the occlusion of the MCA, as indicated. Twenty-four hours
later, the brain infarcts were measured (A and B) after determining the
neurological score (C). In some groups, 10 min after occlusion of the MCA, the
administration of insulin or glucagon was followed by intravenous injection of
glutamate (Glutam) or glucose (Glucose) (20%, 0.75 ml). The mean ⫾ SE of
data from 8 –14 animals/group is shown. *Significant difference from controls
receiving only NS: A, *P ⬍ 0.005; B, *P ⫽ 0.009.
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NEUROPROTECTION BY INSULIN AND GLUCAGON IN DIABETES
decreased blood glucose in the diabetic rats from 186 ⫾ 27.9
to 71 ⫾ 7.41 mg/dl (P ⬍ 0.005, Student’s t-test), whereas
glucagon increased blood glucose from 174 ⫾ 22.14 to 236 ⫾
25 mg/dl (P ⫽ 0.009, one-way ANOVA with Newman-Keuls
post hoc test). As in the case of nondiabetic rats (Figs. 1–3),
injection of glutamate abolished the neuroprotective effect of
both hormones (Fig. 4, A and B), whereas elevated glucose
concentrations of diabetic rats treated with insulin did not
attenuate neuroprotection (Fig. 4A).
To further exclude the possibility that the effect of insulin
and glucagon on glucose levels or any other hormone receptor
mediates neuroprotection, we examined the effect of the glutamate scavenger oxaloacetate (47), which has also been
shown to provide neuroprotection in a TBI model in nondiabetic animals. In this case, the action of glutamate-oxaloacetate
transaminase in the circulation, transforms glutamate into 2-ketoglutarate in the presence of oxaloacetate (47). Oxaloacetate
provided neuroprotection in a tMCAO model in diabetic rats
and had the same therapeutic window as insulin and glucagon
(Fig. 5; P ⬎ 0.004 one-way ANOVA with Newman-Keuls post
hoc test), without affecting the blood levels of glucose (data
not shown). As in nondiabetic animals (47), restoration of
plasma glutamate levels abolished the neuroprotective effect of
oxaloacetate (Fig. 5).
Perspectives and Significance
DISCUSSION
REFERENCES
The finding that insulin, glucagon, and oxaloacetate have a
similar neuroprotective effect and share a therapeutic window
that overlaps with the postischemia increase in brain glutamate,
strongly suggests that the neuroprotective effect of the three
agents is due to their capacity to reduce the concentration of
this excitatory amino acid in the CNS, as reflected by the
changes we measured in the CSF.
Our data are in line with those of others showing a close
correlation between glutamate concentrations in the CSF and
plasma (1, 16, 22, 47) and that decreasing plasma glutamate
leads to decreased concentrations in the CSF (1, 22, 47),
notwithstanding the large differences in the actual concentrations in the two compartments.
Furthermore, our results help to explain the observed dichotomy between the beneficial effects of insulin in various animal
models, when given prior to induction of stroke (29, 45) and its
controversial efficacy in humans when given after the insult (8,
23, 31, 35, 37). We posit that the difference is due to the early
and brief increase in glutamate in the brain after stroke; the
corollary of this concept is the very brief therapeutic window
that has to be taken into consideration in any therapeutic
approach.
The fact that glucagon given prior to brain injury is neuroprotective, suggests that gluconeogenesis may contribute to the
preconditioning response to cerebral injury. The beneficial
effect of glucagon or insulin given prior to traumatic brain
injury suggests that they may also be useful before certain
neurosurgical or cardiac interventions in which the incidence
of perioperative stroke approaches 10% (39).
In any case, a better understanding of the correlation between CSF and plasma glutamate concentrations and the mechanism of the generation and transport of neurotoxic products is
needed to optimize the timing, duration and intensity of the
glutamate-reducing effect of either hormone.
1. Abu Fanne R, Nassar T, Mazuz A, Waked W, Heyman N, Goelman G,
Higazi A. Neuroprotection by glucagon: Role of gluconeogenesis. J
Neurosurg 114: 85–91, 2010.
2. Abu Fanne R, Nassar T, Yarovoi S, Rayan A, Lamensdorf I, Karakoveski M, Vadim P, Jammal M, Cines D, Higazi A. Blood-brain barrier
permeability and tPA-mediated neurotoxicity. Neuropharmacology 58:
972–980, 2010.
3. Adams H, Del Zoppo G, Alberts MJ, Bhatt DL, Brass L, Furlan A,
Grubb RL, Higashida RT, Jauch EC, Kidwell C, Lyden PD, Morgenstern LB, Qureshi AI, Rosenwasser RH, Scott PA, Wijdicks EF;
American Heart Association, American Stroke Association Stroke
Council, Clinical Cardiology Council, Cardiovascular Radiology and
Intervention Council, Atherosclerotic Peripheral Vascular Disease
and Quality of Care Outcomes in Research Interdisciplinary Working
Groups. Guidelines for the early management of adults with ischemic
stroke. Stroke 38: 1655–1711, 2007.
4. Armstead WM, Nassar T, Akkawi S, Smith DH, Chen XH, Cines DB,
Higazi A. Neutralizing the neurotoxic effects of exogenous and endogenous tPA. Nat Neurosci 9: 1150 –1155, 2006.
5. Baird TA, Parsons MW, Phanh T, Butcher KS, Desmond PM, Tress
BM, Colman PG, Chambers BR, Davis SM. Persistent poststroke
hyperglycemia is independently associated with infarct expansion and
worse clinical outcome. Stroke 34: 2208 –2214, 2003.
6. Bederson J, Pitts L, Tsuji M, Nishimura M, Davis R, Bartkowski H.
Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17: 472–476, 1986.
7. Bendotti C, Tortarolo M, Suchak KS, Calvaresi N, Carvelli L, Bastone
A, Rizzi M, Rattray M, TM. Transgenic SOD1 G93A mice develop
reduced GLT-1 in spinal cord without alterations in cerebrospinal fluid
glutamate levels. J Neurochem 79: 737–746, 2001.
8. Bilotta F, Caramia R, Cernak I, Paoloni F, Doronzio A, Cuzzone V,
Santoro A, Rosa G. Intensive insulin therapy after severe traumatic brain
injury: a randomized clinical trial. Neurocrit Care 9: 159 –166, 2008.
9. Blanca Fuentes B, Castillo J, San Jose B, Leira R, Serena J, Vivancos
J, Dávalos A, Gil Nuñez A, Egido J, Díez-Tejedor E. The prognostic
value of capillary glucose levels in acute stroke: the GLycemia in Acute
Stroke (GLIAS) study. Stroke 40: 562–568, 2009.
10. Bômont L, MacKenzie E. Neuroprotection after focal cerebral ischaemia
in hyperglycaemic and diabetic rats. Neurosci Lett 197: 53–56, 1995.
11. Brockman RP, Bergman EN, Joo Pk, Ganns JG. Effects of glucagon
and insulin on net hepatic metabolism of glucose precursors in sheep. Am
J Physiol 229: 1344 –1350, 1975.
ACKNOWLEDGMENTS
A. A. Hijazi designed the research, R. Abu Fanne., T. Nassar, and N. Hijazi
performed the research and collected the data. A. A. Higazi, R. Abu Fanne, T.
Nassar, N. Hijazi, and S. N. Heyman analyzed the data. A. A. Higazi wrote the
paper.
GRANTS
This work was supported by Grants HL077760 and HL82545 from the
National Institutes of Health and Grant 930/04 from the Israeli Science
Foundation.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
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AJP-Regul Integr Comp Physiol • VOL
These data reveal a previously undescribed mechanism for
insulin and glucagon as neuroprotective agents in an experimental model of stroke. The lack of association between blood
glucose and neuroprotection mitigates concerns about the deleterious effects of insulin-induced hypoglycemia, which for
many years was the major reason for avoiding its administration in stroke patients with hyperglycemia (3, 28). Nevertheless, the efficacy and safety profile of glucagon stress its
potential as part of the initial management of stroke.
NEUROPROTECTION BY INSULIN AND GLUCAGON IN DIABETES
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29. LeMay D, Gehua L, Zelenock G, D’Alecy L. Insulin administration
protects neurologic function in cerebral ischemia in rats. Stroke 19:
1411–1419, 1988.
30. Luck JM, Morrison G, Wilbur LF. The effect of insulin on the amino
acid continent of blood. JBC: 151–156, 1928.
31. McCormick M, Hadley D, McLean J, Macfarlane J, Condon B, Muir
K. Randomized, controlled trial of insulin for acute poststroke hyperglycemia. Ann Neurol 67: 570 –578, 2010.
32. Nagy D, Marosi M, Kis Z, Farkas T, Rakos G, Vecsei L, Teichberg V,
Toldi J. Oxaloacetate decreases the infarct size and attenuates the reduction in evoked responses after photothrombotic focal ischemia in the rat
cortex. Cell Mol Neurobiol 29: 827–835, 2009.
33. Natale JE, Stante SM, D’Alecy LG. Elevated brain lactate accumulation
and increased neurologic deficit are associated with modest hyperglycemia
in global brain ischemia. Resuscitation 19: 271–289, 1990.
34. Nelson D, Cox M. Gluconeogenesis. In: Lehninger: Principles of Biochemistry, 4th ed., New York: Freeman, chap. 14, 2005, pp. 543–549.
35. Oddo M, Schmidt J, Carrera E, Badjatia N, Connolly E, Presciutti M,
Ostapkovich N, Levine J, Le Roux P, Mayer A. Impact of tight
glycemic control on cerebral glucose metabolism after severe brain injury:
A microdialysis study. Crit Care Med 36: 3233–3238, 2008.
36. Parsons M, Barber P, Desmond P, Baird T, Darby D, Byrnes G, Tress
B, Davis S. Acute hyperglycemia adversely affects stroke outcome: a
magnetic resonance imaging and spectroscopy study. Ann Neurol 58:
20 –28, 2002.
37. Quinn T, Dawson J, Walters M. Sugar and stroke: Cerebrovascular
disease and blood glucose control. Cardiovasc Ther In press.
38. Sandu O, Song K, Cai W, Zheng F, Uribarri J, Vlassara H. Insulin
resistance and type 2 diabetes in high-fat-fed mice are linked to high
glycotoxin intake. Diabetes 54: 2314 –2319, 2005.
39. Selim M. Perioperative stroke. N Engl J Med 356: 706 –713, 2007.
40. Shafrir E. Animal Models of Diabetes, 2nd ed. CRC Press: London, 328
pp.. 2007.
41. Tai E, Tan M, Stevens R, Low Y, Muehlbauer M, Goh D, Ilkayeva R,
Wenner B, Bain J, Lee J, Lim CS, Khoo C, Shah S, Newgard C. Insulin
resistance is associated with a metabolic profile of altered protein metabolism in Chinese and Asian-Indian men. Diabetologia 53: 757–767, 2010.
42. Takagi K, Ginsberg M, D, Globus M, Y, Dietrich W, D, EM, Kraydieh
S, Busto R. Changes in amino acid neurotransmitters and cerebral blood
flow in the ischemic penumbral region following middle cerebral artery
occlusion in the rat: correlation with histopathology. J Cereb Blood Flow
Metab 13: 575–585, 1993.
43. Tejima E, Katayama Y, Suzuki Y, Kano T, EHL. Hemorrhagic transformation after fibrinolysis with tissue plasminogen activator: evaluation
of role of hypertension with rat thromboembolic stroke model. Stroke 32:
1336 –1340, 2001.
44. Toni D, De Michele M, Fiorelli M, Bastianello S, Camerlingo M,
Sacchetti M, Argentino C, Fieschi C. Influence of hyperglycaemia on
infarct size and clinical outcome of acute ischemic stroke patients with
intracranial arterial occlusion. J Neurol Sci 123: 129 –133, 1994.
45. Warner D, Gionet T, Todd M, McAllister A. Insulin-induced normoglycemia improves ischemic outcome in hyperglycemic rats. Stroke 23:
1775–1789, 1992.
46. Wasserman D, Spalding J, Brooks L, Colburn C, Goldstein R, Cherrington A. Glucagon is a primary controller of hepatic glycogenolysis and
gluconeogenesis during muscular work. Am J Physiol Endocrinol Metab
257: E108 –E117, 1989.
47. Zlotnik A, Gurevich B, Tkachov S, Maoz I, Shapira Y, Teichberg VI.
Brain neuroprotection by scavenging blood glutamate. Exp Neurol 203:
213–220, 2007.
301 • SEPTEMBER 2011 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.5 on June 17, 2017
12. Bruno A, Levine S, Frankel M, Brott T, Lin Y, Tilley B, Lyden P,
Broderick J, Kwiatkowski T, Fineberg S. Admission glucose level and
clinical outcomes in the NINDS rt-PA stroke trial. Neurology 59: 669 –
674, 2002.
13. Bullock R, Zauner A, Woodward J, Young H. Massive persistent
release of excitatory amino acids following human occlusive stroke.
Stroke 26: 2187–2189, 1995.
14. Campos F, Sobrino T, Ramos-Cabrer P, Argibay B, Agulla J, PerezMato M, Rodryguez-Gonzalez R, Brea D, Castillo J. Neuroprotection
by glutamate oxaloacetate transaminase in ischemic stroke: an experimental study. J Cereb Blood Flow Metab 26: 1–9, 2011.
15. Capes S, Hunt D, Malmberg K, Pathak P, Gerstein H. Stress hyperglycemia and prognosis of stroke in nondiabetic and diabetic patients: a
systematic overview. Stroke 34: 2426 –2432, 2001.
16. Castillo J, Dávalos A, Noya M. Progression of ischaemic stroke and
excitotoxic aminoacids. Lancet 349: 79 –83, 1997.
17. Chandra S, White R, Everding D, Feuerstein G, Coatney R, Sarkar S,
Barone F. Use of diffusion-weighted MRI and neurological deficit scores
to demonstrate beneficial effects of isradipine in a rat model of focal
ischemia. Pharmacology 58: 292–299, 1999.
18. Chen J, Sanberg PR, Li Y, Wang L, Lu M, Willing AE, SanchezRamos J, Chopp M. Intravenous administration of human umbilical cord
blood reduces behavioral deficits after stroke in rats. Stroke 32: 2682–
2688, 2001.
19. de Courten-Myers G, Myers RE, Schoolfield L. Hyperglycemia enlarges infarct size in cerebrovascular occlusion in cats. Stroke 19: 623–
630, 1988.
20. Durham H, Truett G. Development of insulin resistance and hyperphagia
in Zucker fatty rats. Am J Physiol Regul Integr Comp Physiol 290:
R652–R658, 2006.
21. Fuentes B, Ortega-Casarrubios M, SanJose B, Castillo J, Leira R,
Serena J, Dávalos J, Gil-Nuñz A, Egido J, Díez-Tejedor E. Persistent
hyperglycemia ⬎155 mg/dl in acute ischemic stroke patients: How well
are we correcting it? Implications for outcome. Stroke 41: 2362–2365,
2011.
22. Gottlieb M, Wang Y, Teichberg V. Blood-mediated scavenging of
cerebrospinal fluid glutamate. J Neurochem 87: 119 –126, 2003.
23. Gray CS, Hildreth AJ, Sandercock PA, O’Connell JE, Johnston DE,
Cartlidge NE, Bamford JM, James OF, Alberti KM. Glucose-potassiuminsulin infusions in the management of post-stroke hyperglycaemia: the
UK Glucose Insulin in Stroke Trial (GIST-UK). Lancet Neurol 6: 397–
406, 2007.
24. Gray CS, Hildreth AJ, Sandercock PA, O’Connell JE, Johnston DE,
Cartlidge NE, Bamford JM, James OF, Alberti KG. Glucose-potassiuminsulin infusions in the management of post-stroke hyperglycaemia: the
UK Glucose Insulin in Stroke Trial (GIST-UK). Lancet Neurol 6: 397–
406, 2007.
25. Haratz S, Tanne D. Diabetes, hyperglycemia and the management of
cerebrovascular disease. Curr Opin Neurol 24: 81–88, 2011.
26. Kaarisalo M, Raiha I, Sivenius J, Immonen-Raiha P, Lehtonen A,
Sarti C, Mahonen M, Torppa J, Tuomilehto J, Salomaa V. Diabetes
worsens the outcome of acute ischemic stroke. Diabetes Res Clin Pract
69: 293–298, 2005.
27. Kernan W, Inzucchi S. Type 2 diabetes mellitus and insulin resistance:
Stroke prevention and management. Curr Treat Options Neurol 6: 443–
450, 2004.
28. Lees KR, Walters MR. Acute stroke and diabetes. Cerebrovasc Dis 20
Suppl 1: 9 –14, 2005.
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