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J. Neurochem. JNC-E-2006-0589.R1 Grünblatt et al.
ORIGINAL ARTICLE
Brain insulin system dysfunction in streptozotocin intracerebroventricularly treated rats
generates hyperphosphorylated tau protein
Grünblatt Edna1, Salkovic-Petrisic Melita2, Jelena Osmanovic2, Riederer Peter1, Hoyer
Siegfried3
1 Institute of Clinical Neurochemistry and National Parkinson Foundation Centre of Excellence
Laboratory, Clinic for Psychiatry and Psychotherapy, Bayrische Julius-Maximilian-University of
Würzburg, Würzburg, Germany.
2 Department of Pharmacology and Croatian Institute for Brain Research, Medical School,
University of Zagreb, Zagreb, Croatia
3 Department of Pathology, University of Heidelberg, Heidelberg, Germany.
Running title: Alzheimer’s disease is an insulin resistant brain state
Keywords: Alzheimer’s disease, Brain, Gene expression, GLUT2, Insulin, Insulin receptor,
Learning / Memory, Protein tyrosine kinase, Streptozotocin, Tau protein.
Word No. Abstract: 200 Word No. Text: 6628 No. Figures: 4
No. Tables: 5 Suppl. Material: 0
Submission date: Friday, 19 May 2023
All correspondence to Dr. Edna Grünblatt:
Bayerische Julius-Maximilians- University Würzburg
Clinic and Policlinic for Psychiatry and Psychotherapy / Neurochemistry Laboratory
Füchsleinstr. 15
D-97080 Würzburg / Germany
Tel: +49-931-20177300
Fax: +49-931-20177220
E-mail: [email protected]
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Abstract
The intracerebroventricular (icv) application of streptozotocin (STZ) in low dosage was used in 3-
month-old rats to explore brain insulin system dysfunction. Three months following STZ-icv
treatment, the expression of insulin-1 and -2 mRNA was significantly reduced to 11% in hippocampus
and to 28% in frontoparietal cerebral cortex, respectively. Insulin receptor (IR) mRNA expression
decreased significantly in frontoparietal cerebral cortex and hippocampus (16% and 33% of control).
At the protein/activity level, different abnormalities of protein tyrosine kinase activity (increase in
hippocampus), total IR -subunit (decrease in hypothalamus), and phosphorylated IR tyrosine residues
(increase) became apparent. The STZ-induced disturbance in learning and memory capacities was not
abolished by icv application of glucose transport inhibitors known to prevent STZ induced diabetes
mellitus. The discrepancy between reduced IR gene expression and increase in both phosphorylated IR
tyrosine residues / protein tyrosine kinase activity may indicate imbalance between
phosphorylation/dephosphorylation of the IR -subunit causing its dysfunction. These abnormalities
may point to a complex brain insulin system dysfunction after STZ-icv application which may lead to
an increase in hyperphosphorylated tau-protein concentration. Brain insulin system dysfunction is
discussed as possible pathological core in the generation of hyperphosphorylated tau protein as a
morphological marker of sporadic Alzheimer’s disease.
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Introduction
Substantial evidence has been gathered in support of the presence of both insulin and insulin
receptors in the brain. The main source of brain insulin is pancreatic beta cells. Insulin is
known to cross the blood brain barrier (BBB) by a saturation transport mechanism. The
transporter is unevenly distributed throughout the brain, with the olfactory bulb having the
fastest transport rate of any brain region (Banks 2004), demonstrating, in addition, regional
differences in transport kinetics. A smaller proportion of insulin is produced in the brain itself
(Plata-Salaman 1991). Insulin gene expression and insulin synthesis have been demonstrated
in both immature and mature mammalian neuronal cells (Schechter et al. 1992; Schechter et
al. 1996; Schechter and Abboud 2001). Insulin mRNA was found to be distributed in a highly
specific pattern with the highest density in pyramidal cells of the hippocampus and high
densities in medial prefrontal cortex, the entorhinal and perihinal cortices, the thalamus and
the granule layer of the olfactory bulb, as well as in hypothalamus. Neither insulin mRNA nor
synthesis of the hormone were observed in glial cells (Devaskar et al. 1994). The release of
insulin from brain synaptosomes is stimulated by glucose (Santos et al. 1999).
It has been demonstrated that insulin receptors are dispersed throughout the brain and also
follow a highly specific pattern with the highest density detected in olfactory bulb,
hypothalamus, cerebral cortex and hippocampus (van Houten et al. 1979; Unger et al. 1989).
Nerve terminals show enriched densities of insulin receptors (van Houten et al. 1980; Abbott
et al. 1999) which bind insulin in a highly specific and rapid manner (Raizada et al. 1988).
Two different types of insulin receptors have been found in the adult mammalian brain: a
peripheral type on glial cells (-subunit 130kDa, -subunit 95 kDa) which is down-regulated
by insulin, and a neuron-specific brain type with high concentration on neurons (-subunit
118kDa, -subunit 91 kDa) which is not down-regulated by insulin (Adamo et al. 1989). The
location of phosphotyrosine-containing proteins corresponds to the distribution of the insulin
receptor (Moss et al. 1990). Insulin receptor mRNA is abundantly present in neuronal somata
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(Schwartz et al. 1992) but the protein shows the highest density in the synaptic neuropil
(Baskin et al. 1994). Besides these brain-related data, detailed effects of insulin on its
peripheral receptor in adipocytes and muscle cells were summarized in several review articles,
as presented below. Binding of insulin to the extracellular -subunit of its receptor induces
autophosphorylation of the intracellular -subunit by phosphorylation of the intrinsic tyrosine
residues 1158, 1162 and 1163 for activation (Combettes-Souverain and Issad 1998). The
receptor’s activation state is regulated by its phosphorylation state. Deactivation may be
induced by the action of both phosphotryosine phosphatase causing dephosphorylation
(Goldstein 1993) and by serine/threonine kinases causing phosphorylation at serine residues
1305 and 1306, and threonine residue 1348 (Häring 1991; Avruch 1998). Interestingly, the
activity of protein tyrosine phosphatase was found to be regulated by insulin (Kenner et al.
1993). Thus, as a general phenomenon insulin receptor signaling dysfunction may be caused
when tyrosine phosphorylation, and/or when tyrosine dephosphorylation fails, and/or when
serine/threonine phosphorylation is increased and maintained at a higher level.
Evidence has been provided that the neuronal glucose metabolism is pre-eminent for
neuronal/brain function. Under normal conditions, this metabolic pathway is the only source
of ATP, and contributes to the formation of acetylcholine, cholesterol and neurosteroids via
the metabolite acetyl CoA, and to UDP-N-glucosamine via the metabolite fructose-6-
phosphate. It may be assumed that the neuronal glucose metabolism is under control of the
neuronal insulin signal transduction system (for review (Hoyer and Frölich 2006). Through
this mediate the insulin signal may effect on neurotrophic and neuromodulatory functions,
synaptic plasticity, and learning and memory capacities (Zhao et al. 1999; Park et al. 2000).
Predominant abnormalities in cerebral glucose metabolism and its control by the neuronal
insulin signal transduction system have been found in sporadic Alzheimer’s disease (SAD)
(Hoyer et al. 1991; Frölich et al. 1998; Hoyer 2002, 2004), putting forward the hypothesis that
SAD is the brain type of diabetes mellitus II (Hoyer 1998). A mismatch of both the insulin
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action and IR function itself, including downstream signaling pathways has been proposed to
be involved in brain insulin system dysfunction in SAD (Salkovic-Petrisic and Lackovic
2005).
Considering the presence of insulin (originating from both periphery and brain) and insulin
receptors in the brain, an experimental rat model was developed by using streptozotocin
(STZ) to induce the brain insulin system dysfunction. In general, STZ is a drug selectively
toxic for insulin producing/secreting cells, as following systemically application STZ enters
the cells via the glucose GLUT2 transporter, mainly localized in pancreatic -cells (to a
certain extent also in hepatocytes and absorptive epithelial cells of the intestine and kidney).
Coupled with glucokinase, GLUT2 participates in a glucose-sensing mechanism in -cells
important for insulin production/secretion. STZ exerts betacytotoxic effects mostly by causing
alkylation of DNA which triggers activation of poly ADP-ribosylation consequently leading
to depletion of cellular NAD+ and ATP, and finally to a permanent diabetes mellitus when
applied in a higher dosage (Szkudelski 2001). In contrast, in moderate to low dosage and in
short-term experiments, STZ caused insulin resistance (Blondel and Portha 1989) by a
decreased autophosphorylation of the insulin receptor (Kadowaki et al. 1984), increased total
insulin receptor number but with little change in phosphorylated IR- subunit one (Giorgino
et al. 1992), and maintained insulin-immunoreactive cells in the pancreas generating a
transient diabetes mellitus (Rajab et al. 1989; Ar'Rajab and Ahren 1993). In this study STZ
was applied intracerebroventricularly (icv) in a dosage up to 100 times lower (per kg b.w.)
than used for systemic application. STZ icv did not cause a systemic diabetes mellitus (Nitsch
and Hoyer 1991; Duelli et al. 1994; Lannert and Hoyer 1998). The pancreatic STZ-transporter
GLUT2 has also been found in mammalian brain (Brant et al. 1993; Leloup et al. 1994;
Ngarmukos et al. 2001). GLUT2 is regionally specifically distributed throughout the rat brain,
especially in the limbic areas and related nuclei, most concentrated in the ventral and medial
regions close to the midline (Arluison et al. 2004b; Arluison et al. 2004a). Localization of
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GLUT2 labeling is more numerous in the vicinity of nerve terminals and/or dendrites or
dendritic spines forming synaptic contacts, which together with neuronal localization
relatively similar to that of glucokinase, support the idea that GLUT2 may be expressed by
some cerebral neurons possibly involved in glucose sensing (Arluison et al. 2004b; Arluison
et al. 2004a). However, other studies have demonstrated that GLUT2 mRNA distribution in
the adult rat brain is not entirely parallel to that of glucokinase at the quantitative level; being
lower, similar or higher than glucokinase in different brain regions, respectively (Li et al.
2003). This may suggest different roles of particular regions in terms of glucose sensing, but
also participation of brain GLUT2 in functions other than glucose sensing, like regulation of
neurotransmitter release and perhaps, in the release of glucose by glial cells (Arluison et al.
2004b; Arluison et al. 2004a). However, direct evidence of STZ entering into particular brain
cells through GLUT2 is still lacking.
After STZ icv administration, severe abnormalities in brain glucose/energy metabolism have
been found; glucose utilization was reduced in 17 of 35 brain areas (Duelli et al. 1994), the
activities of glycolytic key enzymes decreased markedly (Plaschke and Hoyer 1993) finally
causing diminished concentrations of the energy-rich compounds ATP and creatine phosphate
(Nitsch and Hoyer 1991; Lannert and Hoyer 1998). Both energy deficit and reduced activity
of choline acetyltransferase (cholinergic deafferentiation) (Hellweg et al. 1992; Blokland and
Jolles 1993) may form the biological basis for the marked reduction in learning and memory
capacities (Lannert and Hoyer 1998). Furthermore, a direct histopathological evidence of
specific neurotoxic damage caused by STZ icv administration to axons and myelin in the
fornix, anterior hippocampus and periventricular structures that are essential for learning and
spatial memory, have been reported (Shoham et al. 2003; Weinstock and Shoham 2004).
Regarding the brain IR signaling, a recent investigation focusing on the downstream
phosphatidylinositol-3 (PI3)-kinase signaling pathway showed abnormalities of Akt/protein
kinase B level and of both phosphorylated and non-phosphorylated glycogen synthase kinase-
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3/ protein 1 month and 3 months after STZ icv administration (Salkovic-Petrisic et al.
2006). In the latter study, an increase in total tau-protein in the brain and amyloid formation in
leptomeningeal vessels were found.
The data available so far point to different aspects: 1. STZ icv administration causes severe
abnormalities in metabolic pathways being under control of the insulin/insulin receptor
signaling cascade in the rat brain. 2. Observed changes seem to demonstrate great similarities
to cellular and intracellular abnormalities found in the SAD brain. 3. The mechanism of STZ
icv action is not known, however it could be hypothesized that in general it is similar to the
mechanism of STZ peripheral action. Therefore, we were interested in studying abnormalities
of the brain insulin signal transduction cascade at the gene expression and protein
expression/activity levels, which may be induced by STZ icv application. The abnormalities
found served for tentative comparison with changes characteristic for SAD. Using learning
and memory capacities as sensitive markers, the interaction of glucose transporter and STZ
for elucidating possible mechanism of STZ icv action was studied in an additional approach.
Material and Methods
Material
Streptozotocin was purchased from Sigma-Aldrich (Munich, Germany). Protein Tyrosine
Kinase Activity assay kit was purchased from Chemicon International (Hampshire, United
Kingdom; Cat. No. SGT410). For buffer preparations EGTA (Cat. No. E4378), Tris buffer,
Na3VO4 (Cat. No. S6508), EDTA 0.5 M (Cat. No. E7889), Protease Inhibitor mix (Cat. No.
P8340), Sodium Deoxychlorate (Cat. No. D6750), BSA, β-mercaptoethanol (Cat. No. M7154)
and the Insulin Receptor Subunit ELISA (Cat. No. CS00090) were purchased from Sigma-
Aldrich (Munich, Germany). DTT (Cat. No. 43817) was purchased from Fluka (Germany). 5-
thio-D-glucose and 3-0 methyl glucose were purchased from Sigma-Aldrich Chemie (Munich,
Germany). The polyclonal rabbit anti-human tau (K9JA) antibody (recognizes total tau at C-
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terminal part, amino acids 243-441) and monoclonal PHF-1 anti-tau antibody (recognizes tau
phoshorylated at serine S-396 and S-404) were received as a gift from Dr. E-M Mandelkow
(Max-Planck-Gesellschaft, Hamburg, Germany), although the first commercially originated
from DAKOCytomation (Glostrup, Denmark) and the latter was from Dr. Peter Davies
(Albert Einstein College of Medicine, Bronx, New York). Anti-rabbit IgG, HRP-linked
antibody and anti-mouse IgG, HRP-linked antibody were purchased from Cell Signalling
(Beverly, MA, USA). Chemiluminiscent Western blot detection kit was from Amersham
Biosciences (Freiburg, Germany). Gels were from Novex (San Diego, CA, USA), and
nitrocellulose membranes were from Invitrogen (Invitrogen GmbH, Karlsruhe, Germany).
Animals
Three to four-month old male Wistar rats weighing 280-330 g (Department of Pharmacology,
School of Medicine, University of Zagreb) were used throughout the studies. Animals were
kept on standardized food pellets and water ad libitum.
Drug treatments
Rats were randomly divided into groups (5-6 per group) and given general anaesthesia
(chloralhydrate 300 mg/kg, ip), followed by injection of different drugs administered
bilaterally into the left and right lateral ventricle according to the procedure described by
Noble et al. (Noble et al. 1967). Drug concentration and solution volume was adjusted
according to the animal body weight, and a volume of 4 μL per 300 g body weight was
administered (2 μL/ventricle). Control animals received bilaterally an equal volume of vehicle
into the lateral ventricles.
For the IR and tau protein analyses, rats were treated icv with a single STZ injection (1
mg/kg, dissolved in 0.05M citrate buffer pH 4.5). STZ-treated and respective control animals
were sacrificed 3 months following the drug treatment.
In the behavioural experiment, rats were randomly divided in 5 groups and the following drug
icv treatments were applied:
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group I: 0.05M citrate buffer pH 4.5, applied 3 times, on day 1, 3 and 21 (control group,
CTRL)
group II: STZ 1 mg/kg, dissolved in 0.05M citrate buffer pH 4.5; applied once - on day 1, and
citrate buffer applied icv on day 3 and 21 (STZ 1x)
group III: STZ 1 mg/kg, dissolved in 0.05M citrate buffer pH 4.5; applied 3 times – on day 1,
3 and 21 (STZ 3x)
group IV: 3-0-methyl-glucose (30MG) 1 mg/kg followed by STZ 1 mg/kg, both dissolved in
0.05M citrate buffer pH 4.5; applied 3 times by separate consecutive injections – on day 1, 3
and 21 (STZ 3x +30MG 3x)
group V: 5-thio-D-glucose (TG) 375 μg/kg followed by STZ 1 mg/kg, both dissolved in
0.05M citrate buffer pH 4.5; applied 3 times by separate consecutive injections – on day 1, 3
and 21 (STZ 3x + TG 3x)
For the IR and tau protein analyses, brains were quickly removed, and cut into left and right
half. Frontoparietal cortex (IR analysis), hippocampus (IR and tau analyses) and
hypothalamus (IR analysis) were cut out from the brain, immediately frozen and stored at -80
oC. The proportions of the right half of the brain were homogenized as described further.
STZ-icv-treated animals had no symptoms of systemic diabetes and steady-state blood
glucose level did not differ in comparison with control animals.
Young-adult animals were chosen for the study to evaluate also the effect of compensation
after damage. Compensation may be assumed to be facilitated in the young-adult brain (Hoyer
1985). Otherwise, a long-term maintenance of the brain damage starting early in life may
point to abnormalities characteristic of chronic disorders developing later in life (Holness et
al. 2000)
Quantitative real-time RT-PCR
Total-RNA extraction
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Isolation of total RNA was performed using RNeasy Mini Kit (Qiagen GmbH, Germany) for
each animal and brain region separately. An additional step was added to the original protocol
in order to receive a pure DNA free total-RNA. The total RNA was on column pre-treated
with DNase-I and the original protocol was then continued in order to have total RNA
extraction. The RNA quality and quantity was assessed using the Experion electrophoresis
(BioRad Laboratories, Hercules, CA, USA) which analyses the concentration of the total
RNA and quality via the ratio of the 28S / 18S ribosomal RNA. Only intact total RNA
samples (with at least 28S/18S ration of 1.7) were used for the gene expression analysis.
Q-PCR
In order to measure the gene expression profile of insulin-1, insulin-2, and insulin-receptor,
quantitative real-time RT-PCR for mRNA samples isolated from rats' frontal cortex,
hippocampus and hypothalamus were performed. Total RNA (1-0.4 mg) from each sample
was reverse transcribed with random hexamer and oligo-dT primers using iScriptTM cDNA
Synthesis Kit (BioRad Laboratories, Hercules, CA, USA, Cat. No. 170-8890). The genes
measured are listed in Table 1. These were normalized to the house-keeping genes: beta-actin
(ActX), ribosomal 18S (Rnr1), and glyceraldehydes-3-phosphate dehydrogenase (GAPDH)
(Table 1). The house keeping genes were tested for their stability using the geNorm program
(http://medgen.ugent.be/~jvdesomp/genorm/) (Vandesompele et al. 2002). After analysis for
the most stable house-keeping genes, a normalization factor was calculated according to the
program. Absence of DNA contamination was verified by amplifying the house-keeping gene,
ribosomal 18S, and run on gel to observe no product. Minus RT samples were tested
simultaneously with experimental samples by quantitative RT-PCR in order to see whether
the reaction did not yield any amplification below 35 cycles using the PCR protocol. Real-
time PCR was performed in the iCycler iQ system (BioRad Co., Hercules, CA, USA) as
described previously (Svaren et al. 2000; Ugozzoli et al. 2002). Briefly, 30-100 ng of cDNA
and gene specific primers (200nM final concentration) and probes (500nM final
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concentration; when the assay consist probes) (indicated in Table 1) were added to either
QuantiTect SYBR Green PCR Kit (Qiagen Inc., Valencia, CA, USA, Cat. No. 204143) or to
iQ Supermix (BioRad Co., Hercules, CA, USA, Cat. No. 170-8862; in case of the probe
assays). Real-time PCR were subjected to PCR amplification (in general 1 cycle at 950 C for
15 min, 30-45 cycles at 940 C for 15 s, annealing and detecting of specific fluorescent colour
at 550C for 30 s and extension at 760C for 30 s). All PCR reactions were run in triplicate. The
amplified transcripts were quantified using the comparative CT method analyzed with the
BioRad iCycler iQ system program. Standard curves for each amplification product were
generated from 10-fold dilutions of pooled cDNA amplicons, isolated from agarose gel using
MinEluteTM Gel Extraction Kit (Qiagen Inc., Valencia, CA, USA), to determine primer
efficiency and quantization.
Total Insulin-receptor (IR-) subunit & the phosphorylated forms ELISA
Homogenates preparation
Fresh tissue was homogenized one to four volumes of a pre-chilled lysis buffer containing
50mM Tris, pH 8.0, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 1% NP-40, 0.5% Sodium
Deoxychlorate, 0.1% SDS protease inhibitors cocktail and 100 µM Sodium Vanadate. The
homogenate were centrifuged at 12,000 x g for 10 minutes at 40C to pellet the fraction. The
supernatant was than stored at -700C till further analysis in aliquots.
Total protein concentration determination
The amount of protein was assayed according to the method of Bradford (Bradford 1976)
(Sigma, Cat. No. B6916). For calibration curve BSA standard was used (Sigma, Munich,
Germany). The concentration was determined by measurement of the absorbance at 595 nm.
ELISA
The measurement of the phosphorylated and non-phosphorylated IR-β subunit was conducted
according to the kit manual (Sigma, Cat. No. CS0090 for IR-β subunit; Cat. No. PI0100 for
Phospho-IR-β subunit pTyr1158; Cat. No. PI0200 for Phospho-IR-β subunit pTyr1162/1163).
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Homogenates were diluted with buffer diluents one to ten volumes. Standard curve analysis
was run in parallel on each plate. The absorbance was measured in the Multiskan Spectrum
spectrophotometer at OD 450 nm (Thermo Labsystems, Finland). Further analyses of the
results were performed on a PC Excel windows program.
Protein Tyrosine kinase activity assay
The activity of the IR protein Tyrosine Kinase (PTK) was conducted according to the kit
manual (Chemicon, Cat. No. SGT410). Tyrosine Kinase Activity Assay Kit measures the total
activity of the protein tyrosine kinase residues on the insulin receptor. The buffer assay used
in this PTK activity was as recommended in the manual from Upstate Ltd (Dundee, UK,
Kinase Profiler Assay Protocols). The assay buffer contained 50mM Tris, pH 7.5, 0.1 mM
EGTA, 0.1 mM Na3VO4, 0.1% β-mercaptoethanol and 1mg/ml BSA. The homogenate was
used in a one volume to 5.7 volumes of total reaction mix for the reaction. Standard curve
analysis was run in parallel on each plate. The absorbance was measured in the Multiskan
Spectrum spectrophotometer at OD 450 nm (Thermo Labsystems, Finland). Further analyses
of the results were performed on a PC Excel windows program.
Western blot
Tissue preparation.
Hippocampal (1 animal = 1 sample) tissue samples from the left half of the rat brain were
homogenized with 3 volumes of lysis buffer containing 10 mM HEPES, 1 mM EDTA, 100
mM KCl, and 1% Triton X-100, pH 7.5, and protease inhibitors coctail (1:100), and the
homogenates were centrifuged at 600xg for 10 min. The supernatants were further centrifuged
at 45 000 xg for 30 min at 4 oC, and the pellets were resuspended in 100 μL of the lysis buffer.
Finally, the resuspended pellets were mixed with appropriate previous supernatants obtained
after second centrifugation. Protein concentration was measured by Bradford Protein assay.
Samples were frozen and stored at -80 oC.
Immunoblotting.
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Equal amounts of total protein (50 μg per sample) were separated by SDS-PAGE using 8%
polyacrylamide gels and transferred to nitrocellulose membranes (Schneppenheim et al.
1991). The nitrocellulose membranes were blocked by incubation in 5% non-fat milk added to
low salt washing buffer (LSWB) containing 10 mM Tris and 150 mM NaCl, pH 7.5, and
0.5% Tween 20, one hour at room temperature. Blocked blots were incubated overnight at 4
oC with respective primary anti-total tau (1: 10 000), and anti PHF-1-tau antibodies (1:300).
Following the incubation, the membranes were washed three times with LSWB and incubated
for 60 min at room temperature with secondary antibody solution (anti-rabbit IgG, 1:2000,
and anti-mouse IgG, 1:500). The specificity of the signal was checked on the control
membranes that were not incubated with the primary antibody. After washing three times in
LSWB, the membranes were immunostained using chemiluminiscence Western blotting
detection reagents, signal captured and visualised with the Chemi Doc BioRad (UK) video
camera system.
Morris Water Maze Swimming Test
Cognitive functions were tested in Morris Water Maze Swimming Test (Anger 1991) twice, 2
weeks and 2 months after the first drug icv injection. Adaptation of rats to the experimental
environment and behavioural activity was done during two days before the experimental
trials. On the first of these two days animals were subjected to 1 min of freely swimming in a
pool (150x60 cm, 50 cm deep), with water temperature set at 25±1 oC, and on the second day
rats were allowed to freely swim in the pool divided in four quadrants (I-IV). In the
experimental trials, performed from day 1 to day 4, rats were thought to escape from water by
finding a hidden rigid platform submerged about 2 cm below the water surface in quadrant
IV. Stay on the platform was allowed for 15 sec. One trial consisted of three starts, each from
a different quadrant (I – III), separated by a 1-min rest period. Three consecutive trials were
performed per day, separated by a 30-min rest period. After the third trial on day 4, the fourth
trial was performed (starts from quadrants I-III) with a platform being removed from the pool,
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and the time spent in searching for the platform after entering quadrant IV was recorded. The
cut off time was 1 min. Those rats who had no alterations in memory functions (control) were
supposed to remember that the platform had previously been there, and, in line with that, to
spend a long time swimming within quadrant IV, looking for the platform. In case of drug-
induced deterioration of memory functions, rats were supposed to remember less intensively
that the platform had been in quadrant IV, and thus to spend less time in searching for the
platform within this quadrant, in comparison with control rats.
Statistics
The differences of gene expression in the different brain regions were compared using the
one-way- analysis of variance (ANOVA) with Post-Hoc Test of Scheffe, using the StatView
computer program (Stat View 5.0. software, SAS Institute Inc. Cary, NC, USA) on a PC
computer. Statistically significant differences were accepted at p<0.05.
Densitometric values of proteins obtained in Western blot analysis were expressed as
percentage of the control group on the respective gel (mean ± SE), and the significance of
between-group difference was tested by Mann-Whitney U-test. P<0.05 was considered
significant. In the Morris Water Maze Swimming Test, values were expressed as total time
(seconds) spent in searching for the hidden platform in quadrant IV during the three
consecutive starts from quadrant I-III in the last experimental trial. Mean values with a
standard error and standard deviation range were presented. The significance of between-
group differences was tested by Kruskal-Wallis ANOVA median test, followed by Mann-
Whitney U-test, and p<0.05 considered statistically significant for both tests.
Ethics
The experiments carried out on 3 to 4-month-old rats in Zagreb, Croatia, were under the
guidance of the Principles of Laboratory Animal care (NIH Publication No. 86-23, revised in
1985), according to the Croatian Act on Animal Welfare (NN19; 1999) and were approved by
The Ethics Committee of the Zagreb University School of Medicine (No. 04-1343-2006).
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Results
IR analyses
Control rats. In Table 2A, the normalized gene expression profiles of the insulin receptor,
insulin-1 and insulin-2 mRNA and their ratio under control conditions in frontoparietal brain
cortex, hippocampus and hypothalamus are demonstrated. The expression profiles of the IR
gene in frontoparietal brain cortex and hippocampus are in the same range, which seem to be
higher as compared to the hypothalamus. However, statistically significant difference was not
found. Both the expression of the insulin-1 and insulin-2 genes was highest in hippocampus,
followed by frontoparietal brain cortex and hypothalamus. In the latter, insulin-1 gene
expression was significantly decreased as compared to the hippocampus. The ratio of the
expression of the insulin-1 and -2 genes was highest in hippocampus, and nearly in the same
range in frontoparietal brain cortex and hypothalamus.
Table 2B shows the concentration of the total IR-β subunit, the concentrations of
phosphorylated tyrosine residues 1158 (pTyr1158) and 1162/1163 (pTyr1162/1163) per IR-
subunit, the activity of protein tyrosine kinase (PTK) and the ratio of PTK and the two
phosphorylated tyrosine residues. The concentration of the IR-β subunit was highest in
frontoparietal brain cortex (p<0.05) compared to hippocampus and hypothalamus. The
concentration of pTyr1158 per IR- subunit was highest in hippocampus as compared to
frontoparietal brain cortex (p<0.05) and to hypothalamus. The concentration of
pTyr1162/1163 per IR- subunit was found to be less concentrated in both frontoparietal
brain cortex and hippocampus compared to hypothalamus. Both residues may be
phosphorylated to the same degree in all brain areas studied. The highest activity of PTK was
found in frontoparietal brain cortex as compared to hippocampus and hypothalamus. The PTK
activity per phosphorylated tyrosine residue was found to be in the same range in each brain
region studied. However, regional differences became obvious with significantly higher ratio
in frontoparietal brain cortex and lower ratio in hippocampus and hypothalamus (p<0.0005).
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STZ-icv treated rats. The effect of STZ icv on the gene expression profiles of insulin receptor,
insulin-1 and insulin-2 mRNA in frontoparietal brain cortex, hippocampus and hypothalamus
compared to controls is demonstrated in Figure 1. In both frontoparietal brain cortex and
hippocampus a statistically significant insulin receptor mRNA down-regulation was observed
(16% and 33% of control, respectively). In principle, the expression of both insulin-1 and -2
mRNAs demonstrated a trend of down-regulation after STZ icv treatment. A significant down
regulation of insulin-1 mRNA (20% of control) was observed in the hippocampus (p<0.05).
Insulin-2 mRNA expression was significantly down-regulated to 11% of control in the
frontoparietal brain cortex (p<0.05). The ratio of insulin-1 and insulin-2 expression has not
been found statistically significantly changed in STZ icv treated rats either in comparison to
the control group or between the three regions.
The protein content of the functionally important IR- subunit dropped to ~67% (of control)
in the frontoparietal brain cortex, increased in the hippocampus (169% of control) and
decreased in the hypothalamus (74% of control) after STZ icv treatment (Figure 2). STZ icv
caused a significant increase in the concentrations of both pTyr1158 and pTyr1162/1163 in
frontoparietal brain cortex, and in hypothalamus (pTyr1162/1163 only) as compared to
control. The activity of the PTK was unchanged in both frontoparietal brain cortex and
hypothalamus after STZ injection, but increased significantly nearly 3-fold in hippocampus
(Figure 2) (p<0.005). Regional difference was found in the ratio of PTK activity per
pTyr1158 with statistically significant increase (60%) demonstrated in hippocampus and no
change found in the other two regions. Ratio of PTK activity per pTyr1162/1163 was not
significantly different between the regions (Figure 2).
Tau protein
The immunoreaction of total tau (recognizing total tau protein at C-terminal part, amino acids
243-441) and p-tau (recognizing tau phoshorylated at serine S-396 and S-404, ie. GSK-3
related tau phosphorylation) increased significantly (177% and 90%, respectively, P<0.05) in
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J. Neurochem. JNC-E-2006-0589.R1 Grünblatt et al.
the hippocampus of STZ icv treated rats, in comparison to the control animals, as measured 3
months following the drug treatment (Figure 3 A-C).
Learning and memory function
Morris Water Maze Swimming Test discriminated rats that had no alterations in learning and
memory functions from those who have. The former ones remembered where the platform
had previously been, and in line with that, spent a long time swimming within the appropriate
quadrant looking for the platform. Rats with reduction of these functions, remembered less
intensively where the platform had previously been, and in line with that spent less time in
searching for the removed platform, in comparison with control rats. STZ 1x1 mg/kg and 3x1
mg/kg icv treated rats spent significantly less time in search for the removed platform, in
comparison to the control group, found as early as 2 weeks following the first drug treatment
(75.8 sec and 76.85 sec vs. 86.4 sec, respectively, P<0.05)(Figure 4). These STZ icv induced
cognitive deficits were persistent as found also 2 months following the first drug treatment
(66.7 sec and 57.6 sec vs. 110.2 sec, respectively, P<0.05) (Figure 4).
Interestingly, icv pre-administration of glucose transport inhibitors, 30MG and TG followed
by STZ icv administration, also induced significant cognitive deficits in comparison to the
control rats, found as early as 2 weeks after the first drug treatment (50.2 sec and 48.0 sec vs.
86.4 sec, respectively, P<0.05), and persistent as long as 2 months after the first drug
treatment (47.5 sec and 62.0 sec vs. 110.25 sec, respectively, P<0.05) (Figure 4). Therefore,
glucose transport inhibitor pre-treatment did not abolished STZ icv-induced cognitive deficits
(Figure 4).
Discussion
The normal brain
The present study suggests a dispersed gene expression of the IR and the two insulins
throughout the brain thus supporting former findings (Unger et al. 1989; Devaskar et al.
17
J. Neurochem. JNC-E-2006-0589.R1 Grünblatt et al.
1994). The expression of IR mRNA showed a tendency to be higher in frontoparietal brain
cortex and in hippocampus than in hypothalamus. Rats and mice possess two insulin genes (1
and 2) both of which are localized to chromosome 1 (Todd et al. 1985). It is noteworthy that
the expression of both insulin-1 and insulin-2 was found to be relatively high-level in the
hippocampus followed by frontoparietal brain cortex, and by lower values in hypothalamus,
in accordance with literature data. It has been hypothesized that different distribution patterns
of insulin-1 and IR in the brain may suggest that IR in different brain regions may use insulin
from different sources, peripheral versus locally synthesized, for cell-to-cell communication
and neuronal signal transduction (Zhao et al. 2004). Interestingly, in post-mortem aged
control subjects, insulin gene expression was also highest in the hippocampus (Steen et al.
2005).
In this first in vivo-study performed at the protein level, a striking dissociation between IR-β
subunit and total IR mRNA was found among the brain areas studied reported earlier also by
Zhao and colleagues (Zhao et al. 1999). This finding is difficult to understand, and further
studies are needed to clarify whether or not the translational efficiency and/or the IR protein
stability is region-dependent e.g. that the turnover of the complete IR protein may be
regionally different i.e. higher in the metabolically particularly active hippocampus relative to
other brain regions. That differences of receptor concentration and protein turnover are
regionally different has, in principle, been demonstrated between different brain areas in favor
of the hippocampus (Stecher et al. 1997; Zhao et al. 1999).
STZ action in the brain
After systemic application, STZ was found to cause DNA breaks obviously in a dose-
dependent manner (Yamamoto et al. 1981; Kröncke et al. 1995) through intracellular
mechanisms, and after being transported into the cell by the GLUT2-transporter detailed by
Szkudelski (Szkudelski 2001). It is, however, not known as yet whether or not comparable
mechanisms take place in the brain after STZ icv administration, too. As a sensitive marker of
18
J. Neurochem. JNC-E-2006-0589.R1 Grünblatt et al.
STZ action in the brain, the development of long-lasting disturbances in learning and memory
capacities has been repeatedly shown as a consequence of diverse abnormalities in neuronal
glucose metabolism (Mayer et al. 1990; Lannert and Hoyer 1998; Salkovic-Petrisic et al.
2006). In particular, the cholinergic transmission as the basis for learning and memory
capacities has been demonstrated to be severely disturbed inducing gene expression of choline
acetyltransferase (Lester-Coll et al. 2006) the activity of this enzyme (Blokland and Jolles
1993; Ishrat et al. 2006) and its function (Hellweg et al. 1992).
These data clearly indicate that the perturbed function of the neuronal insulin receptor is of
pivotal significance for the development of deficits in learning and memory capacities. This
assumption seems to be in contrast to insulin receptor knockout experiments in which no
alterations in basal brain glucose utilization and learning and memory capacities were found
(Schubert et al. 2004). However, it may have to be considered that the expression of the
insulin-like growth factor-1 (IGF-1) receptor remained unaltered in the latter study indicating
that an important compensation mechanism is intact. IGF-1 is an insulin homologue
abundantly found in the developing brain (Rotwein et al. 1988). The IGF-1 receptor is widely
distributed in the brain (Adamo et al. 1989; Marks et al. 1991b; Marks et al. 1991a). The IGF-
1 receptor and the insulin receptor are also found to be homologous with nearly identical
signal-transducing domains controlling most of the same intracellular pathways (De Meyts et
al. 1994). IGF-1 peaks during postnatal development only and is barely detected in normal
adult brain (Rotwein et al. 1988; Cheng et al. 2000). It is, however, highly induced in glia
cells reacting to damage (Lee et al. 1993). After STZ icv administration, IGF-1 receptor gene
expression was found to be reduced (Grünblatt et al. 2004).
To study the mode of action of STZ icv to brain cells, two different drugs, 5-thio-D-glucose
3-0-methyl glucose, well-known as glucose transport inhibitors, were administered icv. In line
with the fact of STZ entering beta cells by GLUT2, systemic administration of these drugs
19
J. Neurochem. JNC-E-2006-0589.R1 Grünblatt et al.
immediately before STZ has been reported to prevent STZ-induced beta-cell toxicity and
diabetes (Ganda et al. 1976; Wang et al. 1993). Analogously, we have hypothesized that
glucose transport inhibitor icv pre-treatment may avoid STZ transport into the particular brain
cells, and learning and memory capacities served as a sensitive marker of STZ-induced
damage. Surprisingly, icv pre-treatment with two different glucose transport inhibitors did not
abolish the STZ-induced effect on mental capacities which was found to be reduced with STZ
icv treatment alone (Fig. 4), suggesting some differences between the peripheral and central
(icv) mechanism(s) of STZ cytotoxicity. This hypothesis has been further supported by the
fact that although GLUT2 has been demonstrated to be highly expressed in the hypothalamus
what would expect severe damage after STZ icv in this area, the STZ-induced damage on
gene expression in this study was found to be lowest in hypothalamus relative to
frontoparietal brain cortex and hippocampus (Fig. 1 and 2). Since the direct evidence of STZ
either entering the particular brain cells via GLUT2 or its CNS cytotoxicity being strictly
dependent on GLUT2 type of glucose transporter expression (as reported for the pancreatic
islet toxicity of STZ; (Hosokawa et al. 2001) is lacking, interpretation of glucose transport
inhibitor pre-treatment failure in abolishing STZ icv induced effects could be only a
speculative one. Theoretically, 3OMG and TG may also inhibit glucose uptake in all brain
cells expressing any type of glucose transporters leading to extensive intracellular glucose
deprivation. Such condition could be manifested as cognitive deficits, masking thus 3OMG
and TG pre-treatment induced abolishing of STZ icv associated cognitive deficits, if such
abolishment happened. However, there is no information from the literature that these two
substances inhibit all glucose transporter types.
Regionally specific character of brain neurochemical alterations following the STZ icv
administration has been convincingly reported at various levels; IR signaling (Salkovic-
Petrisic et al. 2006), glucose utilization (Duelli et al. 1994), monoaminergic transmission
(Salkovic et al. 1995; Salkovic-Petrisic and Lackovic 2003), suggesting they are not a
20
J. Neurochem. JNC-E-2006-0589.R1 Grünblatt et al.
consequence of a general, non-specific toxicity of STZ. This is further supported by
regionally specific (and partly overlapping) distribution of structures that are known or
assumed to be its target (GLUT2 and IR).
Otherwise, STZ icv has been shown to cause broad effects on metabolic pathways being
under control of the neuronal insulin receptor allowing detailed studies on the
pathophysiology of the insulin/insulin receptor-dependent metabolism (see introduction).
Recent findings clearly demonstrated that STZ icv application caused the down regulation of
gene expression related to insulin signalling such as IGF-1 receptor. In contrast to this down
regulation, an up regulation was found in gene expression related to potassium channels,
GABA receptors and glutamate receptors (Grünblatt et al. 2004). Genes such as GDNF,
BDNF and integrin-alpha-M were up regulated whereas immediate-early-gene-transcription-
factor NGF-IB and methallothionein-1/2 were found to be down-regulated demonstrating that
transcription and inflammatory factors, apoptosis inducers, cell survival and oxidative stress-
involving proteins are altered (Grünblatt et al. 2006). This view is supported by the activation
of the markers of oxidative cell damage (Ishrat et al. 2006). As a consequence of the STZ icv-
induced cascade of genetic and metabolic abnormalities, neuronal damage and loss may
develop accompanied by an astrocytic rescue reaction (Lester-Coll et al. 2006).
This study shows a marked dissociation between severely reduced IR mRNA in frontoparietal
brain cortex and hippocampus, and nearly unchanged concentrations of IR-β subunit in these
areas. Likewise, unchanged or even elevated phosphorylation of the IR tyrosine residues and
PTK activity became obvious. These unexpected findings may point to imbalances between
the generation of proteins and their turnover and between protein phosphorylation and
dephosphorylation under pathological conditions. Receptor proteins localized postsynaptically
may undergo changes of synaptic plasticity, i.e. change from a labile to a stable or even
“regressed” state, the latter being nearly unable to transmit impulses (Changeux and Danchin
1976). It is not clear as yet in which way and to what degree this process influences the
21
J. Neurochem. JNC-E-2006-0589.R1 Grünblatt et al.
turnover of the IR protein. There is also some evidence that failing receptor
dephosphorylation may inhibit autophosphorylation activity (Lai et al. 1989), and that, with
time, tyrosine-phosphorylated receptors become inaccessible to phosphatases, therefore
allowing persistence of tyrosine kinase activity (Paolini et al. 1996).
Indeed, the activity of the protein tyrosine phosphatase decreased after long-term STZ-
damage (Meyerovitch et al. 1989), and induced a drastic reduction of IR dephosphorylation
(Begum et al. 1991). Thus, the diminished activity of the protein tyrosine phosphatase may be
assumed to reduce dephosphorylation of the tyrosine residues of the IR- subunit and may
render it insufficient in function by inhibition of autophosphorylation, whereby regional
differences may come into play in frontoparietal brain cortex and hippocampus. Alternatively,
since a significant increase in brain insulin receptor kinase activity has been found in diabetic
rat brain, it has been hypothesized that brain insulin receptor kinase may be regulated
differently compared to peripheral tissue (Gupta et al. 1992). However, increased protein
tyrosine kinase activity does not necessarily lead to increased IR signal transduction
throughout the cell, if intracellular downstream pathway signaling elements are affected, as
found after STZ icv treatment in PI3 kinase pathway, at the level of Akt/PKB and GSK-3
(Salkovic-Petrisic et al. 2006). Further detailed studies are needed to clarify this issue e.g. the
release of the subunit of the IR from the cellular membrane into the cytosol and the
determination of protein phosphatases activity after STZ.
Bearing in mind that STZ icv treated rats have been proposed as a probable experimental
model of SAD, one could hypothesized that induction of disturbances in the brain insulin
system including insulin receptor signalling may be assumed to be the core of abnormalities
tightly related to SAD, i.e. intracellular fibrillary tangles with hyperphosphorylated tau
protein and extracellular deposits of neuritic plaques with -amyloids (A).
In a recent investigation, both the over production of tau-protein and the accumulation of A
in leptomeningal vessels could be demonstrated after STZ icv (Lester-Coll et al. 2006). In this
22
J. Neurochem. JNC-E-2006-0589.R1 Grünblatt et al.
study we showed, in addition, that tau-protein is hyperphosphrylated as a long-term
consequence of STZ icv administration (Fig. 3). The intracerebral application of STZ in a
higher dosage than used in our study caused neuronal damage and cell loss as well as the
accumulation of A in the brain of pups (Lester-Coll et al. 2006). Bearing in mind the former
data comprising abnormalities in glucose/energy metabolism, acetylcholine formation and
function, insulin signal transduction and learning and memory capacities, it seems likely that
induction of the brain insulin system dysfunction may be relevant for the beginning of SAD.
Further research is needed to clarify this hypothesis.
Acknowledgment
The authors thank the funding that has been provided by the “Alzheimer Forschung Initiative
e.V”, DAAD (project No. A/04/20017), the Dr. Edda Neele Stiftung, and the Croatian
Ministry of Science, Education and Sport (project No. 0108253). Prof. Dr. E-M Mandelkow is
thanked for tau protein antibody donation. Special thank to Miss Miryame Hofmann for her
efficient and exact work in providing these great results. Mrs Bozica Hrzan is thanked for
technical assistance.
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Figure Captions:
Figure 1: Normalized gene expression of IR, insulin-1 & -2 mRNA profiles and the ratio
between insulin-1 to -2 in STZ treated rats in cortex, hippocampus and hypothalamus
compared to controls. One-way ANOVA with Post-Hoc Test of Scheffee; a, p<0.005 vs.
control, b, p<0.05 vs. control.
Figure 2: IR concentration, IR pTyr1158, IR pTyr1162/1163, protein tyrosine kinase
activity, and ratio with pTyr1158 and with pTyr1162/1163 in STZ treated rats in cortex,
hippocampus and hypothalamus compared to control. One-way ANOVA with Post-Hoc
Test of Scheffee; a, p<0.005 vs. control, b, p<0.05 vs. control.
Figure 3: Western blot analysis of total tau visualized by K9JA antibody recognizing C-
terminal part of tau protein (A) and phospho (p) tau visualized by PHF-1 anti-tau antibody
recognizing tau phoshorylated at serine S-396 and S-404 (B) protein in the hippocampus
of rats intracerebroventricularly (icv) treated with streptozotocin (1 mg/kg) 3 months after
drug treatment, and respective statistics (C). CTRL, control group (lanes 1-5); STZ,
streptozotocin icv-treated group (lanes 6-11). *p<0.05 by Mann Whitney U test.
Figure 4: Memory function in Morris Water Maze Swimming Test of rats
intracerebroventricularly (icv) treated with streptozotocin and glucose transporter GLUT2
blockers, 5-thio-D-glucose, and 3-0-methyl glucose. Deficits were measured as the time
spent in search for the removed platform after entering the quadrant where the platform
had been placed in training trials. The better the memory had been preserved, the longer
the rats were searching for the platform, and vice versa. Values are expressed as a mean
with a standard error and standard deviation range. CTRL, control group; STZ1x,
streptozotocin single injection (1 mg/kg, icv) ; STZ3x, streptozotocin triple injection (3x 1
mg/kg, icv); 30MG, 3-0-methyl glucose triple injection (3x 1mg/kg, icv); TG, 5-thio-D-
glucose triple injection (3x 375 μg/kg, icv). *p<0.05 vs. control by Kruskal-Wallis
ANOVA median test followed by Mann Whitney U test.
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J. Neurochem. JNC-E-2006-0589.R1 Grünblatt et al.
Table 1: Quantitative PCR conditions and kits
Gene Gene
Symbo
l
Accession
No.
Firm Kit
description
Probe Primer sequence
(Forwar+Revese)
Produc
t side
(bp)
Annealing
Temperatu
r (0C)
Insulin 1 Ins1 NM_01912
9
Qiagen
QuantiTect
Primer
Assay Cat.
No.
QT0037330
3
--- n.a. 114 55
Insulin 2 Ins2 NM_01913
0
Qiagen
QuantiTect
Primer
Assay Cat.
No.
QT0017738
0
--- n.a. 116 55
Insulin
receptor
IR NM_01707
1
Qiagen
QuantiTect
Primer
Assay Cat.
No.
QT0019896
8
--- n.a. 92 55
Ribosomal
18S
Rnr1 M11188 Qiagen
QuantiTect
Primer
Assay Cat.
No.
QT0019937
4
--- n.a. 103 55
glyceraldehyde
-3-phosphate
dehydrogenase
Gapd NM_01700
8
No kit
(TaqMan
Probe)
atgcccccatgtttgtgatgggtgt Tcaccaccatggagaaggc
gctaagcagttggtggtgca
178 62
Actin beta Actx NM_03114
4
No kit
(TaqMan
tgtccctgtatgcctctggtcgtacc
ac
Agccatgtacgtagccatcc
a
81 65
35
J. Neurochem. JNC-E-2006-0589.R1 Grünblatt et al.
probe) tctccggagtccatcacaatg
Not available, n.a.
36
J. Neurochem. JNC-E-2006-0589.R1 Grünblatt et al.
Table 2: Normalized gene expression profiles (A) and PTK activity and IR-β subunit protein under control conditions in different brain areas.
A) gene Cortex (Mean±SEM , n) Hippocampus (Mean±SEM, n) Hypothalamus (Mean±SEM, n)
IR (F=1.55) 0.321±0.048 (3) 0.370±0.067 (4) 0.166±0.099 (6)
Insulin-1 (F=5.7) 0.980±0.529 (4) 2.618±0.784 (4) 0.246±0.164 a (5)
Insulin-2 (F=2.5) 0.264±0.079 (3) 0.457±0.201 (4) 0.084±0.050 (5)
Insulin-1/insulin-2 3.7±6.69 5.7±3.90 2.9±3.28
B) Protein
IR (ng/ng total protein)
F=4.95.154±0.744 a (5) 2.268±0.764 (5) 3.997±0.386 (6)
IR pTyr1158 (ng/ng IRβ)
(F=7.9)1.138±0.154 a (5) 1.932±0.146 (5) 1.499±0.110 (7)
IR pTyr1162/1163 (ng/ng IRβ)
(F=14.3)1.150±0.101 b (5) 1.215±0.117 b (5) 1.758±0.049 (6)
Protein Tyrosine kinase activity
(ng/µg total protein/30 min)
(F=14.1)
3.009±0.508 a,b (5) 1.312±0.387 (5) 0.603±0.096 (7)
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J. Neurochem. JNC-E-2006-0589.R1 Grünblatt et al.
PTK/IR pTyr1158 (ng/ng
pTyr1158/30 min) (x103)
(F=34.7)
25.9±1.4 a,b (5) 16.1±2.9 b (5) 5.7±0.9 (7)
PTK/IR ptyr1162/1163 (ng/ng
pTyr1162/1163/30 min) (x103)
(F=23.07)
25.9±3.6 b (5) 25.5±3.6 b (5) 4.7±0.9 (7)
One-way ANOVA with Post-Hoc Scheffee-Test: a, p<0.05 vs. hippocampus; b, p<0.05 vs. hypothalamus
2