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Chapter 6
Neurotrophic support, GH/IGF-1 and HPA axes vis-à-vis decreased longevity
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6.1 Introduction
6.2 Materials and methods
6.3 Results
6.4 Discussion
6.5 Summary
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6.1 Introduction
Neurotrophic factors (NTFs) are well known for their roles in supporting and
sustaining development, differentiation, maintenance and plasticity of various brain
regions and their functions throughout life. Any loss in NTF regulation and function
increase the risk of the nervous system for cognitive breakdown, degeneration and
different neuronal malfunctions. There exists a delicate balance between the trophic
support of various neurotrophic factors, which are programmed temporally as well as
spatially. Of these, brain-derived neurotrophic factor (BDNF) is well researched for
its role in neuronal maintenance, survival and growth of new neurons and synapses
(Huang and Reichardt 2001). Pituitary gland has a critical role in controlling many of
the physiological processes like growth, body composition, reproduction, stress-
adaptive responses, balance of sodium and water, lactation, thyroid function and many
others (Veldhuis 2013). Particularly, the anterior pituitary secretes growth hormone
(GH), thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH),
follicle-stimulating hormone (FSH), luteinizing hormone (LH) and prolactin, the
hormones involved in a plethora of monitoring and regulatory functions (Veldhuis, et
al. 2013, Hohl, Ronsoni et al. 2014, Otto, Franca et al. 2014, Nicolaides, Kyratzi et al.
2015).
Insulin-like growth factor 1 (IGF-1) has anabolic effects in adults and has a
molecular structure similar to insulin. It is produced mainly as an endocrine hormone
in liver and acts in a paracrine or autocrine way in the target tissues after being
stimulated by GH. IGF-1 production is decreased by undernutrition, lack of GH
receptors, GH insensitivity or problems in downstream signaling pathways post GH
receptor including SHP2 and STAT5B. GH secretion has been shown to decreased in
old rats (Sonntag, Steger et al. 1980) as compared to the young ones. Insulin/IGF-1
like receptor pathway contributes significantly to the biologic ageing process.
Insulin/IGF-1-like signaling is conserved from worms like C. elegans to humans.
Indeed, in vitro experiments have shown that mutations (Kenyon, Chang et al. 1993)
reducing insulin/IGF-1 signalling extend life (Bartke 2011) by decelerating the
degenerative, ageing process. GH/IGF-1 axis is in fact, known to play an important
role in modulating the ageing process across species (Junnila, List et al. 2013).
BDNF helps neurons in their survival, growth and differentiation of new
neurons and synapses (Acheson, Conover et al. 1995). It is found in high
120
concentrations in hippocampus, cerebral cortex and basal forebrain and is reported to
be involved in learning and memory (Yamada and Nabeshima 2003, Bekinschtein, et
al. 2008). Its levels show significant changes with ageing (Greising, Ermilov et al.
2014) and lack of BDNF has been reported to underlie / to be associated with various
neurodegenerative outcomes and depression (Calabrese, Guidotti et al. 2013, Dalby,
Elfving et al. 2013, Dwivedi 2013). BDNF is also known to regulate brain energy
metabolism and cardiovascular health (Rothman, Griffioen et al. 2012, van Praag,
Fleshner et al. 2014). Hence a study of BDNF in CNS and periphery is very important
in evaluating its role, if any, in obesity and ageing.
Any disruption of the homeostasis through physical or psychological stressors
is defined as stress. The body tries to manage the situation through various
mechanisms and one of them is the neuroendocrine (NE) system, which plays a
critical role in responding to stress. The NE system includes hypothalamus, pituitary
and adrenal glands. On being activated by corticotropin-releasing hormone and
arginine-vasopressin, pituitary gland secretes ACTH that cascades in to the secretion
of cortisol / corticosterone and other glucocorticoids from adrenal glands. The
corticoids released as a stress response, work in close collaboration and terminate the
stress situation through a negative feedback loop to the hypothalamus. ACTH is an
important component of the Hypothalamo-Pituitary-Adrenal (HPA) axis and is often
produced in response to biological stress (Papadimitriou and Priftis 2009, Nicolaides,
Kyratzi et al. 2015). Hypothalamus also releases corticotropin-releasing hormone
simultaneously. ACTH regulates the levels of the steroid hormone cortisol /
corticosterone, which is released from the adrenal glands.
Melatonin is one of the universal / most fundamental hormone in the
evolution of cellular functions. It is identified primarily due to its antioxidant activity
(Hardeland 2005) and the protection it gives from the Sun’s radiation (Hardeland,
Pandi-Perumal et al. 2006, Tan, Zheng et al. 2014). It also acts to control sleep and its
deficiency has been implicated in sleep disorders (Chang, Wu et al. 2009). In small
animals, it is involved in energy metabolism and body weight control. Indeed studies
have shown that chronic melatonin supplementation reduces abdominal fat and body
weight (Wolden-Hanson, Mitton et al. 2000) and hence it is proposed as an approach
to treat obesity, basically due to its ability to regulate brown adipose tissue
metabolism (Tan, Manchester et al. 2011). There is also support for its anti-ageing
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effects (Brown, Young et al. 1979, Touitou 2001) and it has been proposed as a
biomarker for the intrinsic process of brain ageing (Sharma, Palacios-Bois et al.
1989). Melatonin also restores the basal concentrations of pituitary hormones and
pituitary responsiveness to the levels observed in young rats (Diaz, Pazo et al. 2000).
Hence quantification of ACTH, melatonin and corticosterone in the WNIN/Ob obese
rat provides a platform to study the basal stress response and systemic inflammation
and their probable role in the development of various pathologies including decreased
longevity.
6.2 Materials and methods
6.2.1 Maintenance of animals
The animal feeding and maintenance protocol used for the studies is same as
that reported in chapter 2 of the thesis. Therefore, only the deviations if any from it
are given herewith. Three groups of rats (n=5-7) were taken for the study i.e. parental
WNIN control (WN), WNIN/Ob obese (OO) and WNIN/Ob lean littermates (OL) of
3, 12 and 15 months of age.
6.2.2 Blood drawing
Blood was drawn from the ophthalmic venous plexus (orbital sinus) using a
thin walled heparinised capillary tube. The animal was held on to a platform, and the
skin on the head was stretched a little for proper positioning of the eye. The capillary
tube was positioned at the inner corner of the eye (beside the eye ball); by gentle and
firm push the fragile ophthalmic venous plexus was ruptured. By capillary action the
blood entered the capillary tube and the blood flow was started by slight rotation of
the capillary tube (Donovan and Brown 2006). Approximately 1.5 mL of blood
(Diehl, Hull et al. 2001) was collected in pre-coated EDTA vacuettes (Shanghai
International Holding Corp. GmbH (Europe), Hamburg, Germany). The capillary tube
was removed and the pressure was released from the animal.
6.2.3 Collection of plasma samples
The anticoagulant EDTA containing tubes were used for collecting blood for
plasma preparation. After collecting blood, the tubes were gently mixed for
homogeneity and kept on ice in an Esky box. The tubes were centrifuged (Eppendorf
Centrifuge 5810R; Swinging-bucket rotor A-4-62, Eppendorf AG, Hamburg,
Germany) at 2,230 rpm (or 1000x g) for 12 minutes at 4 0C. The supernatant plasma
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from the EDTA tubes was carefully collected without disturbing the lower layers and
stored as aliquots at -20 0C for 4 hours and then at -80 0C till they were further
analyzed.
6.2.4 Collection of cerebrospinal Fluid (CSF) samples
CSF concentrations of growth factors, various metabolites and many other
compounds are used as a surrogate measure of their CNS availability. CSF
concentration is logically, a good perceptive marker of biological availability to the
CNS for hydrophilic or large molecular weight compounds with poor to moderate
permeability (Shen, Artru et al. 2004). Hence, in order to check the bioavailability of
BDNF to CNS, we collected CSF from the cisterna magna by the procedure described
below. According to a report (Consiglio and Lucion 2000), cisterna magna can be
used to tap the largest volume (i.e. 50 – 120 µl) of CSF from the adult rat CNS.
6.2.4.1 Construction of collection syringe
The plastic part of the 24G needle (Hindustan Syringes & Medical Devices
Ltd., Faridabad, India) was cut and the stainless steel needle was taken. PE-50 tubing
(of internal diameter 0.023” and outer diameter 0.038”) (BPE-T50; Harvard
Apparatus Inc. (Holliston, USA) of 30 cm length was used to cover most part of the
stainless steel needle, excepting the first 5 mm portion so that the needle does not
penetrate deep during the procedure of CSF collection. 1 tuberculin mL syringe
(Hindustan Syringes & Medical Devices Ltd., Faridabad, India) was then attached to
the other end to develop the suction pressure.
6.2.4.2 Anesthetizing rats and collecting CSF
The rat was placed in an induction chamber and anesthetized using 5 %
halothane. Checking the eye reflex using a wet cotton bud did confirmation of the
complete anaesthetization of the animal. After complete loss of the consciousness, the
animal underwent shaving of fur on the back of neck region using electric shaver and
scrubbed with 70 % ethyl alcohol for disinfection. During the procedure, a mask was
kept near the nostrils of the rat and the halothane concentration was maintained at ≈ 2
– 3 %. The anesthetized rat was secured with the ear bars in the stereotaxic frame
(51600; Stoelting Co., Wood Dale, IL, USA) while keeping a 450 angle between head
and body axes. On the back of neck, a depressible surface was felt between occipital
protuberance and the atlas spine using the fingertip. After disinfecting with 70 % ethyl
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alcohol the needle of the collecting syringe was inserted horizontally and medially at
the cisterna magna up to the limit of cessation of resistance. Then the plunger of the
syringe was withdrawn to allow the flow of CSF in the PE-50 tubing under a
moderate to slow suction pressure. CSF sample was colorless and the blood
contamination was closely monitored. In case of blood contamination, the PE tubing
was cut little above the area of contamination and rest of the CSF was aspirated in to
the syringe. The collected CSF was rapidly transferred in to a polypropylene
microfuge tubes and stored at -80 0C until used for various assays.
6.2.5 Estimation of IGF-1, GH, ACTH, Melatonin, Corticosterone in plasma and
BDNF in plasma, CSF and various brain parts
IGF-1 was estimated in plasma using Milliplex MAP Rat / Mouse IGF-1 kit
(Item # RMIGF187K, Millipore Corporation, MA, USA), whereas GH, ACTH and
BDNF were estimated using Milliplex MAP Rat Pituitary kit (Item # RPT86K).
Melatonin and Corticosterone were determined using Milliplex MAP Rat Stress
Hormone kit (Item # RSH69K).
Different kits used for these estimations follow the same principle and
procedure and hence to avoid repetitions, details are given only for Rat Pituitary
Hormones analysis.
Principle:
BioPlex assays are bead based multiplex capture sandwich immunoassays in
which an antibody to the target protein is covalently bound to internally dyed beads
known as microspheres. The microspheres are internally colour-coded with two
fluorescent dyes. Through precise concentrations of these dyes, 100 distinctly
coloured beads are created, each of which is coated with a specific capture antibody.
The analyte in the sample binds to the specific capture antibody on the coated bead; a
biotinylated antibody detects the bound analyte. The above reaction mixture is then
incubated with Streptavidin-Phycoerythrin conjugate, the reporter molecule to
complete the reaction on the surface of each microsphere. These microspheres are
allowed to pass rapidly through a laser that excites the internal dyes marking the
microsphere set and a second laser excites the Phycoerythrin, the fluorescent dye on
the reporter molecule. High-speed digital-signal processors identify each individual
microsphere and quantify the result of its bioassay based on fluorescent reporter
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signals. Multiple conjugated beads (multiplex) are added to each sample to obtain
multiple results at a time.
Materials and reagents:
11. Rat Pituitary Standard (LRPT-8086) – reconstituted with 250 µl of deionized
water to get a concentration of 10,000 pg/mL (for BDNF, ACTH) and 50,000
pg/mL (for GH).
• Preparation of Working standard – 50 µl of reconstituted standard was
made up to 200 µl with assay buffer to prepare 2,000 pg/mL, etc.
concentration of working standard (Standard-5). Serial dilutions of the
working standard were made by taking 50 µl of previous standard and adding
it to 200 µl of assay buffer to get next dilution.
12. Rat Pituitary quality controls 1 and 2 (LRPT-6086) – reconstituted with 250 µl
of deionized water.
13. Assay Buffer (LE-ABGLP).
14. Serum matrix (LRPT-SM) – reconstituted with 2 mL of deionized water first
and then made up to 6 mL of final volume by adding 4 mL of assay buffer.
15. Rat Pituitary Detection Antibodies (LRPT-1086).
16. Streptavidin – Phycoerythrin (L-SAPE).
17. Bead Diluent.
18. 10x wash Buffer (L-WB) – diluted to 1x with deionized water.
19. Set of a 96-well filter plate with few sealers.
20. Mixing bottle.
21. Anti-BDNF beads, Anti-GH beads and Anti-ACTH Beads. Each individual
antibody-bead vial was sonicated for 25 seconds and vortexed for 1 minute.
150 µl of each antibody-bead was added to the mixing bottle and the final
volume was made up to 3 mL with bead diluent and vortexed again to prepare
the antibody-immobilized beads.
22. Luminex Sheath Fluid (#40-50000).
Procedure:
18. All the reagents were warmed to room temperature (20-25 0C) before use
in the assay.
19. 96-well filter plate was placed on a plate holder at all times to avoid the
bottom of the plate touching any surface.
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20. To prewet the filter plate, 200 µl of assay buffer was added to the filter
plate, sealed and mixed on a plate shaker for 10 minutes at room
temperature.
21. After the incubation, the assay buffer was removed by vacuum and the
excess assay buffer from the bottom of the plate was removed by blotting
on a tissue paper.
22. 25 µl of each of the standard, control and assay buffer (used as background
/ Standard 0) was added to the appropriate wells on the filter plate.
23. 25 µl of assay buffer was added to the sample wells.
24. 25 µl of serum matrix was added to the background, standards, and control
wells.
25. 25 µl of the sample (diluted 1:3 with serum matrix) was added to the
sample wells.
26. The mixing bottle containing the bead mix was vortexed and 25 µl of the
bead mix was added to each well, sealed with a plate sealer, covered with
the lid and incubated with agitation on a plate shaker overnight (16-18
hours) at 4 0C.
27. After overnight incubation, the fluid was gently removed by vacuum.
28. The filter plates were washed 3 times by adding 200 µl of wash buffer /
well. The wash buffer was removed by vacuum filtration between each
wash and the excess buffer from the bottom of the plate was removed by
blotting on a tissue paper.
29. 50 µl of detection antibody was added to each well, and then the filter
plate was sealed, covered with the lid and kept for incubation with
agitation on a plate shaker for 30 minutes at room temperature.
30. After incubation, 50 µl of Streptavidin – Phycoerythrin was added to each
well and then the filter plate was sealed, covered with the lid and kept for
incubation with agitation on a plate shaker for 30 minutes at room
temperature.
31. The contents were gently removed by vacuum.
32. The filter plates were washed 3 times by adding 200 µl of wash buffer /
well. The wash buffer was removed by vacuum filtration between each
wash and the excess buffer from the bottom of the plate was removed by
blotting on a tissue paper.
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33. 100 µl of Sheath fluid was added to each well and the beads were re-
suspended by agitation on a plate shaker for 5 minutes.
34. The filter plate with the re-suspended beads was run on a Luminex 200
system with BioPlex Manager, version 4.1, build 431, Bio-Rad
Laboratories, Inc., CA, USA.
Calculations:
The Median Fluorescent Intensity data using a weighted 5-parameter logistic
curve fitting method was used for calculating the analyte concentrations in samples.
One derivation of the 5PL equation is expressed below as an example:
where:
a = estimated response at zero concentration
b = slope factor
c = mid-range concentration (C50)
d = estimated response at infinite concentration
g = asymmetry factor
Statistical analyses
The data were analysed by Graphpad Prism 6.0f for Mac OS X (Graphpad
Software Inc., San Diego, CA, USA). Significance of difference among the 3 groups
of rats for each parameter studied at 3 age points was analysed by two-way analysis of
variance (ANOVA) followed by post hoc Tukey’s multiple comparison tests
appropriately. A p-value <0.05 was considered significant for the f ratio obtained by
two-way ANOVA and the t values obtained in the post hoc Tukey’s multiple
comparison tests between different groups.
y = d + [(a – d) / {1 + (x/c)b}g]
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6.3 Results
6.3.1 IGF-1 and GH axis
The plasma IGF-1 level (Table 6.1) in WNIN/Ob obese rats (OO) was
significantly higher than that in parental WNIN (WN) and WNIN/Ob lean littermates
(OL) at 3 and 15 months of age but not at 12 months. There were no such changes in
the normal rats across the ages. The pooled value representing the overall levels of an
animal group also was significantly higher (P<0.0001) in the OO than the two control
groups (WNIN and WNIN/Ob lean). On the other hand plasma levels of GH (Table
6.2) were significantly lower (P<0.0001) than that in both the controls groups. In line
with available literature the control rats were observed to show significant decreased
levels as an ageing effect.
Table 6.1 IGF-1 levels (ng/mL) in plasma of WNIN (WN), WNIN/Ob lean (OL)
and WNIN/Ob obese (OO) at 3, 12 and 15 months of age.
Age
Group 3 m 12 m 15 m Pooled Value
WN 266±9.4α 274±8.2 306±12.8α 282±12
OL 256±9.9aα 278±8.5ab 320±17.4bα 284±19
OO 360±16.6β 325±11.5 383±16.6β 356±17****
Across groups F(2, 54) = 32, P <0.0001
Across time points F(2, 54) = 11, P <0.0001
Interaction F(4, 54) = 1.5, P = 0.221
Data analysed using two-way ANOVA and represented as mean±S.E.M. The differences were
considered significant at minimum of P<0.05. The superscripts a, b and c represent the significant
differences (ageing changes) in an animal group across a row; α, β and γ represent the significant
differences among groups at a given age point, across a column. The pooled values (mean±S.E.M.)
represent the overall plasma level of IGF-1 in an animal group irrespective of age. Significant changes
among the groups for the pooled values are shown with asterisks (****P<0.0001).
128
Table 6.2 GH levels (pg/mL) in plasma of WNIN (WN), WNIN/Ob lean (OL) and
WNIN/Ob obese (OO) at 3, 12 and 15 months of age.
Age
Group 3 m 12 m 15 m Pooled Value
WN 6196±234aα 4357±284bα 3546±198bα 4700±784
OL 5882±453aα 3751±164bα 2679±195cα 4104±941
OO 832±165β 578±91β 368±79β 592±134****
Across groups F(2, 54) = 273, P <0.0001
Across time points F(2, 54) = 64, P <0.0001
Interaction F(4, 54) = 10, P <0.0001
Data analysed using two-way ANOVA and represented as mean±S.E.M. The differences were
considered significant at minimum of P<0.05. The superscripts a, b and c represent the significant
differences (ageing changes) in an animal group across a row; α, β and γ represent the significant
differences among groups at a given age point, across a column. The pooled values (mean±S.E.M.)
represent the overall plasma level of GH in an animal group irrespective of age. Significant changes
among the groups for the pooled values are shown with asterisks (****P<0.0001).
6.3.2 BDNF as the neurotrophic support in central and peripheral systems
Plasma BDNF levels decreased (Table 6.3) with increasing age in normal rats.
Interestingly, plasma BDNF levels were significantly lower in young OO rats than
both the control groups and the lower BDNF levels continued at all age points
studied. However the decrease in CSF BDNF levels (Table 6.4) was only significant
at 15 months age in all the animal groups as compared to the levels at 3 months of
age. Also there were no changes in BDNF levels in CSF across the groups as such.
Still the pooled value for OO showed a significant decrease as compared to both of
the control groups.
129
Table 6.3 BDNF levels (pg/mL) in plasma of different groups of animals, WNIN
(WN), WNIN/Ob lean (OL) and WNIN/Ob obese (OO) at the age of 3, 12 and 15
months.
Age
Group 3 m 12 m 15 m Pooled Value
WN 2610±155aα 1984±219aα 1647±162bα 2080±282
OL 2478±132aα 1844±158abα 1596±167bα 1973±263
OO 495±75β 597±93β 556.6±42β 550±30****
Across groups F(2, 54) = 107, P <0.0001
Across time points F(2, 54) = 13, P <0.0001
Interaction F(4, 54) = 4.3, P = 0.005
Data analysed using two-way ANOVA and represented as mean±S.E.M. The differences were
considered significant at minimum of P<0.05. The superscripts a, b and c represent the significant
differences (ageing changes) in an animal group across a row; α, β and γ represent the significant
differences among groups at a given age point, across a column. The pooled values (mean±S.E.M.)
represent the overall plasma level of BDNF in an animal group irrespective of age. Significant changes
among the groups for the pooled values are shown with asterisks (****P<0.0001).
130
Table 6.4 BDNF levels (pg/mL) in CSF of different groups of animals, WNIN
(WN), WNIN/Ob lean (OL) and WNIN/Ob obese (OO) at the age of 3, 12 and 15
months.
Age
Group 3 m 12 m 15 m Pooled Value
WN 50.5±3.18a 40.7±3.51ab 32.5±1.92b 41.24±5.200
OL 48.8±2.85a 38.6±3.89ab 35.3±2.17b 40.91±4.063
OO 44.7±3.05a 33.5±2.39ab 24.5±1.93b 34.22±5.834***
Across groups F(2, 54) = 5.8, P = 0.005
Across time points F(2, 54) = 28, P <0.0001
Interaction F(4, 54) = 0.44, P = 0.78
Data analysed using two-way ANOVA and represented as mean±S.E.M. The differences were
considered significant at minimum of P<0.05. The superscripts a, b and c represent the significant
differences (ageing changes) in an animal group across a row. The pooled values (mean±S.E.M.)
represent the overall CSF level of BDNF in an animal group irrespective of age. Significant changes
among the groups for the pooled values are shown with asterisks (***P<0.001).
131
The BDNF levels in cerebral cortex (Table 6.5) were not different across
various groups but showed a trend of decreasing levels during ageing, albeit the
pooled BDNF levels in cerebral cortex of OO were significantly lower (P<0.001) than
those in the control groups. In hippocampus (Table 6.6) the BDNF levels were
significantly lower at the young age in OO than the controls and so were the pooled
values (P<0.0001). Similar to cerebral cortex, there was a slight decrease in BDNF
levels in the hippocampus during ageing in all the groups. In hypothalamus (Table
6.7), BDNF levels were very high as compared to other brain regions (e.g. cerebral
cortex and hippocampus). Here again, the levels were significantly lower in OO at the
young age compared that in age-matched controls and a similar change was seen in
the pooled BDNF levels (P<0.0001).
Table 6.5 BDNF levels (pg/mg total protein) in the cerebral cortex of different
groups of animals, WNIN (WN), WNIN/Ob lean (OL) and WNIN/Ob obese (OO)
at the age of 3, 12 and 15 months.
Age
Group 3 m 12 m 15 m Pooled Value
WN 7.79±0.596a 7.72±0.430ab 5.93±0.287b 7.15±0.606
OL 7.99±0.506 8.00±0.617 6.09±0.476 7.36±0.636
OO 6.66±0.780a 5.57±0.491b 4.67±0.294b 5.63±0.574***
Across groups F(2, 54) = 9.9, P = 0.0002
Across time points F(2, 54) = 11, P <0.0001
Interaction F(4, 54) = 0.42, P = 0.792
Data analysed using two-way ANOVA and represented as mean±S.E.M. The differences were
considered significant at minimum of P<0.05. The superscripts a, b and c represent the significant
differences (ageing changes) in an animal group across a row. The pooled values (mean±S.E.M.)
represent the overall BDNF level in cerebral cortex in an animal group irrespective of age. Significant
changes among the groups for the pooled values are shown with asterisks (***P<0.001).
132
Table 6.6 BDNF levels (pg/mg total protein) in the hippocampus of different
groups of animals, WNIN (WN), WNIN/Ob lean (OL) and WNIN/Ob obese (OO)
at the age of 3, 12 and 15 months.
Age
Group 3 m 12 m 15 m Pooled Value
WN 21.4±1.58aα 19±0.7ab 15.5±0.71b 18.7±1.70
OL 19.6±1.07α 19.2±1.02 15.1±0.48 18±1.43
OO 9.8±0.81aβ 17.9±1.90b 13.7±0.66ab 13.8±2.34****
Across groups F(2, 54) = 18, P <0.0001
Across time points F(2, 54) = 9.9, P = 0.0002
Interaction F(4, 54) = 8.3, P <0.0001
Data analysed using two-way ANOVA and represented as mean±S.E.M. The differences were
considered significant at minimum of P<0.05. The superscripts a, b and c represent the significant
differences (ageing changes) in an animal group across a row; α, β and γ represent the significant
differences among groups at a given age point, across a column. The pooled values (mean±S.E.M.)
represent the overall BDNF level in hippocampus in an animal group irrespective of age. Significant
changes among the groups for the pooled values are shown with asterisks (****P<0.0001).
133
Table 6.7 BDNF levels (pg/mg total protein) in the hypothalamus of different
groups of animals, WNIN (WN), WNIN/Ob lean (OL) and WNIN/Ob obese (OO)
at the age of 3, 12 and 15 months.
Age
Group 3 m 12 m 15 m Pooled Value
WN 39.4±4.69α 33.4±2.66 31.5±2.63α 34.8±2.38
OL 31.1±3.11α 27.1±2.37 26.1±2.07αβ 28.1±1.54
OO 14.8±1.11β 22.5±2.93 18.4±2.17β 18.6±2.23****
Across groups F(2, 54) = 26, P <0.0001
Across time points F(2, 54) = 1.0, P = 0.372
Interaction F(4, 54) = 2.0, P = 0.107
Data analysed using two-way ANOVA and represented as mean±S.E.M. The differences were
considered significant at minimum of P<0.05. The superscripts α, β and γ represent the significant
differences among groups at a given age point, across a column. The pooled values (mean±S.E.M.)
represent the overall BDNF level in hypothalamus in an animal group irrespective of age. Significant
changes among the groups for the pooled values are shown with asterisks (****P<0.0001).
134
6.3.3 Hormones in stress response
Plasma ACTH levels (Table 6.8) of OO rats were significantly higher than
WN as well as OL control groups at all the time points studied. Also there was a
significant increase in plasma ACTH levels at the age of 15 months in control groups
indicating the effect of ageing. Interestingly, these levels were higher in 3 months old
OO than in 15 months old WN and 3OL group animals. The pooled ACTH levels also
showed a significant increase of comparable magnitude. (P<0.0001) in OO rats.
Table 6.8 ACTH levels (pg/mL) in plasma of different groups of animals, WNIN
(WN), WNIN/Ob lean (OL) and WNIN/Ob obese (OO) at the age of 3, 12 and 15
months.
Age
Group 3 m 12 m 15 m Pooled Value
WN 193.1±10.16aα 273.0±32.68aα 460.6±43.00bα 308.9±79.29
OL 237.8±13.80aα 345.4±27.64aα 592.7±44.65bα 392.0±105.1
OO 1480.7±73.91β 1441.73±60.95β 1646.3±106.61β 1523±62.72****
Across groups F(2, 54) = 468, P <0.0001
Across time points F(2, 54) = 20, P <0.0001
Interaction F(4, 54) = 0.90, P = 0.468
Data analysed using two-way ANOVA and represented as mean±S.E.M. The differences were
considered significant at minimum of P<0.05. The superscripts a, b and c represent the significant
differences (ageing changes) in an animal group across a row; α, β and γ represent the significant
differences among groups at a given age point, across a column. The pooled values (mean±S.E.M.)
represent the overall plasma level of ACTH in an animal group irrespective of age. Significant changes
among the groups for the pooled values are shown with asterisks (****P<0.0001)
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Plasma melatonin levels (Table 6.9) were significantly lower in 3 months old
OO rats than the corresponding controls. Further, Plasma melatonin levels were
observed to decrease with age in all groups of rats. The pooled value of melatonin
levels also showed a significant decrease (P<0.0001) in OO rats compared to the
controls.
Table 6.9 Melatonin levels (pg/mL) in plasma of different groups of animals,
WNIN (WN), WNIN/Ob lean (OL) and WNIN/Ob obese (OO) at the age of 3, 12
and 15 months.
Age
Group 3 m 12 m 15 m Pooled Value
WN 84.6±6.27aα 60.0±3.08b 48.6±3.76b 64.4±10.6
OL 83.1±6.95aα 67.8±3.84a 41.9±4.10b 64.3±12.0
OO 54.1±6.86β 40±3.5 32.8±2.59 42.3±6.24****
Across groups F(2, 54) = 21, P <0.0001
Across time points F(2, 54) = 35, P <0.0001
Interaction F(4, 54) = 1.7, P = 0.163
Data analysed using two-way ANOVA and represented as mean±S.E.M. The differences were
considered significant at minimum of P<0.05. The superscripts a, b and c represent the significant
differences (ageing changes) in an animal group across a row; α, β and γ represent the significant
differences among groups at a given age point, across a column. The pooled values (mean±S.E.M.)
represent the overall plasma level of melatonin in an animal group irrespective of age. Significant
changes among the groups for the pooled values are shown with asterisks (****P<0.0001).
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Plasma corticosterone levels (Table 6.10) were significantly higher in OO rats
at 15 months of age but not earlier. Compared to the values at 3 months of age,
plasma corticosterone levels decreased at 12 months, but at 15 months the values
increased significantly in all three groups. However the pooled values were
comparable among the three groups.
Table 6.10 Corticosterone levels (pg/mL) in plasma of different groups of
animals, WNIN (WN), WNIN/Ob lean (OL) and WNIN/Ob obese (OO) at the
age of 3, 12 and 15 months.
Age
Group 3 m 12 m 15 m Pooled Value
WN 54.6±4.46 47.5±2.45 72.3±12.79 58.1±7.39
OL 34.7±6.68a 25.8±5.60a 72.9±7.73b 44.5±14.5
OO 37.7±1.79a 33.0±5.57a 86.6±10.71b 52.4±17.1
Across groups F(2, 54) = 2.7, P = 0.08
Across time points F(2, 54) = 29, P <0.0001
Interaction F(4, 54) = 1.5, P = 0.21
Data analysed using two-way ANOVA and represented as mean±S.E.M. The differences were
considered significant at minimum of P<0.05. The superscripts a, b and c represent the significant
differences (ageing changes) in an animal group across a row. The pooled values (mean±S.E.M.)
represent the overall plasma level of corticosterone in an animal group irrespective of age.
6.4 Discussion
WNIN/Ob obese rat presents a unique animal model combining the features of
obesity and accelerated ageing. Considering their established role in / association with
ageing, we endeavoured to evaluate various neurotrophic factors, anterior pituitary
hormones, and stress hormones in the WNIN/Ob obese rats that are established rat
models of obesity and metabolic syndrome and show reduced longevity / accelerated
ageing. Accumulation of proinflammatory cytokines during ageing increases the risk
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of brain for cognitive decline and dementia (Cotman 2005) by generating a state of
neurotrophic resistance. Under these circumstances even if the levels of neurotrophic
factors are normal there is a greater probability of their dysfunction in the ageing
brain.
GH secreted from anterior pituitary, circulates all over body to exercise
important actions on growth and metabolism. It signals the secretion of IGF-1 that
mediates growth-promoting functions of GH. The role of GH/IGF-I axis is well
recognized in the longevity of organisms ranging from Caenorhabditis elegans to
mice (Bartke, Sun et al. 2013, Ding, Sackmann-Sala et al. 2013). GH/IGF-1 axis plays
an important role in modulating the ageing process across species from worms to
mammals (Holzenberger 2004, Junnila, List et al. 2013). Therefore, to start with we
checked the levels of GH and IGF-1 in the plasma of WNIN/Ob obese, WNIN/Ob
lean and parental WNIN control rats of different ages and observed that the levels of
GH to decrease with age (Sonntag, Steger et al. 1980) in the WN as well as OL
groups. In line with previous observations (Sonntag, Steger et al. 1980), GH levels
were significantly low (p<0.001) at 3 months of age in OO rats, and the levels
remained so throughout its ageing. On the other hand, levels of IGF-1 were
significantly increased in the WNIN/Ob obese rats compared to controls at 3 months
of age. This increase became less significant at 12 months of age, but a significant
increase was observed again at 15 months of age. IGF-1 levels are high in children as
it is required for their normal growth and development of various tissues. Although
IGF-1 level is known to decrease in adults and during normal ageing (Nessi, De Hoz
et al. 1995, Kuwahara, Kesuma Sari et al. 2004, Bartke 2005), a few studies have
reported that it increases during ageing where high risks of cancer are involved
(Bartke 2008, Bartke 2009). Considering that high levels of plasma IGF-1 have been
reported in cancer patients (Yu, Spitz et al. 1999, Otake, Takeda et al. 2010, Llanos,
Brasky et al. 2013, Guevara-Aguirre and Rosenbloom 2014), the high plasma IGF-1
levels in WNIN/Ob obese rats may explain / be associated with the greater incidence
of different types of tumours and increased DNA damage (Harishankar, et al. 2011,
Reddy, et al. 2014, Sinha, et al. 2014b). Deficiency in GH signaling is known to delay
ageing and remarkably extend longevity in laboratory mice (Brown-Borg, Borg et al.
1996, Flurkey, Papaconstantinou et al. 2001, Bartke, Sun et al. 2013) and similarly
humans with similar mutations are also reported to be benefitted (Guevara-Aguirre,
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Balasubramanian et al. 2011). Reduced levels of GH is consistent in elderly people
(Rudman 1985, Giustina and Veldhuis 1998, Muniyappa, Sorkin et al. 2007, Veldhuis
2008) and hence it has been proposed as a result or symptom or even as a ‘biomarker’
of ageing (Bartke, Sun et al. 2013). So the drastically low levels of GH may be an
important cause of the disturbed physiology of the WNIN/Ob obese rats.
BDNF is indispensable for neuronal maintenance, survival and growth of new
neurons and synapses (Lewin and Barde 1996, Huang and Reichardt 2001). Its
involvement is also reported in mood and eating disorders (Nakazato, Hashimoto et
al. 2006, Monteleone, Castaldo et al. 2008), mental health or psychiatric disorders
(Weickert, Hyde et al. 2003, Kim, Lee et al. 2007, Roth, Lubin et al. 2009, Castren
2014, Mitchelmore and Gede 2014), depression, cognition (Roth, Lubin et al. 2009),
neurodegeneration and ageing (Sohrabji and Lewis 2006, Komulainen, Pedersen et al.
2008, Erickson, Prakash et al. 2010, Driscoll, Martin et al. 2012). Considering that
BDNF is essential for survival and function of hippocampal, cortical, basal forebrain,
and entorhinal cortex neurons, our finding of extremely low BDNF levels in plasma,
hypothalamus and hippocampus at an early age of 3 months suggests strongly about
the lack of essential microenvironment for the normal maintenance of the neurons in
different parts of the brain. That the levels didn’t improve much even at later age
points probably corroborates the above inference that the deficiency of BDNF may be
correlated with / underlie the accumulation of different negative factors in the brain.
Also, our study is in line with some recent reports that through epigenetic
programming obesity reduced the expression of BDNF that had long-term deleterious
effects in the brain. Further this could contribute to the early onset of cognitive
decline during ageing (Greising, Ermilov et al. 2014, Wang, Freire et al. 2014).
Epigenetic studies in the WNIN/Ob obese rats would help to find the plausible causes
of the disturbed physiology.
The anterior pituitary (or adenohypophysis) regulates several biological
processes like stress, growth, reproduction, and lactation (Veldhuis 2013). It contains
five specialized hormone-secreting cell types that have many regulatory functions and
maintains homeostasis (Nicolaides, Kyratzi et al. 2015). They are: (a) Corticotropes
produce ACTH that acts on the adrenal gland, (b) Gonadotropes secreting FSH and
LH that regulate gonadal functions, (c) Lactotropes producing prolactin that acts on
the mammary glands, (d) Somatotropes secreting GH that targets the liver and bone,
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and (e) Thyrotropes secreting TSH that targets the thyroid gland (Watkins-Chow and
Camper 1998, Savage, Yaden et al. 2003, Gumbel, Patterson et al. 2012) In terms of
stress and its response, ACTH plays important role. After being activated by
corticotropin-releasing hormone and arginine-vasopressin, pituitary gland secretes
ACTH that signals adrenal glands to release cortisol / corticosterone and other
glucocorticoids and this cocktail of corticoids lays off the condition of biological
stress and hence ACTH secretion is considered important to check the initiation of
stress management (Papadimitriou and Priftis 2009, Nicolaides, Kyratzi et al. 2015).
As compared to the controls rats the levels of corticosterone (Sakamuri, et al. 2011)
and ACTH in WNIN/Ob obese rats were significantly higher at all the ages studied.
These findings clearly indicate increased oxidative stress in the animal including its
brain and hence increased plasma levels of ACTH and corticosterone in them in
response to the increased oxidative stress, probably to cope up with the stress
situation. In our studies, the ACTH levels were observed to increase slowly but not
significantly with age in control as well as WNIN/Ob obese rats. These findings
suggest the probable utility of plasma ACTH as a marker to assess the effectiveness /
efficacy of anti-oxidants in future studies, by determining whether or not the pituitary
gland is getting the signal of decreased biological stress and responding accordingly
by modulating the expression of ACTH and corticosterone. Although corticosterone
levels were higher in WNIN/Ob obese rats than WNIN controls at 15 months of age
around which the morbidity rate is high, but not at younger age. As reported in
chapter 2 regarding CRP levels that are known to put individuals at high risk of CVDs
and morbidity, corticosterone levels were also observed to follow similar trend. These
results suggest increased biological stress in the rats and hence in the same context we
evaluated the levels of melatonin as it is known to have strong anti-oxidant activity
(Hardeland 2005). In parental WNIN controls we observed a significant decrease in
the melatonin levels with age whereas the WNIN/Ob obese rats the levels were
significantly low right from the age of 3 months. The low levels of melatonin in
WNIN/Ob obese rats perhaps represent a compromised situation where the stress is
high and antioxidants are low, which can disturb sleep-wake cycle causing sleep
disorders (Chang, Wu et al. 2009) and may be associated with neurodegeneration and
ageing (Videnovic, Lazar et al. 2014). Considering the reports that rats supplemented
with melatonin had decreased abdominal fat and body weight (Wolden-Hanson,
Mitton et al. 2000), monitoring melatonin levels may be a useful approach to treat
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obesity as melatonin can regulate brown adipose tissue metabolism (Tan, Manchester
et al. 2011), is considered as having anti-ageing effects (Brown, Young et al. 1979,
Touitou 2001) and its deficiency is considered a biomarker for the intrinsic process of
the brain ageing (Sharma, Palacios-Bois et al. 1989). In view of the literature referred
above, our findings that are in line with these reports indicate that many of the
important biological processes were affected in WNIN/Ob obese rats at an early age
of 3-6 months. The neural circuits regulating food intake converge at the
paraventricular nucleus that also contains corticotrophin releasing hormone (CRH)
and urocortin containing neurons. Considering that hypothalamo-pituitary-adrenal or
HPA axis regulates biological stress responses as well as feeding behavior of an
animal (due to sharing same neuroanatomy), it becomes evident that both systems can
influence each other in stimulating a response (Maniam and Morris, 2012). Future
studies with supplementation of melatonin in WNIN/Ob obese rats appears to be a
possible way out for modulating obesity / early ageing in them as it has been shown
that melatonin reinstates pituitary responsiveness and base levels of pituitary
hormones in old rats to the levels observed in young ones (Diaz, Pazo et al. 2000).
6.5 Summary
• Significantly lower levels of GH and increased levels of IGF-1 in WNIN/Ob
rats points to an altered GH/IGF-1 axis, which is an important determinant of
longevity. This finding is suggestive of the increased susceptibility of
WNIN/Ob obese rats towards various types of tumors and cancers.
• BDNF levels were significantly low in plasma, hippocampus and
hypothalamus which shows poor neurotrophic support to brain regions,
thereby leading to neurodegeneration and accelerated ageing phenotype.
• Increased levels of ACTH and corticosterone, and concomitant decreased
levels of melatonin in plasma of young WNIN/Ob obese rats clearly reveals
increased biological stress and decreased antioxidant like activity, probably
accelerating the ageing process.
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Chapter 7
General discussion and conclusion
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7.1 General discussion
7.2 Conclusions
7.3 Limitations of the study
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7.1 General discussion
Lifespan is the duration of time an organism lives and longevity is the
expected average lifespan under ideal conditions. Lifespan is for an individual and
differs as per the prevailing conditions; but longevity covers mathematical terms like
average lifespan of a given population that is normally observed in absence of certain
disorders like cancer, stroke, etc. Ageing is a part of the lifespan that constitutes the
second half of the life story and is indeed very important (Sinha and Ghosh, 2010). As
the ageing population is on rise all over the globe, we need to be prepared with more
knowledge of the other co-existent disorders with different mechanisms playing their
roles to further deteriorate the normal ageing process. These include CVDs,
Alzheimer’s disease, Parkinson’s disease, diabetes, macular degeneration, various
cancers and obesity among others. These are well known to accelerate the ageing
process and cause reduced longevity of various organisms (Butterfield et al., 2001).
Numerous strategies have been tried in the past and continue to be tried almost
everywhere mainly to increase the longevity of human beings in general and a healthy
ageing in particular. It is very important to understand the basic mechanisms of
normal ageing and also the changes in different pathways in different disorders that
may be a reason or causally related to the ageing process. Accelerated ageing is even
more important to study because the mechanisms work out much faster and in an
unregulated way (Oliver et al., 1987). If the points where this disarrangement or
mismanagement occurs can be found out, that would be a great milestone in ageing
research. For such a kind of research, good animal models are required representing
real human situations or afflictions.
The factors that determine the longevity of an organism can be divided into
genetic and environmental (modifiable) ones. Obesity is one of the co-existing health
problems, which is known to cause an early death. Different metabolic pathways are
being researched upon to unravel the mystery but still the actual mechanism is
elusive. In such a scenario, it is of immense importance to develop appropriate animal
models that show the characteristics of both the co-morbid health situations. At NIN,
researchers have developed the WNIN/Ob obese rat strain using selective back
crossing, which shows morbid obesity (Giridharan, 1998) as well as significantly
reduced longevity. These are the first inbred mutant obese rat model which is also the
heaviest known till date (maximum ~1.47 Kg). Studies on these rats show the
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presence of various complications like retinal degeneration, cataract, compromised
immunity, hyperinsulinemia, hypercholesterolemia, hyperleptinemia, infertility,
polycystic ovaries and other kinds of tumors, etc. (Reddy et al., 2009, Harishankar et
al., 2011, Bandaru et al., 2013). Recently the mutation has been shown to be in 4.3cM
region on chromosome 5 upstream of the leptin receptor (Kalashikam et al., 2013) and
also altered leptin gene promoter methylation (Kalashikam et al., 2014). To the best of
our knowledge, no studies have been undertaken to establish them as an appropriate
model of reduced longevity / accelerated ageing and also the associated / underlying
mechanisms specially in their brain, considering the pivotal role the brain plays in
modulating a variety of physiological functions including ageing. Therefore, these
studies were conducted in the WNIN/Ob, the obese mutant rat with reduced longevity.
Evaluation of growth characteristics, survival and regional brain volume
changes
WNIN/Ob obese rat is the obese rat model with significantly decreased
longevity and hence could be a very useful model to study obesity associated
accelerated ageing. Although WNIN/Ob obese rats have been known to have
decreased lifespan, it has not been established / validated statistically nor reported
scientifically. In order to find out the probable mechanisms underlying the early death
in these rats, it is essential to establish them first, as models for accelerated ageing.
Therefore we attempted to scientifically establish and statistically validate the
accelerated ageing / decreased longevity of the WNIN/Ob obese rats. One of the
established experiments to examine the effects of genetic manipulations and various
chemical compounds on ageing is the measurement of lifespan. This has traditionally
been accomplished by survival analysis over the lifetime (Yang et al., 2011) because
one can derive interesting and useful information by appropriate statistical survival
analysis of the survival data (Valenzano et al., 2006, Harrison et al., 2009, Honjoh et
al., 2009, Yang et al., 2011). Using Kaplan – Meier estimation, we found a
significantly lower mean lifespan of 420 days in WNIN/Ob obese rats as compared to
the parental WNIN normal as well as WNIN/Ob lean littermates where it was 1067
and 1035 days respectively, which is around 60% reduction. Considering the
calculations of George Sacher using least squares regression of log lifespan (Sacher,
2008), there is relationship between brain weight and lifespan, and hence we checked
the brain wet weights of these rats. We found significantly decreased brain weights in
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WNIN/Ob obese rats as compared to both the control groups at all the time points
studied. It was of interest that the brain weights of WNIN/Ob obese rats at 3 months
of age were lower than that of WNIN control and WNIN/Ob lean littermates at the
age of 18 months, indicating very premature occurance of age related decrease in the
brain weight in the WNIN/Ob obese rats (at a very young age itself) probably
suggesting accelerated ageing in them. It is reported that with advancing age, there
was a reduction in the volume of prefrontal and cingulated cortices’ (Shamy et al.,
2011), thinning of prefrontal and superior temporal cortices (Alexander et al., 2008),
reduction in gray and white matter volume (Wisco et al., 2008), thinning of
somatosensory and motor cortices and thickening of superior temporal and cingulate
cortices (Koo et al., 2012). As the brain weights were significantly lower in
WNIN/Ob obese rats at 3 months of age itself, we looked for the possibility of
neuronal loss and / or shrinkage of various brain regions reported in brain during old
age. Therefore the regional brain volumetric analysis was performed using MRI.
Although there was decreasing trend observed in different brain volumes, there were
no significant changes. Nevertheless it is possible that even in the absence of any
volumetric change in the brain, there could be a compromised microenvironment in
the brain causing the molecular pathways to go awry (Miranda et al., 2012).
Therefore, we conducted cellular studies to examine different cellular subsets i.e. glial
and neuronal evaluations.
Cellular (neuronal and glial) changes in the brain vis-à-vis decreased longevity
During normal ageing, many structural and functional changes occur in the
brain and for evaluating the gross cytoarchitechture changes in brain, Nissl staining is
widely used (Huang et al., 2013). Therefore to evaluate cytostructural changes we
used Nissl staining and observed the paucity of neurons in the cerebral cortical layers
in the brain of WNIN/Ob obese rats compared to WNIN controls and WNIN/Ob lean
littermates , which is line with previous reports of similar nature (Brizzee et al., 1980).
To our surprise, increased neuronal populations were seen in the peri-ventricular
region where various hypothalamic nuclei are present. As WNIN/Ob obese rats are
hyperphagic and orexin-A (Ox-A) is known to increase food intake by delaying the
onset of normal satiety sequence (Rodgers et al., 2002), we quantitated by counting
the absolute numbers of Ox-A positive neurons and found it to be significantly higher
in lateral and dorsomedial areas of hypothalamus of WNIN/Ob obese rats as
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compared to the normal rats. These findings indicate that the hyperphagy seen in these
rats could be due to increased activity of the orexigenic neurons.
C-reactive protein (CRP) is known to increase the blood-brain-barrier
permeability and enter the brain at high levels and is implicated in obesity and
inflammation (Hsuchou et al., 2012). Therefore we checked the CRP levels in the
plasma of these rats and found it be increased in the WNIN/Ob obese rats pointing its
possible role in the complex mechanisms of obesity and the associated ageing. High
CRP levels are known to cause astrogliosis (Hsuchou et al., 2012) and hence we
checked its marker GFAP in the brain of these rats. In immunohistochemical analysis
of the brain sections, we found an increased expression of GFAP in the hippocampus
of 6 months of old WNIN/Ob obese rats but at the age of 12 months it was observed
to decrease as compared to the control rats. This may be due to the operation of some
age dependent compensatory mechanisms in the brains of WNIN/Ob obese rats. To
further confirm this finding we did quantitative analysis of GFAP using Western
blotting and found similarly increased GFAP protein expression in the WNIN/Ob
obese rat brains compared to the WNIN and WNIN/Ob lean littermates of comparable
age. Also there was an increased GFAP expression observed in the lateral
hypothalamus and arcuate nucleus in the obese rats suggesting the probable damage to
neuronal integrity of these hypothalamic nuclei that are involved in regulation of
feeding behavior and hormonal control. This also seems to suggest that altered
signaling pathways in the brain cause / underlie obesity and reduced longevity of
WNIN/Ob obese rats. In view of these findings, it was considered pertinent to assess
the alterations if any in the various neurochemicals and brain metabolites (Haga et al.,
2009, Rothman et al., 2011) in order to analyze the changes in the microenvironment
of the brains of these rats. Therefore, as the next step we evaluated the neurochemical
profile and estimated the brain metabolism of these rats using 13C-glucose and NMR
spectroscopy.
Evaluation of neurochemical profile and brain metabolism of WNIN/Ob obese
rats using Nuclear Magnetic Resonance Spectroscopy
Neurochemical profiling using NMR is a sensitive approach to simultaneuosly
measure numerous key neurochemicals that are present in minute amounts. During
ageing, concentration of most of the metabolites in the brain reflecting structural and
functional properties of the specific region are altered to a significant level
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(Boumezbeur et al., 2010, Duarte et al., 2012, Duarte et al., 2014). Therefore the
complex communication among the neurometabolites gets modified either due to
ageing or in the process of adapting to the changing microenvironment of the nervous
system. This makes them good canditates to be considered as biomarkers of different
patterns of health and disease (Duarte et al., 2014). It is well known that the level of
different neurometabolites such as N-acetylaspartate (NAA), total creatine (Cr),
phosphorylethanoloamine (PE), taurine (Tau), glutamate (Glu), glutamine (Gln),
gamma-aminobutyrate (GABA), alanine (Ala), get altered under disturbed
physiological conditions, and could be reliably measured using in vivo and in vitro
MRS (Dedeoglu et al., 2004, Duarte et al., 2014, Harris et al., 2014). As compared to
in vivo MRS, a wider range of neurochemicals can be quantitated using in vitro MRS
(Dedeoglu et al., 2004) and hence we did the in vitro study to find the changes in the
neurochemical profile in cerebral cortex of WNIN/Ob obese rats as compared to the
controls rats. The neurochemical profile and the brain metabolism (of 13C-glucose)
have been determined in the cerebral cortex considering its importance in the process
of ageing (Villa et al., 2012). Glu is the main excitatory neurotransmitter and NAA is
an indicator of neuronal integrity. Tau is well known for its neuroprotective and
antioxidant-like activities in the brain. A decrease in the levels of these
neurometabolites indicates neuronal dysfunction and compromised state of
neuroprotection (Duarte et al., 2012). In this study, the concentrations of Glu, NAA
and Tau were significantly lower in the cerebral cortex of WNIN/Ob obese rats (than
that of controls), which suggests the altered microenvironment in the cerebral neurons
posing a damaging situation where neuronal death and associated changes like
astrogliosis can occur. The neurochemicals considered as markers of astrogliosis viz,
Gln and Inositol (Ino; also shows osmotic stress dysfunction) have been reported to be
increased during ageing (Zahr et al., 2014). We observed increased concentrations of
Gln and Ino in the cerebral cortex of these obese rats of 3 months age (compared to
the corresponding controls) again confirming the negative balance of the
neurochemicals favouring neurodegeneration and ageing. As these observations were
done in the obese rats at 3 months of age, our findings indicate that at a young age
only the neurochemical profile of these animals is getting altered and resembles the
changes seen in the control rat brains at later age (e.g. 15 – 18 months)(Duarte et al.,
2014), corroborating the accelerated ageing of the WNIN/Ob obese rats.
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In terms of metabolism, the energy needs of brain are very high. According to
the neuronal activity (or energy needs) this demand is normally fulfilled with stringent
regulatory mechanisms to deliver sufficient energy substrates (Hyder et al., 2006).
Therefore different metabolite concentrations reveal the activity of various metabolic
processes. To evaluate this, we infused 13C-labeled glucose in these rats through vein
and measured amount of 13C being incorporated in the brain metabolites involved in
neurotransmitter cycling. Neurons release neurotransmitters Glu and GABA (based on
the type of neuron i.e. either glutamatergic or GABAergic) into the synaptic cleft.
From the cleft, astrocytes take up the neurotransmitters and convert into Gln and
release to the neurons. Inside neurons Gln is hydrolyzed to Glu or GABA, re-
packaged into vesicles and be ready for the next release at the arrival of action
potential. Considering that GluC4 and GABAC2 labeling occurs via glucose
metabolism through the TCA cycle and their determination provides an estimate of
TCA cycle flux associated with glutamatergic and GABAergic neurons respectively,
the GlnC4 labeling represents the total neurotransmitter cycling associated with
glutamatergic and GABAergic neurons. Hence we evaluated the concentrations of
GluC4, GABAC2 and GlnC4 from the 1H-[13C] NMR spectra of the cortical extract and
found these levels to be significantly decreased in the WNIN/Ob obese rats compared
to controls, indicating hypoactivity and decreased metabolism. This hypometabolism
concomitant to probable neuronal damage (as evident from the observed changes in
neuronal and glial profile) and increased lipid peroxidation (shown by decreased
NAA and Tau) probably indicates insufficient supply of the energy to the obese rat
brain even at a young age of three months. That these microenvironmental alterations
and metabolic changes normally seen in the ageing brain (Duarte et al., 2014) were
observed in the young age in the obese rats seems to further strengthen the inference
that changes associated with normal ageing appear to be occurring much earlier in the
brain of the WNIN/Ob obese rats and could therefore underlie their accelerated ageing
/ decreased longevity. Considering that despite no changes observed in the volume of
different brain regions in the WNIN/Ob obese rat, the neurochemical profile and
metabolism were altered in the WNIN/Ob obese rat brain suggestive of accelerated
ageing and since ageing in general is known to be associated with stress (e.g.
oxidative and corticosteroid), it was considered imperative to assess the level of
oxidative stress, antioxidant enzymes’ activities and the attendant damage of
macromolecules in the brain of these rats.
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Extent of DNA damage, oxidative stress status and activity of antioxidant
enzymes in the brain of WNIN/Ob obese rats
Ageing is well known to be associated with increased oxidative stress (Finkel
and Holbrook, 2000, Marosi et al., 2012), different types of macromolecular damage
and accumulation of DNA damage, especially in the brain (Freitas and de Magalhaes,
2011, Moskalev et al., 2013). When these events are set in to motion prematurely, it is
termed as accelerated ageing, causing an early build-up of deteriorating factors
normally seen at an advanced age. Factors contributing to accelerated ageing include
genetic conditions, chronic disorders and unbalanced lifestyle related disorders like
obesity, cardiovascular diseases and type 2 diabetes mellitus (Tzanetakou et al., 2012,
Zhu and van der Harst, 2014). In addition, obesity is well demonstrated to be
associated with ageing (Harrington and Lee-Chiong, 2009, Tzanetakou et al., 2012,
Alfadda et al., 2013) with the probable mechanism being through chronic
inflammation caused by altered adipokine signaling (Michaud et al., 2013) and
increased oxidative stress in the body. Obesity has also been correlated with increased
oxidative stress (Furukawa et al., 2004). Enhanced and uncontrolled production of
reactive oxygen species (ROS) and an inefficient machinery of antioxidant enzymes
in turn results in DNA damage (Barzilai and Yamamoto, 2004). The brain has been
observed to be more vulnerable to age-related DNA damage compared to other tissues
(Price et al., 1971, Mori and Goto, 1982). DNA damage in the form of single strand
breaks has been found to be maximum in neurons of cerebral cortex in aging rat brain
(Mandavilli and Rao, 1996). Thus, brain can be considered as the appropriate tissue to
decipher role of oxidative stress and DNA damage in ageing.
Early accumulation of deteriorating factors (especialy in terms of
macromolecular damage) in the brain (Barzilai and Yamamoto, 2004) can cause
accelerated ageing in organism. Therefore, we checked the oxidative stress markers:
levels of lipid peroxidation and protein carbonyls in the cerebral cortex and
hippocampus of these young (3 month age) obese rats and found it to be significantly
higher than age matched parental WNIN and lean littermates. Indeed these levels
were as high as what was seen at an later time point (15 months) in the control rats,
indicating an early increase of oxidative stress in the obese rat brain. Considering the
signifcant increase observed in lipid peroxidation and protein carbonyl levels in the
obese rat brain, it was of interest to check for the damage of DNA if any in these
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brains because the study of Rutten et al. suggests that neurons show age-related
increase in the accumulation of DNA damage in mouse brain (Rutten et al., 2007).
The extent of DNA damage in the brain of these rats was assessed in terms of single-
and double-stranded breaks (SSBs and DSBs respectively) using Comet assay (Singh
et al., 1988). To our surprise these results were also in the line of our previous
observations, i.e. significantly higher DNA damage both in terms of SSBs and DSBs,
in both regions of brain of the WNIN/Ob obese rats than controls. Considering the
fact that macromolecular damage is a part of normal wear-and-tear processes occuring
in a cell which is constantly rejevunated by the activity of antioxidant enzymes and
other factors which prevent / repair the damage caused by oxidative stress, we
checked if the activites of superoxide dismutase and catalase, the two important
antioxidant enzymes in the brain and found it to be significantly lower in the obese
rats than controls. These observations clearly suggest the early accumulation of DNA
damage due to increased oxidative stress in the brain of these rats could be due to the
compromised antioxidant capacity of the brain. In addition it could also be due to the
lack of sufficient neurotrophic support and neuroprotection (due to low levels of
taurine as observed in the neurochemical profiling studies) causing these rats to die
earlier. Therefore, we checked the availability of brain-derived neurotrophic factor
(BDNF) in the brain and also evaluated the axes involved in longevity pathways i.e.
GH/IGF-1 and HPA axes in the brains of WNIN/Ob obese rats and the controls.
Neurotrophic support, GH/IGF-1 & HPA axes vis-à-vis decreased longevity
Importance of neurotrophic factors (NTFs) in the development, differentiation,
maintenance and plasticity of various brain regions is well known. There is always a
spatial and temporal balance of trophic support required in the CNS for normal
functioning. BDNF is well known for its role in neuronal maintenance, survival and
growth of new neurons and synapses (Huang and Reichardt, 2001). BDNF support is
known to decrease as the age of an animal advances (Lommatzsch et al., 2005,
Ziegenhorn et al., 2007, Sen et al., 2008, Tapia-Arancibia et al., 2008, Greising et al.,
2014) and this forms one of the reasons for decreased probability of neuronal
viability. Considering this we checked BDNF levels at 3, 12 and 15 months of age, in
various brain regions mostly affected during ageing (i.e. cerebral cortex, hippocampus
and hypothalamus) as well as the periphery (i.e. plasma and cerebrospinal fluid
(CSF)) of the WNIN/Ob obese rats and compared it with those of age matched
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parental WNIN control and lean littermates. We found a significantly lower BDNF
concentration in plasma at all the age points of these rats. In CSF, there was a
significant decrease during normal ageing but we saw only a decreasing trend in
WNIN/Ob obese rats where the levels were not significantly lower. When we checked
BDNF concentration in brain regions, we found a decreasing trend in the cerebral
cortex, whereas these levels were significantly lower in hippocampus and
hypothalamus. At the age of 3 months the obese rats showed more depressed BDNF
levels both in hippocampus and hypothalamus, which indicates an insult at an early
age changing the microenvironment, which is required for normal development and
maintenance of neural tissues. At 12 and 15 months of age the BDNF levels were
significantly lower than control rats but among obese rats, it improved as compared to
that observed at 3 months of age. This probably shows a compensatory mechanism
trying to improve the microenvironment by helping in the maintenance of the neural
tissue.
GH/IGF-1 axis plays an important role in modulating longevity across species
(Holzenberger et al., 2003). IGF-1 also plays a major role in mediating the effects of
longevity genes on ageing and life span (Bartke et al., 2003). GH secretion has been
shown to decrease in old rats (Sonntag et al., 1980) as compared to the young ones.
Insulin/IGF-1 like signaling pathway contributes significantly to the biologic ageing
process. Insulin/IGF-1-like signaling is conserved from worms like C. elegans to
humans. Indeed, in vitro experiments have shown that mutations (Kenyon et al., 1993)
reducing insulin/IGF-1 signalling extend life (Bartke, 2011) by decelerating the
degenerative, ageing process. GH/IGF-1 axis is in fact, known to play an important
role in modulating the ageing process across species and hence it is very important to
measure the levels of GH and IGF-1 in animal models to check if these are also
involved in reducing longevity (Junnila et al., 2013) of WNIN/Ob obese rats. In line
with available literature, we observed that the levels of GH to decrease with age
(Sonntag et al., 1980) in the rats of control groups and it was significantly low at 3
months of age in obese rats, and the levels remained so throughout its ageing. On the
other hand, levels of IGF-1 were significantly increased in the WNIN/Ob obese rats
compared to controls at 3 months of age. This increase became less significant at 12
months of age, but a significant increase was observed again at 15 months of age.
IGF-1 levels are high in children as it is required for their normal growth and
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development of various tissues. Although IGF-1 level is known to decrease in adults
and during normal ageing (Nessi et al., 1995, Kuwahara et al., 2004, Bartke, 2005), a
few studies have reported that it increases during ageing where high risks of cancer
are involved (Bartke, 2008, 2009). Considering that high levels of plasma IGF-1 have
been reported in cancer patients (Yu et al., 1999, Otake et al., 2010, Llanos et al.,
2013, Guevara-Aguirre and Rosenbloom, 2014), the high plasma IGF-1 levels in
WNIN/Ob obese rats may explain / be associated with the greater incidence of
different types of tumours and increased DNA damage (Harishankar et al., 2011,
Reddy et al., 2014, Sinha et al., 2014b) observed / reported in them. Reduced levels of
GH is consistent in elderly people (Rudman, 1985, Giustina and Veldhuis, 1998,
Muniyappa et al., 2007, Veldhuis, 2008) and hence it has been proposed as a result or
symptom or even as a ‘biomarker’ of ageing (Bartke et al., 2013). So the drastically
low levels of GH may be an important cause of the disturbed physiology of the
WNIN/Ob obese rats. Future studies are required to check the downstream signalling
and see if there are any other alterations in this longevity determinant-signalling
pathway.
As we observed an increased oxidative stress in the brain of WNIN/Ob obese
rats at an early age (Sinha et al., 2014b), we wanted to see if signalling from brain is
affecting biological stress (if any) in the body. Hypothalamo-pituitary-adrenal (HPA)
axis forms an important direct influencing and feedback interaction neuroendocrine
system controlling reactions to stress and regulating body processes like digestion,
energy storage and expenditure, immunity, sexuality and other psychological status of
an organism. In case of any kind of stress in the body, it tries to manage the situation.
On being activated by corticotropin-releasing hormone (from hypothalamus) and
arginine-vasopressin, pituitary gland secretes ACTH that cascades in to the secretion
of corticosterone and other glucocorticoids from adrenal glands in rodents. The
corticoids released as a stress response, work in close collaboration and terminate the
stress situation through a negative feedback loop to the hypothalamus. ACTH is an
important element of the HPA axis and is often produced in response to biological
stress (Papadimitriou and Priftis, 2009, Nicolaides et al., 2015). As compared to the
controls rats the levels of corticosterone (Sakamuri et al., 2011) and ACTH in the
plasma of WNIN/Ob obese rats were significantly higher at all the ages studied
showing the activation of HPA axis in response to the stress in body. In our studies,
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the ACTH levels were observed to increase slowly but not significantly with age in
control as well as WNIN/Ob obese rats. These findings suggest the probable utility of
plasma ACTH as a marker to assess the effectiveness / efficacy of anti-oxidants in
future studies, by determining whether or not the pituitary gland is getting the signal
of decreased biological stress and responding accordingly by modulating the
expression of ACTH and corticosterone. Plasma corticosterone levels were indeed
higher in WNIN/Ob obese rats than WNIN controls at 15 months of age around which
the morbidity rate is high, but not at younger age.
Melatonin is one of the most fundamental hormones in the evolution of
cellular functions and is identified primarily due to its antioxidant activity (Hardeland,
2005) and the protection it gives from the Sun’s radiation (Hardeland et al., 2006, Tan
et al., 2014). It also acts to control sleep and its deficiency has been implicated in
sleep disorders (Chang et al., 2009). In small animals, it is involved in energy
metabolism and body weight control. It has been proposed as a biomarker for the
intrinsic process of brain ageing (Sharma et al., 1989). We observed a significant
decrease in the levels of melatonin in the plasma of WNIN/Ob obese rats as compared
to the lean littermates as well as parental WNIN control rats at an early age of 3
months and these decreased levels always remained low compared to the control rats
during ageing. In such a scenario where we see most of the damaging / deteriorating
factors to be present in a young age of WNIN/Ob obese rats concomitant with lack of
antioxidants and neuroprotective factors, it becomes very clear that the genetic output
or easily incitable epigenetic factors either get activated at 3 months of age or earlier.
Chronic melatonin supplementation is known to reduce abdominal fat and body
weight (Wolden-Hanson et al., 2000) and hence it is proposed as an approach to treat
obesity, basically due to its ability to regulate brown adipose tissue metabolism (Tan
et al., 2011). There is also support for its anti-ageing effects (Brown et al., 1979,
Touitou, 2001) and also that it restores the basal concentrations of pituitary hormones
and pituitary responsiveness to the levels observed in young rats (Diaz et al., 2000).
Therefore future supplementation studies may be done in these rats to check the
different pathways getting rectified.
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7.2 Conclusions
• WNIN/Ob obese rats can be useful model for decreased longevity and
accelerated ageing.
• Changes seen in normally ageing control WNIN rats (around 15 months of
age) are seen in the WNIN/Ob obese rats at a much younger age of 3-6 months
of age, confirming the accelerated ageing in them.
• That neuronal loss and astrogliosis, altered neurochemical profile, increased
oxidative stress, decreased neurotrophic support, the factors that underlie
normal ageing also were seen in WNIN/Ob obese rats albeit at a much
younger age suggest that they also underlie the accelerated ageing in
WNIN/Ob obese rats.
7.3 Limitations of the study
• Since this study is the first attempt to establish the WNIN/Ob obese rats as an
appropriate model to study accelerated ageing / reduced longevity, we had
started with the minimum age point as 3 months. As it is evident from this
study that most of the accumulation of ageing factors appeared by the age of 3
months, in our future studies we would start experiments from an earlier age
point like the weaning age or may be embryos, to see how early the deleterious
changes begin to appear in these rats.
• As WNIN/Ob obese rats are infertile, have reduced longevity and high
susceptibility to opportunistic infections, obtaining the available number of
male rats for various studies remained a big hurdle. Considering that these
studies were planned in male rats that are difficult to procure, in few of our
experiments the number of animals was less than six. As per the clues we have
obtained from the present studies, we will plan and perform future
experiments taking this factor into account.
• It is important to do rehabilitation studies (e.g. calorie restriction,
supplementation with various antioxidants and therapeutic substances like
resveratrol, etc.) in order to prove the causal relationship (proof-of-concept) of
various deleterious factors to accelerated ageing in WNIN/Ob obese rats. As
mentioned earlier, that these are the first exploratory studies on ageing in these
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rats, future studies will be executed taking care of all the factors including
present knowledge and evidences obtained.
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