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Diet-induced obesity and low testosterone increase neuroinflammation and impair neural function

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Background Low testosterone and obesity are independent risk factors for dysfunction of the nervous system including neurodegenerative disorders such as Alzheimer¿s disease (AD). In this study, we investigate the independent and cooperative interactions of testosterone and diet-induced obesity on metabolic, inflammatory, and neural health indices in the central and peripheral nervous systems.Methods Male C57B6/J mice were maintained on normal or high-fat diet under varying testosterone conditions for a four-month treatment period, after which metabolic indices were measured and RNA isolated from cerebral cortex and sciatic nerve. Cortices were used to generate mixed glial cultures, upon which embryonic cerebrocortical neurons were co-cultured for assessment of neuron survival and neurite outgrowth. Peripheral nerve damage was determined using paw-withdrawal assay, myelin sheath protein expression levels, and Na+,K+-ATPase activity levels.ResultsOur results demonstrate that detrimental effects on both metabolic (blood glucose, insulin sensitivity) and proinflammatory (cytokine expression) responses caused by diet-induced obesity are exacerbated by testosterone depletion. Mixed glial cultures generated from obese mice retain elevated cytokine expression, although low testosterone effects do not persist ex vivo. Primary neurons co-cultured with glial cultures generated from high-fat fed animals exhibit reduced survival and poorer neurite outgrowth. In addition, low testosterone and diet-induced obesity combine to increase inflammation and evidence of nerve damage in the peripheral nervous system.Conclusions Testosterone and diet-induced obesity independently and cooperatively regulate neuroinflammation in central and peripheral nervous systems, which may contribute to observed impairments in neural health. Together, our findings suggest that low testosterone and obesity are interactive regulators of neuroinflammation that, in combination with adipose-derived inflammatory pathways and other factors, increase the risk of downstream disorders including type 2 diabetes and Alzheimer¿s disease.
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R E S E A R C H Open Access
Diet-induced obesity and low testosterone
increase neuroinflammation and impair neural
function
Anusha Jayaraman, Daniella Lent-Schochet and Christian J Pike
*
Abstract
Background: Low testosterone and obesity are independent risk factors for dysfunction of the nervous system
including neurodegenerative disorders such as Alzheimers disease (AD). In this study, we investigate the
independent and cooperative interactions of testosterone and diet-induced obesity on metabolic, inflammatory,
and neural health indices in the central and peripheral nervous systems.
Methods: Male C57B6/J mice were maintained on normal or high-fat diet under varying testosterone conditions
for a four-month treatment period, after which metabolic indices were measured and RNA isolated from cerebral
cortex and sciatic nerve. Cortices were used to generate mixed glial cultures, upon which embryonic cerebrocortical
neurons were co-cultured for assessment of neuron survival and neurite outgrowth. Peripheral nerve damage was
determined using paw-withdrawal assay, myelin sheath protein expression levels, and Na
+
,K
+
-ATPase activity levels.
Results: Our results demonstrate that detrimental effects on both metabolic (blood glucose, insulin sensitivity) and
proinflammatory (cytokine expression) responses caused by diet-induced obesity are exacerbated by testosterone
depletion. Mixed glial cultures generated from obese mice retain elevated cytokine expression, although low
testosterone effects do not persist ex vivo. Primary neurons co-cultured with glial cultures generated from high-fat
fed animals exhibit reduced survival and poorer neurite outgrowth. In addition, low testosterone and diet-induced
obesity combine to increase inflammation and evidence of nerve damage in the peripheral nervous system.
Conclusions: Testosterone and diet-induced obesity independently and cooperatively regulate neuroinflammation in
central and peripheral nervous systems, which may contribute to observed impairments in neural health. Together, our
findings suggest that low testosterone and obesity are interactive regulators of neuroinflammation that, in combination
with adipose-derived inflammatory pathways and other factors, increase the risk of downstream disorders including
type 2 diabetes and Alzheimers disease.
Keywords: Central nervous system, Diet-induced obesity, Glia, Neuroinflammation, Peripheral nervous system,
Testosterone
Background
Normal aging is associated with a wide range of physio-
logical changes that independently and cooperatively im-
pact the functioning of the nervous system. One such
age change is the depletion of testosterone in men. Age-
related testosterone loss is linked to dysfunction and dis-
ease in several androgen-responsive tissues including
adipose tissue and brain [1,2]. In brain, low testosterone
is associated with significant impairment in select as-
pects of cognition in aging men [3,4], which rodent
studies suggest could reflect the loss of testosterone
regulation of behaviors [5,6], synapse formation [7], and
neuron survival [8,9]. Further, low testosterone is a risk
factor for Alzheimers disease (AD) as defined by both
clinical [10-13] and neuropathological [14,15] diagnoses.
In the peripheral nervous system, experimentally-induced
low testosterone levels in male rats are associated with
decreased expression of myelin sheath protein, which
contribute to several demyelinating disorders [16].
* Correspondence: cjpike@usc.edu
Davis School of Gerontology, University of Southern California, 3715
McClintock Avenue, Los Angeles, CA 90089, USA
JOURNAL OF
NEUROINFLAMMATION
© 2014 Jayaraman et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.
Jayaraman et al. Journal of Neuroinflammation 2014, 11:162
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Testosterone and its derivatives have been shown to be
protective against experimental diabetic neuropathy by
reversing several of the detrimental effects of low tes-
tosterone and diabetes in male rats [17]. In men with
diabetes, low levels of testosterone correlate signifi-
cantly with increases in neuropathy as compared to
those with normal testosterone levels [18].
A second age-related change associated with poor
neural outcomes is increasing adiposity. Waist circum-
ference, body mass index, and prevalence of obesity
have been shown to increase with age [19]. Obesity is a
significant risk factor for development of metabolic syn-
drome, a collective term that includes dyslipidemia,
hyperinsulinemia, and glucose intolerance [20,21]. Fur-
ther, obesity is associated with inflammatory responses
[22] as well as endocrine changes leading to lower tes-
tosterone levels [23]. Obesity and metabolic syndrome
are also associated with increased risk for disorders in-
cluding type 2 diabetes (T2D) and AD [24-27]. In the
brain, high-fat diet has been shown to accelerate cogni-
tive decline and increase insulin-resistance [28]. In the
peripheral nervous system, obesity is shown to be an
important factor for development of neuropathy in T2D
patients [29].
Interestingly, testosterone and obesity are interactive
factors that may cooperatively regulate a wide range of
health measures, including nervous system function. For
example, epidemiological studies have shown that men
with low testosterone have higher risk of developing
metabolic syndrome [30,31] and T2D [32,33]. On the
other hand, central obesity and T2D reduce testosterone
levels [34-37]. Moreover, testosterone therapy has been
shown to reduce adiposity and T2D [38,39]. Conversely,
androgen deprivation therapy for prostate cancer treat-
ment increases the risk for metabolic syndrome and
T2D [40-43].
Given the significant independent contributions of
obesity and low testosterone on neural outcomes, it is
important to consider the downstream effects of both
these risk factors when present together. In this study,
we investigate interactions between obesity and low tes-
tosterone levels on metabolic indices and neuron health
in both central (CNS) and peripheral nervous system
(PNS) using hormone and diet manipulations in wild-
type male mice. We also examine potential contributions
of inflammatory pathways in hormone and diet-induced
changes in the treated animals.
Methods
Materials
Testosterone was purchased from Steraloids (Newport,
RI, USA), solubilized in 100% ethanol, and stored at 80°C.
Glucose (Life Technologies, Carlsbad, CA, USA) was dis-
solved in sterile water to a concentration of 0.2 g/mL.
Irradiated control (10% kcal fat; Cat#D12450Bi) and
high-fat (60% kcal fat; Cat#D12492i) diets were pur-
chased from Research Diets, Inc. (New Brunswick, NJ,
USA). EDTA, bovine serum albumin, NaCl, and
Na
2
HPO
4
were purchased from Thermo Fisher Scien-
tific (Hudson, NH, USA). Imidazole was purchased from
SantaCruzBiotechnology,Inc.(Dallas,TX,USA).All
other chemicals and reagents were purchased from
Sigma-Aldrich (St. Louis, MO, USA).
Animal procedures
For in vivo studies and primary glia cultures, male
C57BL6 mice were purchased gonadectomized (GDX)
and sham-GDX at 3 months of age (The Jackson
Laboratory, Sacramento, CA, USA). All animals were
housed individually with ad libitum access to food and
water under a 12-h light/dark cycle. All animal pro-
cedures were conducted under a protocol that was ap-
proved by the USC Institutional Animal Care and Use
Committee and in accordance with National Institute
of Health standards.
For testosterone treatment, GDX male mice were im-
planted subcutaneously with a 30 mm length Silastic
capsule (1.47 mm ID x 1.96 mm OD; Dow Corning,
Midland, MI, USA) packed with dry testosterone to a
length of 20 mm and capped on both ends with 5 mm
of silicone glue. This capsule length has been previously
demonstrated to deliver physiological levels of testoster-
one in male mice [44]. In a separate group of male mice,
we found that this treatment yielded serum testosterone
levels of 4.2 ± 0.3 ng/mL. The vehicle-treated animals
were implanted with an empty capsule with the same di-
mensions. The diet treatments were started 1 week after
GDX surgery and continued for 4 months. Prior to start-
ing the diet treatments, base-line body weight and over-
night fasting blood glucose measurements were recorded
for all animals. Thereafter, body weights and food intake
were measured weekly, and fasting blood glucose levels
were measured every 4 weeks. Behavioral tests were con-
ducted 1 week prior and glucose tolerance test 2 days
prior to the end of the treatment period.
At the end of 4 months, mice were euthanized by
CO
2
inhalation. Brains were removed and hemisected:
one-half cortex was used to generate primary glia cul-
tures, the other half was snap-frozen on dry ice for
RNA extraction and RT-PCR analyses. The sciatic
nerves were dissected from both legs and snap frozen
for RNA extraction, cryosectioning (for immunostaining),
and for preparing lysates (for sodium potassium ATPase
(Na
+
,K
+
-ATPase) assay).
Glucose tolerance test
After overnight fasting, mice received a bolus of D-
glucose (2 g/kg body weight) through oral gavage. Baseline
Jayaraman et al. Journal of Neuroinflammation 2014, 11:162 Page 2 of 14
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fasting blood glucose was recorded prior to D-glucose ad-
ministration and subsequent blood glucose levels were re-
corded 15, 30, 60, and 120 minutes after D-glucose
administration. Area under the curve (AUC) was calcu-
lated using GraphPad Prism Software v5.02.
RNA isolation and real-time PCR
For RNA extractions, cortex and sciatic nerve from each
treated animal and primary glia cultures were homoge-
nized using TRIzol reagent (Invitrogen Corporation,
Carlsbad, CA, USA) and processed for total RNA extrac-
tion as per manufacturers protocol, as previously de-
scribed [45]. Purified total RNA (1 μg) was used from
each sample for reverse transcription using the iScript
cDNA synthesis system (Bio-Rad, Hercules, CA, USA)
and the resulting cDNA was used for real-time quanti-
tative PCR carried out using Bio-Rad CFX Connect
(Bio-Rad). Relative quantification of mRNA levels from
various treated samples was determined by the ΔΔCt
method [46] after normalizing with the corresponding
β-actin levels from samples. In addition, the PCR prod-
ucts were qualitatively analyzed by electrophoresis
using 1% agarose gels. The following primer pairs were
used: tumor necrosis factor alpha (TNFα), forward: 5-
GCCTGTAGCCCACGTCGTAG-3,reverse:5-TTG
GGCACATTGACCTCAGC-3; interleukin-1β(IL-1β),
forward: 5-CCCAAGCAATACCCAAAGAA-3,re-
verse: 5-GCTTGTGCTCTGCTTGTGA-3;P0,for-
ward: 5-TGTGGTTTACACGGACAGGG-3,reverse:
5-AGAGCAACAGCAGCAACAG-3;β-actin, for-
ward: 5-A G CCATGTACGTAGCC ATCC- 3 ,reverse:
5-CTCTCAGCTGTGGTGGTGAA-3.
Primary glia cultures and neuron-glia co-cultures
Adult primary mixed glia were obtained according to
previously described protocol [47] from the cortex of
each individual mouse. Dissected cortices were mechan-
ically dissociated, then plated onto poly-D-lysine coated
flasks containing DMEM-F12/20% FBS and placed in a
humidified incubator at 37°C with 5% CO
2
. The medium
was changed every three days until the cultures were
grown to confluency. Confluent cultures were re-plated
onto poly-D-lysine-coated 24-well plates. The cultures
were shifted to serum-free DMEM/F12 1 to 3 days prior
to use in experiments. For neuron-glia co-culture studies,
timed-pregnant female C57BL6 mice (Harlan Laboratories
Inc., Livermore, CA, USA) were killed via CO
2
inhalation
and embryonic day 16 to 17 pups were collected for prep-
aration of neuronal cultures. Primary cortical neurons
were plated on the mixed glia at a density of 2.5 × 10
4
cells/cm
2
for cell viability assays, and 0.5 × 10
4
cells/cm
2
for neurite outgrowth studies. A parallel set of primary
cortical neurons were plated at similar densities directly
on poly-D-lysine coated 24-well plates and maintained in
conditioned media collected from the mixed glial cultures.
Cell viability and neurite outgrowth
For neuron viability and neurite outgrowth experiments,
cells were fixed with ice-cold 4% paraformaldehyde 24 to
48 h after plating neurons. The fixed cells were immu-
nostained with the neuron-specific marker β-tubulin III
antibody (5 μg/mL; R&D Systems, Minneapolis, MN,
USA) overnight at 4°C then processed with standard
avidin: biotinylated enzyme complex immunocytochem-
istry using the Vector Elite ABC kit (Vector Laborato-
ries, Burlingame, CA, USA); labeling was visualized by
diaminobenzidine. Stained cultures were rinsed and
stored in ice cold PBS until quantitation. For determin-
ing neuron survival in both neuron-glia co-cultures and
neuron-only cultures, stained cells were counted in four
separate fields (in a predetermined, regular pattern) per
well, three wells per condition. For determining the ex-
tent of neurite outgrowth and average neurite length of
neurons, 50 cells per condition were examined. The
total number of neurites per cell and the average neur-
ite length in each cell were determined using Neuron J
software v.1.4.2.
Macrophage infiltration
Twenty 40-mm length pieces of sciatic nerve from each
treated animal were fixed in 4% PBS-buffered paraformal-
dehyde for 2 h at 4°C, rinsed with PBS, and then stored
overnight in 20% sucrose solution. These tissues were then
embedded in optimal temperature cutting compound and
rapidly frozen. Seven-micron thick transverse-sections
were cut with cryostat microtome CM1800 (Leica Micro-
systems Inc., Buffalo Grove, IL, USA) and mounted on
microscope slides. Sections were immunostained with an
antibody against the macrophage/microglia-specific Iba1
protein (1:500, Wako Chemicals, Richmond, VA, USA)
overnight at C and processed for immunohistochemistry
as described in the previous section. Slides were rinsed,
dehydrated through a graded series of alcohols, and cover-
slipped with permanent mounting medium.
Thermal nociceptive threshold
The nociceptive threshold to heat was measured using a
paw withdrawal assay. A plexiglass chamber was placed
over a hotplate and the temperature was maintained at
20°C. After placing the animal in the chamber, the
temperature was gradually increased at the rate of 5°C/
min until reaching a 50°C maximum. The threshold was
measured as the temperature at which the animal shows
the first sign of discomfort (i.e., paw withdrawal or lick-
ing of hind paw). For paw withdrawal latency measure-
ments, the hotplate was maintained at 50°C and the
latency was measured as the time from placement in the
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chamber until the animal displayed the first signs of dis-
comfort. Animals were tested twice for each measure-
ment with an interval of 5 min between repeats and an
interval of at least 30 m in between threshold measure-
ments and latency measurements.
Na
+
,K
+
-ATPase assay
Na
+
,K
+
-ATPase activity was measured using sciatic
nerve samples homogenized in a chilled solution of
0.25 M sucrose, 6 mM EGTA, and 10 mM Tris, at
pH 7.5. Na
+
,K
+
-ATPase activity was determined colori-
metrically at 700 nm using Spectramax 250 microplate
reader (Molecular Devices, Sunnyvale, CA, USA) as pre-
viously described [48]. Optical density values were ana-
lyzed using SoftMax Pro 5 software. Protein content in
homogenates was determined by bicinchoninic acid
method (Promega) with bovine serum albumin as
standard.
Statistical analyses
Raw data were statistically analyzed using two-way
ANOVA to identify simple main effects of diet and hor-
mone status and diet X hormone interactions. Signifi-
cant main effects were subsequently analyzed using
Bonferroni test to compare between-group differences.
Significance was indicated by P0.05.
Results
Low testosterone and high-fat diet increases metabolic
indices
To determine the effects of high-fat diet and low testos-
terone on obesity and T2D, we investigated several
metabolic indices. There was a significant main effect of
diet (F
1,40
= 138.3; P<0.001) but not testosterone (F
2,40
=
0.48; P= 0.62) in mice where high-fat diet was associated
with a significant increase in body weight (Figure 1A).
Fasting blood glucose levels showed significant effects
for both diet (F
1,40
= 73.0; P<0.01) and testosterone
(F
2,40
= 8.5; P= 0.001). Interestingly, GDX caused in-
creases in fasting blood glucose in high-fat diet treat-
ment as compared to the respective sham-GDX groups,
effects that were significantly reversed by testosterone
treatment. A similar non-significant trend was also seen
in the control diet animals (Figure 1B). Fasting insulin
levels significantly increased with diet (F
1,38
= 16.2;
P<0.001) and not testosterone (F
2,38
= 1.6; P= 0.22),
with higher fasting insulin values in the high-fat diet-fed
sham-GDX as compared to the sham-GDX control diet
animals. We also observed a non-significant trend of re-
duced fasting insulin levels by testosterone in both the
diet groups (Figure 1C). Similar results were observed
with HOMA index, a measure of insulin resistance
based upon glucose and insulin levels where only a sig-
nificant main effect of diet (F
1,38
= 21.1; P<0.001) was
seen between the sham groups, and testosterone treat-
ment showed a non-significant reversal of this effect
(Figure 1D). In glucose tolerance test, both diet (F
1,39
=
14.2; P<0.001) and hormone (F
2,39
= 9.7; P<0.001)
showed significant changes. The high-fat diet sham ani-
mals showed a significant increase in AUC as compared
to the control diet sham animals. Castration caused a
non-significant increase in AUC that was reversed
by testosterone treatment in the high-fat diet group
(Figure 1E). No significant interactions between diet and
hormone status simple main effects were observed in
any of the metabolic measures.
Low testosterone and high-fat diet increase proinflammatory
cytokines in cortex
To determine whether diet and hormone manipulations
affect markers of neuroinflammation, we examined the
effects of diet and hormone status on cerebrocortical
mRNA levels of the proinflammatory cytokines TNFα
and IL-1βacross all groups. We observed a statistically
significant main effect of diet on mRNA levels of TNFα
(F
1,36
= 18.2; P<0.001) and IL-1β(F
1,38
= 28.2; P<0.001),
with high-fat diet associated with elevated levels relative
to matched control diet groups (Figure 2AC). We also
observed significant changes in the mRNA levels
of TNFα(F
2.36
= 5.8; P<0.01) and IL-1β(F
2,38
= 12;
P<0.001) due to hormone status, with GDX causing ele-
vated levels in both diet groups, which was reversed by
testosterone treatment (Figure 2AC). There was no sig-
nificant interaction between diet and hormone simple
main effects for TNFαlevels. However, a significant
interaction was observed for IL-1βmRNA levels (F
2,38
=
3.5; P= 0.04).
Low testosterone and high-fat diet increase proinflamma-
tory cytokines in primary glia cultures
To determine whether diet and hormone manipula-
tionshaveaneffectonneuroinflammationinmixed
glial cultures, we examined the effects of diet and hor-
mone changes on mRNA levels of proinflammatory cy-
tokines TNFαand IL-1βin the primary glial cultures
prepared from the cortices of the treated animals. We
observed statistically significant main effects of only
diet (F
1,12
=33.4; P<0.001) and not hormone (F
2,12
=
2.1; P= 0.17) on mRNA levels of TNFαin comparing
the levels in the glial cultures from both diet groups.
However, there was a significant decrease in TNFα
mRNA levels with testosterone treatment in the high-
fat animals (P<0.05) (Figure 3A). IL-1βmRNA levels
were also significantly affected by diet (F
1,12
= 49.5;
P<0.001) but not hormones (F
2,12
=0.2; P=0.82)
(Figure 3B). Moreover, no significant interactions were
observed between diet and hormone status simple
main effects for either TNFαor IL-1βmRNA levels.
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High-fat diet alters glia-mediated neuron survival
We investigated whether the changes in the level of in-
flammatory markers observed in the glial cultures de-
rived from cortices of animals under the two diets
affected neuron survival differently. Primary cortical
neurons from E17 mouse pups were co-cultured with
mixed glia cultured from treated animals. We ob-
served a significant main effect of diet alone on
neuron viability in the neuron-glia co-cultures (F
1,12
=
43.4; P<0.001) (Figure 4AC). Interestingly, primary
neurons growing in the conditioned media from these
high-fat derived glia also showed lower viability as
compared to those growing in the conditioned media
from control-diet derived glia (F
1,12
= 22.7; P<0.001)
(Figure 4D). In both co-culture and conditioned media
experiments, there were no significant changes seen
due to hormone status (Figure 4C,D). No interaction
between diet and hormone status simple main effects
was seen either in the co-cultures or with conditioned
media.
High-fat diet alters neurite outgrowth number and length
To determine whether high-fat diet affects neurite
number and neurite length, neurons were examined
under co-culture and conditioned media paradigms.
We observed that neurons growing on high-fat diet
Figure 1 Low testosterone and high-fat diet increases metabolic indices. (A) Graphical representation of the percentage differences in
average body weight of animals in sham GDX (Sham), GDX, and GDX mice with testosterone treatment (GDX + T) in both control-diet and
high-fat diet. (B, C). Graphical representations of average fasting blood glucose and insulin in each treated group. ((D) Graphical representation of
HOMA index, which is representative of level of insulin resistance in each group. (E) Graphical representation of area under the curve (AUC) for
glucose tolerance test (0 to 120 min) from each group of animals. Statistical significance is based on ANOVA followed by Bonferroni. * P0.05
between the diet groups; ** P0.05 between hormone groups; N 6.
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derived glia (F
1,18
= 121.2; P<0.001) or the conditioned
mediaobtainedfromthem(F
1,18
= 102.5; P<0.001) had
fewer numbers of neurites as compared to those grow-
ing on control-diet derived glia/conditioned media
(Figure 5AD). Moreover, the average length of neu-
rites was significantly shorter in neurons growing
on high-fat diet-derived glia (F
1,27
=7.6; P<0.05)
(Figure 5E). Interestingly, there were no differences in
average neurite lengths in neurons growing in either of
the conditioned media (Figure 5F). As in the case of
neuron survival, hormone status of the animals from
which the glia were derived did not have an effect on
neurite numbers and lengths (Figure 5CF). No signifi-
cant interaction was observed in neurite numbers or
average neurite lengths in co-cultures and conditioned
media.
Figure 2 Low testosterone and high-fat diet increase
proinflammatory cytokines in cortex. (A) Representative agarose
gel of RT-PCR products shows the relative levels of TNFα,IL-1β,and
β-actin mRNAs in sham GDX (Sham), GDX, and GDX mice with
testosterone treatment (GDX+ T) in both control-diet and high-fat diet.
(B) Quantitative real-time PCR data show the mean SEM) expression
levels compared to the Sham control group for TNFαmRNA.
(C) Quantitative real-time PCR data show the mean (±SEM) expression
levels compared to the Sham control group for IL-1βmRNA. All data
are normalized with corresponding β-actin values. Statistical
significance is based on ANOVA followed by Bonferroni. * P0.05
between diet groups; ** P0.05 between hormone groups; N 6.
Figure 3 High-fat diet increase proinflammatory cytokines in
primary glial culture. (A) Quantitative real-time PCR data show the
mean (±SEM) expression levels compared to the Sham control
group for TNFαmRNA. (B) Quantitative real-time PCR data show the
mean (±SEM) expression levels compared to the Sham control
group for IL-1βmRNA. All data are normalized with corresponding
β-actin values. Statistical significance is based on ANOVA followed
by Bonferroni. * P0.05 between diet groups; ** P0.05 between
hormone groups; N 6.
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Low testosterone and high-fat diet increase macrophage
infiltration in the sciatic nerve
To investigate the effect of diet and hormone manipu-
lations in the PNS, sciatic nerve sections from treated
animals were examined for macrophage infiltration,
which is one of the key signs of peripheral inflammatory
response. We observed significant main effects of both
diet (F
1,19
= 11.6; P<0.01) and hormone status (F
2,19
=
4.8; P<0.05) on macrophage infiltration in the sciatic
nerve, with high-fat diet and GDX independently in-
creasing the number of macrophages in the sciatic nerve
sections (Figure 6A,B,D,E,G). Testosterone treatment in
both the diet groups reversed the macrophage levels al-
though non-significantly (Figure 6C,F,G). The interaction
between diet and hormone status simple main effects
was also not significant.
Low testosterone and high-fat diet increase proinflammatory
cytokines and decrease myelin sheath marker in sciatic
nerve
To determine whether the low testosterone levels and
high-fat diet alter proinflammatory cytokines in the PNS
as they did in the cortex, mRNA levels of TNFαand IL-
1βwere observed in the sciatic nerve samples. Our
results showed a significant increase in TNFαmRNA
expression by both diet (F
1,30
=13.4; P= 0.001) and
hormone (F
2,30
= 12.4; P<0.001), which was reversed
by testosterone treatment in both the diet-fed animals
(P<0.05) (Figure 7A,B). Similarly, IL-1βmRNA was
significantly affected by diet (F
1,12
=12.5; P<0.05) and
hormone (F
2,12
=4.2; P<0.05). Testosterone treatment
decreased IL-1βmRNA expression in both diet groups,
with significant effects in the control-diet animals
(P<0.001). In addition, we investigated the effect of
low testosterone and high-fat diet on myelin sheath
protein (P0) in the sciatic nerve, which is shown to de-
crease in case of diabetic neuropathy [49]. We ob-
served that P0 mRNA levels significantly changed with
hormone status (F
2,18
= 18.5; P<0.001), with GDX
decreasing the P0 mRNA levels, which was restored
with testosterone treatment in both the diet groups
(Figure 7C). We also observed a decrease in P0 mRNA
in high-fat diet sham animals as compared to the con-
trol diet sham animals (P<0.01). There was significant
interaction observed between diet and hormone status
for TNFαmRNA levels (F
2,30
=3.3; P= 0.05) and P0
mRNA levels (F
2,18
=9.4; P= 0.0016). However, no
such interaction was seen for IL-1βmRNA expression.
Figure 4 High-fat diet derived glia cultures support reduced neuron viability. Primary cortical neurons were plated either by themselves or
on confluent primary glial cultures derived from the cortices of sham GDX (Sham), GDX, and GDX mice with testosterone treatment (GDX + T)
in both control-diet and high-fat diet. Top panels show representative pictures of neurons co-cultured with control diet (A) and high-fat diet
(C) derived glia. (B) Quantitative graph for percentage of neurons per treatment group in the neuron-glia co-culture. (D) Quantitative graph
for percent neuron viability in conditioned glial media from each group. Data show mean cell viability (±SEM) of a representative experiment.
*P0.05 between diet groups; ** P0.05 between hormone groups; N = 3.
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Low testosterone and high-fat diet promote hyperalgesia
and Na
+
,K
+
-ATPase activity in sciatic nerve
To investigate whether low testosterone and high-fat
diet promote other markers of peripheral nerve damage,
we did a hot plate paw withdrawal assay one week prior
to the end of the treatment period. We observed that
diet (F
1,38
= 6.2; P<0.05), but not hormone (F
2,38
= 1.8;
P= 0.18), induced significant changes in the threshold to
heat between different animal groups. Interestingly,
hyperalgesia was induced in the respective groups of
animals as observed by significant main effects of both
diet (F
1,38
= 28.9; P<0.001) and hormone (F
2,38
= 6.3;
P<0.005) on latency to heat in these animals (Figure 8A,
B). Testosterone treatment was able to reverse these in
both diet conditions (Figure 8A,B). We also looked at
the Na
+
,K
+
-ATPase activity in the sciatic nerve samples
from the different animal groups. We observed that
hormone status altered the Na
+
,K
+
-ATPase activity levels
(F
2,32
= 10.1; P<0.001), with GDX lowering the enzyme
activity levels in both diet conditions which was rescued
by testosterone treatment (Figure 8C). Diet and hor-
mone status together had no interactive effect on thresh-
old and latency to heat as well as Na
+
,K
+
-ATPase
activity.
Figure 5 High-fat diet-derived glia cultures yield reduced neurite outgrowths. Primary cortical neurons were plated either by themselves or
on confluent primary glial cultures derived from the cortices of sham GDX (Sham), GDX, and GDX mice with testosterone treatment (GDX + T) in
both control-diet and high-fat diet. Top panels show representative pictures of neurite outgrowths on neurons co-cultured with (A) control
diet- and (B) high-fat diet-derived glia. Quantitative graphs show mean (C) number of neurites and (E) length of neurites per neuron across
treatment groups in the neuron-glia co-cultures. Quantitative graphs show mean (D) number of neurites and (F) length of neurites per neuron
in conditioned glial media from each group. Data show mean values (±SEM) of a representative experiment. * P0.05 between diet groups;
** P0.05 between hormone groups; N = 3.
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Discussion
Prior work has identified obesity and low testosterone
as risk factors for the development of the metabolic
syndrome [50-53] and T2D [54-57]. Interestingly, both
obesity and low testosterone are also risk factors for
neural dysfunction, including cognitive impairment
[58-61] and development of AD [10,11,14]. Levels of
obesity and testosterone are often inversely correlated,
suggesting the possibility that they are interactive fac-
tors [62]. In this study, we investigated the individual
and combined effects of obesity and testosterone status
on neural outcomes. Our results demonstrate that diet-
induced obesity causes significant metabolic disturbances
and impairs central and peripheral nervous systems. Tes-
tosterone status also affected metabolic and neural mea-
sures. In general, obesity-related changes were worsened
by low testosterone and improved by testosterone treat-
ment; however, this relationship was not statistically sig-
nificant in several instances. Further, our data suggest that
a common pathway that may contribute to obesity and
testosterone effects is regulation of inflammation.
We observed that metabolic measures were affected by
both diet-induced obesity and testosterone status. In some
cases, fasting blood glucose levels were independently and
additively increased by GDX-induced testosterone deple-
tion and high-fat diet. Importantly, testosterone treatment
significantly reduced fasting glucose under both the
normal and high-fat diets, demonstrating potential
therapeutic efficacy of testosterone supplementation.
For measures of fasting insulin, insulin resistance
(HOMA index), and glucose tolerance, low testoster-
one tended to exacerbate and or testosterone treat-
ment improved outcomes. Because testosterone status
did not significantly affect body weight, testosterones
effects likely do not indicate an indirect result on adi-
posity but rather regulatory action(s) on other aspects
of metabolic homeostasis. Prior work in rodents has
shown diet-induced obesity induces insulin resistance
in rat brain [63] and that testosterone replacement im-
proves insulin sensitivity in obese rats [64]. Our findings
are consistent with the human literature, which indicates
that (i) testosterone levels are inversely correlated to
Figure 6 Low testosterone and high-fat diet increase macrophage infiltration in sciatic nerve. (AF) Top panels are the representative
pictures showing amount of macrophage infiltration in sciatic nerve sections in each treatment condition. (G) Quantitative graph showing the
percentage of macrophages present in the sciatic nerve sections from each treatment group. * P0.05 between diet groups; ** P0.05 between
hormone groups; N 6.
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insulin resistance and T2D in healthy [30,65] as well as
obese men [66], and (ii) androgen therapy can improve
some metabolic measures in overweight men with low tes-
tosterone [67-69].
In addition to impairing metabolic function, both
obesity and low testosterone are linked with promotion
of inflammatory pathways [70-72] and exert harmful ac-
tions on the central [73-75] and peripheral [29,76] ner-
vous systems. To investigate these relationships and
their potential interactions, we first examined the effects
of experimentally-induced low testosterone and obesity
on brain levels of two established pro-inflammatory
markers, TNFα[77] and IL-1β[78]. Our data demon-
strate that low testosterone and obesity independently
increased cerebrocortical mRNA levels of both TNFα
and IL-1β. Although there was not a statistically signifi-
cant additive effect of the two factors on cytokine ex-
pression, testosterone treatment significantly lowered
TNFαand IL-1βexpression to near basal levels even in
obese mice, indicating a protective benefit of testoster-
one across diet conditions. Similar to the observations
on metabolic outcomes, our findings on cytokine expres-
sion are consistent with both individual and inter-related
effects of testosterone and obesity. Because many benefi-
cial effects of testosterone, including inhibition of proin-
flammatory cytokine expression [79] and neuroprotection
[80,81], are dependent upon androgen receptors, the ob-
served effects of testosterone in this study may involve an-
drogen receptor activation. However, testosterone can be
converted by the enzyme aromatase into estradiol, which
is also known to exert anti-inflammatory [82] and neuro-
protective [83] actions. Additional research will be needed
to elucidate the relative contributions of androgen and es-
trogen pathways to the observed relationships between
testosterone, obesity, inflammation, and neural outcomes.
Because glia are the primary sources of proinflammatory
molecules in the CNS, we considered whether glia may
contribute to the established neural effects of low testos-
terone and obesity. We generated mixed glial cultures
from treated mice, an established paradigm in which glial
phenotype established in vivo is retained in culture
[47,84,85], then plated upon them naïve embryonic cere-
brocortical neurons. An advantage of this approach is that
it allows isolated assessment of glial effects on neuronal
health without influence of systemic alterations associated
with obesity (e.g., alterations in glucose and insulin signa-
ling). We observed significantly poorer survival of neurons
grown on glia from mice maintained on high-fat diet as
well as significant reductions in the numbers and lengths
of neurites. Since testosterone can affect glial function
[86] and improve neuronal growth and survival [87-89], it
was unexpected that testosterone status exhibited rather
modest effects on neural health indices with the only
significant response being an increase in survival in the
Figure 7 Low testosterone and high-fat diet increase
proinflammatory cytokines TNFαand IL-1b mRNA and decrease
myelin sheath marker P0 mRNA in sciatic nerve. (A) Quantitative
real-time PCR data show the mean (±SEM) expression levels compared
to the Sham control group for TNFαmRNA. (B) Quantitative real-time
PCR data show the mean (±SEM) expression levels compared to the
Sham control group for IL-1βmRNA. (C) Quantitative real-time PCR
data show the mean (±SEM) expression levels compared to the
Sham control group for P0 mRNA. All data are normalized with
corresponding β-actin values. Statistical significance is based on
ANOVA followed by Bonferroni. * P0.05 between diet groups;
** P0.05 between hormone groups; N 6.
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testosterone-treated, high-fat diet group. Note that tes-
tosterone status was controlled in vivo but not in vitro,
suggesting the possibility that its effects on glia and
neurons require maintained exposure to the hormone.
Importantly, the inhibitory effects of cultured glia on
neurons were largely reproduced by exposing neuron
cultures to media conditioned by glial cultures generated
from mice maintained on a high-fat diet. Both neuron
survival and neurite number were reduced to similar
levels in neurons either co-cultured with glia from fat-
fed mice or conditioned media from the fat-fed glial cul-
tures. This finding suggests that soluble factors, possibly
including toxic proinflammatory cytokines secreted by
astrocytes and/or microglia, adversely affected neuronal
viability. Consistent with this possibility, we observed
significantly increased expression of TNFαand IL-1βin
glia cultures derived from obese mice. In prior work, it
has been shown that TNFαhas inhibitory effects on
neuron survival, differentiation, and neurite outgrowth
[90-92]. Similarly, IL-1βtreatment has been shown to
induce synapse loss and inhibit differentiation of neu-
rons [93,94]. Interestingly, mean neurite length was not
reduced by the conditioned media, indicating a signifi-
cant contribution of glial cell surface components in the
regulation of neurite length.
We also considered the possible effects of low testos-
terone and obesity on health and functioning of the
PNS. A major PNS-related complication of T2D is dia-
betic neuropathy. Peripheral diabetic neuropathy in-
volves several changes, including increased induction of
inflammatory cytokines, macrophage infiltration, de-
creased expression of P0, thermal nociception, and bio-
chemical changes in the nerves [95]. In our diet-induced
pre-diabetes model, we investigated some of these
changes in the sciatic nerves of treated animals. Testos-
terone status and diet-induced obesity were associated
with significant regulation of macrophage infiltration,
mRNA levels of IL-1β, TNFα, and P0, and activity levels
of Na
+
,K
+
-ATPase. For example, GDX-induced low tes-
tosterone and diet-induced obesity each independently
decreased the expression levels of P0. Although there
was no an additive effect of these factors, testosterone
treatment in both control and high-fat diet groups
Figure 8 Low testosterone and high-fat diet promote hyperalgesia
and Na
+
,K
+
-ATPase activity in sciatic nerve. (A) Quantitative graph
shows the mean (±SEM) temperature threshold for sham GDX (Sham),
GDX, and GDX mice with testosterone treatment (GDX + T) in both
control-diet and high-fat diet fed mice. (B) Quantitative graph show the
mean (±SEM) latency to thermal nociception (at 50°C) in different animal
groups. (C) Quantitative graph shows the mean (±SEM) activity levels
of Na
+
,K
+
-ATPase in the sciatic nerve of animals in the different
treatment groups. Statistical significance is based on ANOVA
followed by Bonferroni. * P0.05 between diet groups; ** P0.05
between hormone groups; N 6.
Jayaraman et al. Journal of Neuroinflammation 2014, 11:162 Page 11 of 14
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significantly increased P0 expression to values observed
in the sham-GDX control-diet group, demonstrating
protection effect of testosterone across conditions. Simi-
larly, testosterone reduced IL-1βand TNFαlevels and
improved the activity of Na
+
,K
+
-ATPase that was re-
duced by both GDX and high-fat conditions. These find-
ings are in accordance with previous studies that have
correlated the decreased expression of P0 and Na
+
,
K
+
-ATPase activity to the onset of peripheral neuropathy
in several animal models of diabetes [17,96] and in per-
sons with T2D [97,98].
One clinical feature of peripheral neuropathy that is also
observed in many animal models of diabetes is hyperalge-
sia [99,100]. For this functional endpoint, we observed
threshold hyperalgesia in GDX animals that was exacer-
bated by high-fat diet. Importantly, testosterone prevented
and/or restored thermal nociception in both diet groups.
The underlying mechanism for changes in thermal noci-
ception has been linked to inflammatory pathways. For ex-
ample, up-regulation of TNFαin both CNS and PNS
results in development of hyperalgesia [101,102]. Similarly,
IL-1βhas also been shown to induce pain hypersensitivity
by activating nociceptors [103]. Our observations of IL-1β
and TNFαchanging in parallel with thermal nociception
are consistent with prior observations of a mechanistic
link between inflammation and hyperalgesia.
Conclusions
Our study provides novel insights into the individual and
interactive effects of low testosterone- and diet-induced
obesity on nervous system function. The most significant
observation is that the combination of low testosterone
and obesity worsen several metabolic and inflammatory
indices, which in turn are largely reversed by testosterone
treatment. The inverse relationship between various end-
points and proinflammatory cytokine levels suggests a
possible mechanism by which obesity and testosterone
levels may affect the health of both CNS and PNS. Low
testosterone and obesity are commonly present together
in many middle-age and aged men. Our findings suggest
that these factors have individual and combined effects.
Continued investigation of the interplay between these
two factors in the nervous system would be beneficial not
only in increasing our understanding of their long-term
impact, but also in designing and optimizing potential
hormone-based approaches as a therapeutic strategy to
overcome some of their negative outcomes.
Abbreviations
AD: Alzheimers disease; AUC: Area under the curve; CNS: Central nervous
system; GDX: Gonadectomized; IL-1β: Interleukin-1β;Na
+
,K
+
-ATPase: Sodium
potassium ATPase; P0: Myelin protein 0; PNS: Peripheral nervous system;
T2D: Type 2 diabetes; TNFα: Tumor necrosis factor α.
Competing interests
The authors declare that they have no competing interests.
Authorscontributions
AJ was primarily responsible for the animal work, culture studies and
associated assays, immunohistochemistry, behavioral test, biochemical assay,
statistical analyses, and drafting the manuscript, and contributed to the RNA
work and experimental design. DLS contributed to RNA isolation and PCR.
CJP conceived the study and contributed to both the experimental design
and manuscript preparation. All authors read and approved of the
manuscript.
Acknowledgements
The authors thank Drs. Amy Christensen, Joo-Won Lee, Radhika Palhar, and
David McKemy for technical assistance. This study was supported by NIH
grant AG034103 (CJP).
Received: 18 March 2014 Accepted: 28 August 2014
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doi:10.1186/s12974-014-0162-y
Cite this article as: Jayaraman et al.:Diet-induced obesity and low
testosterone increase neuroinflammation and impair neural function.
Journal of Neuroinflammation 2014 11:162.
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... Analyzed from a possible molecular mechanism standpoint, potential factors related to brain changes and cognition may be explained by the release of different secretory factors from different fat deposits (55,56). These different fat deposits release different secreted factors that can cross the blood-brain barrier (BBB) and cause damage, increase cognitive impairment and accelerate AD progression (55). ...
... Analyzed from a possible molecular mechanism standpoint, potential factors related to brain changes and cognition may be explained by the release of different secretory factors from different fat deposits (55,56). These different fat deposits release different secreted factors that can cross the blood-brain barrier (BBB) and cause damage, increase cognitive impairment and accelerate AD progression (55). Pro-inflammatory factors secreted by adipocytes, such as leptin, IL-6 and TNF-α, can cross the BBB and lead to neuroinflammation, thus playing a role in cognitive impairment and AD (56). ...
... Most of these changes occur in the volume of different brain regions and the level of cortical proteins. This series of changes points to commonalities between metabolic diseases and cognitive changes in brain injury (13,19,20,41,(55)(56)(57). Another study found that long-term high-sugar and high-fat diets can induce metabolic syndrome in experimental animals, and their brain tissue can exhibit typical characteristic changes of AD (99). ...
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