Biomedicine & Pharmacotherapy 140 (2021) 111734

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Original article

Pioglitazone rescues high-fat diet-induced depression-like phenotypes and

hippocampal astrocytic deficits in mice
Ying-Yiu Lam a, 1, Sheng-Feng Tsai b, c, 1, Pei-Chun Chen b, d, Yu-Min Kuo b, c, Yun-Wen Chen a, *
a
Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
b
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan
c
Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan
d
Department of Physiology, College of Medicine, National Cheng Kung University, Tainan, Taiwan

A R T I C L E I N F O A B S T R A C T

Keywords: The prevalence of diabetes is rapidly increasing worldwide and is highly associated with the incidence of
Astrocyte depression. Pioglitazone, a Peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist, is widely used
Antidepressant for treating patients with type 2 diabetes. However, whether pioglitazone alleviates metabolic disorder-related
Diabetes
depression and astrocytic deficits remains unclear. Here we showed that 12 weeks of high-fat diet (HFD)
Obesity
Thiazolidinedione
feeding (from 8- to 20-week-old) induced not only obesity and insulin resistance, but also depression-like be­
haviors in mice. Astrocytic activation, a sign closely associated with depression, was also evident in the ventral
hippocampus. Four weeks of pioglitazone (10 or 20 mg/kg, daily, from 20- to 24-week-old) treatment alleviated
the HFD-induced glucose-metabolic dysfunctions, upregulation of ventral hippocampal GFAP, reduction of the
total process lengths and the number of branch points of the ventral hippocampal CA1 GFAP-immunoreactive
astrocytes and depressive phenotypes but had no effect on anxiety-like behaviors or hippocampus-related
learning and memory in mice. These findings suggest that pioglitazone could be a potential therapeutic agent
for metabolic disorders and associated depression.

1. Introduction
Diabetes mellitus is a chronic disease characterized by hyperglyce­
mia with disturbances of carbohydrate, protein, and fat metabolism [1].
Major depressive disorder (MDD) is a mental illness characterized by at
least 2 weeks of low mood or anhedonia [2]. MDD displays high rates of
comorbidity with diabetes. Patients with MDD have a higher incidence
of type 2 diabetes mellitus (T2DM) when compared to the general
populations [3,4]. Patients with depression have a 32–41% increased
risk of diabetes and 8–15% of diabetic patients are diagnosed with
depressive disorders [5]. Several animal studies have shown that animal
models of diabetes and/or obesity are associated with an exhibition of
depression-like and/or anxiety-like behaviors [6–8]. But the patho­
physiological mechanisms underlying the concurrence of T2DM and
MDD are unclear.
Pioglitazone (PIO) is a thiazolidinedione antidiabetic drug that acts
as a PPARγ agonist. PPARγ gonists have been known to improve varied
neurological disorders, including Alzheimer’s disease, multiple
sclerosis, and depression [9–11]. PPAR-γ agonizts have been hypothe­
sized to influence mood status through inhibiting the expression of in­
flammatory genes and improving insulin sensitivity. PIO improves
learning and memory and reduces neuroinflammation [12,13].
Recently, it has been reported that PIO mediated the exhibition of
anti-depressant-like behaviors in mice [14]. Moreover, clinical studies
suggest that PIO could be a potential therapy for treating depression
[15]. However, we still know very little about the mechanisms under­
lying the antidepressant-like effects of PIO.
Astrocytes are abundant throughout the brain and involved in many
fundamental processes, such as synaptic transmission, neurovascular
coupling, and blood-brain barrier maintenance [16]. Astrocytes also
play an important role in the control of neurons activity and the con­
nections between synapses [17]. Astrocytes modulate both short-term
and long-term synaptic plasticity, maintain stability of synaptic scaling
and regulate hippocampal neurotransmission [18]. Reactive astrocytes
show signs of hypertrophy and increase of glial fibrillary acidic protein
(GFAP) expression under different pathological situations [19].

* Correspondence to: Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan.
E-mail address: yunwen_chen@mail.ncku.edu.tw (Y.-W. Chen).
1
These authors contributed equally.

https://doi.org/10.1016/j.biopha.2021.111734
Received 15 February 2021; Received in revised form 7 May 2021; Accepted 11 May 2021
Available online 19 May 2021
0753-3322/© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y.-Y. Lam et al.
Fig. 1. Details of number of animals used in each experiment. (a) Experimental procedures and number of animals used in each experiment of HFD study. (b)
Experimental procedures and number of animals used in each experiment of HFD-PIO study.
Numerous studies have demonstrated that altered changes in
morphology and number of astrocytes are closely associated with severe
depression [20,21]. Hippocampal astrocyte atrophy was observed in a
mouse depression model induced by corticosterone [22]. Previous
studies have demonstrated that metabolic disorders disturb the structure
and function of astrocytes in the hypothalamus. Short-term high-fat diet
(HFD) feeding triggers an increase number of astrocytes [23]. Long-term
HFD feeding induces astrocyte shrinkage and subsequently impairs
GABA reuptake capacity of these astrocytes [24]. In line with these re­
sults, we recently reported that HFD induces exhibition of
depression-like behaviors accompanied with an astrocytic activation
and a reduced astrocytic process arborization in the hippocampus of
mice [8]. Nevertheless, whether PIO is able to alleviate the HFD-induced
behavioral and cellular abnormalities remains unclear.
In the present study, we sought to examine the effects of PIO on the
metabolic disorder-related depression and the ventral hippocampal
astrocytic changes in mice. We fed the mice with a 12-week HFD
regimen to induce metabolic dysregulations. Body weights and systemic
insulin resistance were used as indicators of metabolic impairments. PIO
was daily exposed to mice via oral gavage during 4 weeks after 12-week
feeding period. The effects of HFD and PIO on the exhibition of
depression-like behaviors and the morphological properties of ventral
hippocampal astrocytes were determined. Since the hippocampus is
involved in regulating anxiety-like behaviors and memory processes, we
also examined the HFD and PIO effects on the performances of these
behaviors.
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Biomedicine & Pharmacotherapy 140 (2021) 111734
2. Materials and methods
2.1. Animals
The 8-week-old male C57BL/6N mice were purchased from National
Cheng Kung University Laboratory Animal Center. Under the care of
National Cheng Kung University Institutional Animal Care, mice were
housed (4–5 per cage) in a temperature- (temperature: 25 ± 2 ◦ C; hu­
midity: around 60–80%) and light-control environment under a 12:12 h
light-dark cycle (lights on at 6:00 AM). All experimental procedures of
animal studies were approved by the Institutional Animal Care and Use
Committees (IACUC) of National Cheng Kung University (IACUC
approval number: 104135). Mice randomly assigned to normal diet (ND)
and high-fat diet (HFD) feeding groups. The ND mice were fed with a
standard diet (Cat. #: 5010, LabDiet, St. Louis, MO, USA), and the HFD
mice were fed with a diet containing 60% kcal fat (Cat. #: 58Y1, Test­
Diet, St. Louis, MO, USA). PIO (Cat. #: 047903, Matrix Scientific,
Columbia, SC, USA) at doses of 10 mg/kg or 20 mg/kg or vehicle (0.25%
carboxymethylcellulose, CMC; Cat. #: C5678, Sigma-Aldrich, St. Louis,
MO, USA) was administered to ND and HFD mice by oral gavage once
daily for 4 weeks (from 20 to 24 weeks old). The mice were grouped
during experimentation. The experimental timelines could be found in
the figures. The details of number of mice used in each experiment were
list in Fig. 1.
Y.-Y. Lam et al.
2.2. Intraperitoneal glucose tolerance test
Intraperitoneal glucose tolerance test (IPGTT) was performed as
previously described [25]. Mice were intraperitoneally injected with
40% sterile glucose solution dissolved in saline at a dose of 2 g/kg after a
12-h fasting. Blood samples were obtained from tail of mice at 0, 30, 60,
90, 120 min after glucose injection. Glucose level were examined by
using OneTouch® Ultra test strips and OneTouch® UltraEasy blood
glucose meter (LifeScan, Milpitas, CA, USA).
2.3. Intraperitoneal insulin tolerance test
Intraperitoneal insulin tolerance test (IPITT) was performed as pre­
viously described [25]. Mice were intraperitoneally injected with insulin
(0.75 U/kg) after 4-h fasting. Blood samples were collected from tails of
mice at 0, 30, 60, 90 and 120 min after insulin injection. Glucose levels
were measured by using OneTouch® Ultra test strips and OneTouch®
UltraEasy blood glucose meter (LifeScan).
2.4. Measuring fasting plasma levels of glucose and insulin
After a 12-h fasting, the mice were anesthetized with pentobarbital
(1 g/kg, i.p.) and blood samples were collected from the cardiac punc­
ture with heparinized capillary tubes. Plasma was collected after
centrifuging the blood at 3000g for 10 min. Plasma glucose levels were
determined by using a commercial glucose-oxidase kit (Cat. #: 11538,
BioSystems, Barcelona, Spain). Plasma insulin levels were determined
by using a commercial mouse insulin ELISA kit (Cat. #: 10-1247-01,
Mercodia, Uppsala, Sweden).
2.5. Calculating homeostasis model assessment insulin resistance index
The homeostasis model assessment insulin resistance (HOMA-IR)
index was calculated as followed formula: fasting glucose (mM) ×
fasting insulin (mU/L)/22.5 [26].
2.6. Sucrose preference test
The sucrose preference test (SPT) was performed as described [27].
To avoid the confounding effects resulted from isolation housing and
switching cagemates, the SPT was performed by the cage. Total 4 cages
of mice (18 mice) were subjected. Two identical water bottles were
provided to mice for 24 h. One is filled with distilled drinking water and
the other is filled with a palatable 1% sucrose solution. To minimize the
side preference, these 2 bottles were switched at 12 h. Consumptions of
sucrose solution and water were examined by measuring the changes in
weight of fluid consumed. Sucrose preference was defined as the ratio of
the consumed weight of sucrose solution to the total consumed weight of
sucrose solution and pure water during 24 h.
2.7. Forced swimming test
The forced swimming test (FST) was performed as described [28].
Mice were placed in a cylindrical transparent tank (height 30 cm,
diameter 20 cm and depth 15 cm) filled with temperature-controlled
water (23–25 ◦ C). The scoring of active (swimming and climbing) or
passive (immobility) behavior was recorded over a 6-min test period but
only the last 4-min data were analyzed.
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Biomedicine & Pharmacotherapy 140 (2021) 111734
2.8. Tail suspension test
The tail suspension test (TST) was performed as described [27]. Mice
were suspended 50 cm above the floor of a white plastic chamber. The
behavior was recorded over a 6–min test period but only the last 4-min
data were analyzed.
2.9. Tissue preparations
One day after the conclusion of experiments, the anesthetized mice
(pentobarbital, 1 g/kg, i.p.) were perfused from the left ventricle with
chilled saline, and their brains were removed and divided into two
hemispheres used for immunoblotting and immunohistochemistry,
respectively. For immunoblotting, the ventral hippocampus was quickly
dissected out, frozen in liquid nitrogen, and stored at 80 ◦ C until use. For
immunohistochemistry, the brain hemispheres were fixed in 4% para­
formaldehyde in 0.1 M phosphate buffer for 2 days at 4 ◦ C. The brain
specimens were then dehydrated in graded sucrose solutions (10%, 20%,
30%, and 35%, dissolved in 0.1 M phosphate buffer), and embedded
with frozen section media (Cat. #: 3801480, Leica Biosystems, Wetzlar,
Hessen, Germany). The brains were coronally sliced into 25-μm sections
and stored in cryoprotectant at − 20 ◦ C.
2.10. Immunoblotting
The ventral hippocampal tissue specimens of mice were lysed with
ice-cold commercial tissue protein extraction reagent (Cat. #: 78510,
Thermo Fisher Scientific, Waltham, MA, USA) containing complete
protease inhibitor cocktail tablets (Cat. #: 04693132001, Roche, Basel,
Switzerland). Protein concentrations were determined by a Bio-Rad
protein assay. Equal amount of protein lysates was separated by SDS-
PAGE, and then transferred to nitrocellulose membrane (Cat. #:
S80209, Pall Life Science, Port Washington, NY, USA). Membranes were
blocked in blocking buffer, and then incubated with rabbit anti-GFAP
antibodies (1:1000, Cat. #: Z0334, Dako-Agilent, Santa Clara, CA,
USA) and mouse anti-β-actin (1:10,000, Cat. #: A5441, Sigma-Aldrich),
washed, and incubated with the corresponding horseradish peroxidase-
conjugated secondary antibodies (Cat. #: 115-035-003 and 111-035-
003, Jackson ImmunoResearch, West Grove, PA, USA), and visualized
with western blotting luminol reagent (Cat. #: sc-2048, Santa Cruz
Biotechnology, Inc.). Bands in the immunoblots were quantified by
using ImageQuant LAS 4000 (GE Healthcare, Chicago, IL, USA).
2.11. Immunohistochemistry
The ventral hippocampal sections were collected in the range, ste­
reotaxic reference point, from 2.30 mm to 3.88 mm posterior to the
Bregma. The collected brain sections were washed with phosphate-
buffered saline containing 0.3% Triton X-100 to remove the embed­
ding frozen section media, immersed in 3% H2O2 to abolish endogenous
peroxidase activity, and blocked with 3% normal goat serum for 1 h at
room temperature. The free-floating brain sections were stained using
rabbit anti-GFAP (1:1000, Cat. #: Z0334, Dako-Agilent) antibody in 4 ◦ C
overnight., washed, and then incubated with secondary antibodies (Cat.
#: 111-035-003, Jackson ImmunoResearch) for 2 h at room tempera­
ture. The brain sections were washed and applied for 3,3´-dia­
minobenzidine kit (SK-4100, Vector Laboratories, Burlingame, CA,
USA).
Y.-Y. Lam et al. Biomedicine & Pharmacotherapy 140 (2021) 111734

Fig. 2. Effects of HFD on body weight, systemic glucose metabolism and exhibition of depression-like behaviors in mice. (a) Experimental timeline. (b) Quantitative
results of body weight of mice during the feeding period. (c) Quantitative results of body weight of mice after the end of regimen (d) Quantitative results of energy
intake of mice. (e) Alterations of blood glucose levels of mice in the IPGTT were shown in left panel and the quantitative results of area under curves (AUC) were
shown in the right panel. (f) Alterations of blood glucose levels of mice in the IPITT were shown in left panel and the quantitative results of AUC were shown in the
right panel. (g) Quantitative results of fasting plasma levels of glucose in mice. (h) Quantitative results of fasting plasma levels of insulin in mice. (i) Quantitative
results of HOMA-IR index. (j) Quantitative results of SPT. (k) Quantitative results of FST. (l) Quantitative results of TST. *p < 0.05, **p < 0.01, ***p < 0.001,
****p < 0.0001, vs. ND, unpaired Student’s t-test in scatter plots and repeated measure two-way ANOVA in line graphs. Numbers given in the parentheses indicates
the sample sizes. Values represent mean ± S.E.M. from three independent experiments.

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Y.-Y. Lam et al. Biomedicine & Pharmacotherapy 140 (2021) 111734

Fig. 3. Effects of PIO on body weight and systemic glucose metabolism in HFD-induce obese mice. (a) Experimental timeline. (b) Quantitative results of body weight
of mice during the feeding period. (c) Quantitative results of body weight of mice after the end of regimen. (d) Quantitative results of energy intake of mice. (e)
Alterations of blood glucose levels of mice in the IPGTT were shown in left panel and the quantitative results of AUC were shown in the right panel. (f) Alterations of
blood glucose levels of mice in the IPITT were shown in left panel and the quantitative results of AUC were shown in the right panel. (g) Quantitative results of fasting
plasma levels of glucose in mice. (h) Quantitative results of fasting plasma levels of insulin in mice. (i) Quantitative results of HOMA-IR index. *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001, vs. respective ND group. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, vs. respective CMC group, ordinary two-way
ANOVA in scatter plots and repeated measure two-way ANOVA in line graphs. Numbers given in the parentheses indicates the sample sizes. Values represent
mean ± S.E.M. from three independent experiments.

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Y.-Y. Lam et al.
Fig. 4. Effects of PIO on the plasma levels of lipids in HFD-induced obese mice. (a) Quantitative results of plasma levels of triglyceride in mice. (b) Quantitative
results of plasma levels of free fatty acid in mice. (c) Quantitative results of plasma levels of LDL/VLDL cholesterol in mice. *p < 0.05, **p < 0.01, ***p < 0.001,
****p < 0.0001, vs. respective ND group. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001vs. respective CMC group, ordinary two-way ANOVA. Values
represent mean ± S.E.M. from three independent experiments.
2.12. Analysis of astrocytic morphology
Photomicrographs were taken using a digital camera connected to a
computer equipped with Axiovision 4.8 software (Carl Zeiss, Oberko­
chen, Germany). To minimize the batch-to-batch variation, each set of
immunohistochemical experiment was performed at the same time. The
GFAP-immunoreactive (GFAP+) signals were identified by the pixels
display higher intensity than the background threshold using the ImageJ
Fiji software. The background intensity threshold was fixed and applied
to all sections. The ImageJ plugin, NeuronJ, was used to analyze the
process arbors (Sholl analysis), process lengths and branch points of the
GFAP+ astrocytes located in the CA1 subregion of the ventral hippo­
campus. Total 25 astrocytes (5 astrocytes/brain section, 5 brain sec­
tions) obtained from each mouse were measured. The averaged value of
measured parameters obtained from the 25 selected astrocytes was
presented as one data point. Five mice in each group were analyzed. The
experimenter who analyzed the morphologies of the hippocampal as­
trocytes was blinded from the experimental grouping.
2.13. Object recognition test
The object recognition test (ORT) was used to determine the
hippocampus-related non-spatial learning and memory in mice as pre­
viously described [29]. Each mouse was placed an open field
(40 cm × 50 cm × 50 cm brown plywood box) and allowed free
exploration for 5 min every day for 3 consecutive days (habituation
phase). On the fourth day, the mouse was introduced in the same box
consisting of two identical objects (A1, A2, towers of Lego bricks with
diameter 8 cm and height 9.5 cm) positioned separately at 10 cm from a
wall for 5 min (acquisition phase). Short-term memory test was per­
formed after 120 min. The mouse was placed in the same box with one of
the old object A1 that had been replaced with a new object B (trans­
parent glass bottles with diameter 5 cm and height 9.5 cm) for 5 min.
Long-term memory test was performed after 24 h. The mouse was
introduced in the same box with object B that had been replaced with a
new object C (white plastic box 3 cm × 7.5 cm × 8 cm) for 5 min. The
time spent to explore each object was recorded. The exploring behavior
was defined as “mice touching the object with the nose or sniffing to­
ward the object within a distance of 1 cm.” The ratio of “new object
exploring time divided by total exploring time” to represent the memory
function.
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Biomedicine & Pharmacotherapy 140 (2021) 111734
2.14. Open field test
The open field test (OFT) was performed as previously described
[30]. Each mouse was placed in a white plastic box
(50 cm × 40 cm × 40 cm) for 15 min. The time spent in the central
area (25% of surface area), the total travel distance and the locomotor
immobility were measured and analyzed using the EthoVision video
tracking system (Noldus, Wageningen, GE, Netherlands).
2.15. Elevated plus maze test
The elevated plus maze test (EPM) was conducted as previously
described [31]. A plus-shaped apparatus that included 2 open and 2
enclosed arms (50 cm × 5 cm × 40 cm) was used in this test. The mice
were first placed in the central zone of the maze with open access to any
arm and allowed to freely explore for 10 min. The frequency and
duration of entering the open arms and enclosed arms within 10 min
were recorded by using the EthoVision video tracking system (Noldus).
2.16. Statistical analysis
Total number (N) of observations in each group was indicated in
Figures. All data were plotted and reported as mean ± standard devia­
tion. Significance was set at p < 0.05. Student’s t-test was adopted to
analyze the data sets with a single factor (HFD effect). The body weight
and energy intake of mice were analyzed using repeated measured two-
way ANOVA. The process arborization of individual astrocytes (Sholl
analysis) was analyzed using mixed-model two-way ANOVA with
concentric rings and HFD as the two main effects. Ordinary two-way
ANOVAs were used to analyze the results with two factors (HFD and
PIO). Sidak’s post-hoc test was used to perform multiple comparison
analysis after the two-way ANOVAs. Statistical analyses were performed
with GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA).
3. Results
3.1. HFD concurrently induces metabolic disorders and exhibition of
depression-like behaviors in mice
We fed male C57BL/6N mice with HFD with 60% of kcals from fat at
the age of 8 weeks for 12 weeks to generate obesity mouse model
(Fig. 2a), which is a widely used animal model of T2DM, showing insulin
Y.-Y. Lam et al. Biomedicine & Pharmacotherapy 140 (2021) 111734

Fig. 5. Effects of PIO and HFD on exhibition of depression-like behaviors in mice. (a) Quantitative results of SPT. (b) Quantitative results of FST. (c) Quantitative
results of TST. ****p < 0.0001, vs. respective ND group. ###p < 0.001, ####p < 0.0001, vs. respective CMC group, ordinary two-way ANOVA. Values represent
mean ± S.E.M. from three independent experiments.

resistance [32]. We compared the body weight gain and energy intake of
the ND and HFD mice and found that HFD gradually increased body
weight gain (Fig. 2b). At the end of the 12-week feeding, the body
weights of the HFD group increased 50% more than those of the chow
group (Fig. 2c). There was no significant difference on energy con­
sumption of mice fed with HFD when compared with mice fed with
chow (Fig. 2d). We next analyzed the capacity of the mice to break down
glucose by IPGTT and examined the glucose-lowering effects of insulin
by IPITT. As a result, HFD mice displayed glucose intolerance (Fig. 2e)
and impaired insulin sensitivity (Fig. 2f). The HFD feeding induced in­
sulin resistance in mice, by increasing the levels of fasting plasma
glucose (Fig. 2g) and insulin (Fig. 2h), and the HOMA-IR index (Fig. 2i).
A growing number of studies indicate an association existing be­
tween metabolic disorders and depression [33,34]. Hence, we further
evaluated whether these obese mice exhibit depression-like behaviors
by performing SPT, FST, and TST. Results showed that HFD induced
depression-like phenotypes in mice by decreasing sucrose consumption
in SPT (Fig. 2j) and increasing time fractions of immobility in FST
(Fig. 2k) and TST (Fig. 2l). These data indicated that a 12-week HFD
induces obesity, systemic insulin resistance, as well as exhibition of
depression-like behaviors in mice.
3.2. Pioglitazone ameliorates HFD-induced metabolic dysfunctions in
mice
Next, we examined whether PIO improves HFD-induced impair­
ments of glucose metabolism. PIO (10 or 20 mg/kg) was treated to mice
during 4 weeks after the 12-week feeding period. The HFD regimen was
continued within the period of 4-week PIO treatment (Fig. 3a). The mice
administrated with the equal volume of vehicle, CMC, served as control
groups. Results showed that both doses of PIO did not affect the body
weight gain as well as energy intake in the ND and HFD mice (Fig. 3b–d).
In the IPGTT, the HFD mice treated with either 10 mg/kg or 20 mg/kg
PIO cleared glucose faster than the HFD mice treated with CMC, indi­
cating PIO improved glucose intolerance in the HFD mice (Fig. 3e)
Consistent with this, IPITT showed that glucose-lowering effects of in­
sulin were improved in the HFD mice treated with 20 mg/kg PIO
compared with the HFD mice supplemented with CMC (Fig. 3f).
Furthermore, 20 mg/kg PIO reduced plasma glucose levels (Fig. 3g),
fasting plasma insulin levels (Fig. 3h), as well as HOMA-IR index
(Fig. 3i). Additionally, 20 mg/kg PIO reduced the HFD-induced eleva­
tions of plasma levels of triglyceride (Fig. 4a), free fatty acid (Fig. 4b)
and LDL/VLDL cholesterol (Fig. 4c). Our results showed that 20 mg/kg
PIO treatment improves the HFD-induced impairments of glucose and
lipid metabolism in mice. Therefore, we focused on 20 mg/kg PIO
treatment for the rest of the studies.
3.3. Pioglitazone improves HFD-induced depressive phenotypes in mice
We further evaluated whether PIO improves depression-like pheno­
types in mice. Results showed that HFD induced the exhibition of
depression-like behaviors in the CMC mice by lowering sucrose con­
sumption in the SPT (Fig. 5a) and increasing the time fractions of
immobility in the FST (Fig. 5b) and TST (Fig. 5c). A 4-week 20 mg/kg
PIO treatment reversed the HFD-induced reduction of sucrose con­
sumption in the SPT (Fig. 5a) and HFD-induced elevations of time
fractions of immobility in the FST (Fig. 5b), TST (Fig. 5c). Our results
showed that the 20 mg/kg PIO treatment exerts potential therapeutic
efficacy on the HFD-induced exhibition of depression-like behaviors in
mice.
3.4. Pioglitazone reverses the HFD-induced astrocyte activation and
remodeling of astrocytic morphology in the hippocampi of mice
Recently, we demonstrated that HFD induces not only exhibition of
depression-like behaviors but also astrocyte activation and remodeling
of the astrocytic morphology in the hippocampi of mice [8]. Here, we
examined the effects of PIO treatment on the HFD-induced astrocytic
alterations. In line with our previous findings [8], HFD upregulated the
ventral hippocampal GFAP expression in the CMC mice, suggesting HFD
induced astrocyte activation (Fig. 6a and b). PIO (20 mg/kg) abolished
the HFD-induced increase of ventral hippocampal GFAP expression
(Fig. 6a and b). Activation of astrocytes is known to be associated with
morphological changes, including hypertrophy and process remodeling,
so we further examined the effects of PIO on the fine structure of the
astrocytes. Results showed that the total process lengths, the number of
branch points, and the number of process intersections with concentric
rings in the Sholl analysis were significantly decreased in the GFAP+
astrocytes located in the ventral hippocampal CA1 of HFD mice of CMC
groups (Fig. 6c–g). HFD-induced process remodeling of the ventral
hippocampal CA1 astrocytes were reversed by the PIO treatment
(Fig. 6c–g), suggesting that PIO increases astrocytic process arborization
in HFD mice.
3.5. Pioglitazone has no effect on the exhibition of anxiety-like behaviors
and the hippocampus-related learning and memory in mice
Since the hippocampus also mediates anxious phenotypes and
memory functions, we further assessed the effects of PIO treatment on
the performances of these behaviors. The OFT and EPM tests were
adopted to estimate the exhibition of anxiety-like depression. In the
OFT, both HFD and PIO did not affect the measured parameters,
including the total travel distances, time fraction of immobility, time
spend in the center, number of entries into the center, and travel

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(caption on next page)

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Y.-Y. Lam et al. Biomedicine & Pharmacotherapy 140 (2021) 111734

Fig. 6. Effects of PIO and HFD on GFAP expression and astrocytic fine structures in the hippocampus of mice. (a) Representative immunoblotting micrographs of
GFAP in the hippocampus of mice. (b) Quantitative results of GFAP levels in the hippocampus of mice. (c) Representative immunomicrographs of the GFAP+ as­
trocytes in the hippocampus (upper four panels). Representative process tracing graphs of selected GFAP+ astrocyte (red frames in the upper four panels) are shown
in the respective lower four panels. (d) Quantitative results of total process length. (e) Quantitative results of number of branch points. (f) Quantitative results of Sholl
analysis. (g) Quantitative results of total number of intersections obtained from the Sholl analysis. *p < 0.05, **p < 0.01, ****p < 0.0001, vs. respective ND group.
###p < 0.001, ####p < 0.0001, vs. respective CMC group, ordinary two-way ANOVA in scatter plots and mix-model repeated measure two-way ANOVA in line
graphs. Numbers given in the parentheses indicates the sample sizes. Values represent mean ± S.E.M. from three independent experiments. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)

distance within the center (Fig. 7a–e). Similarly, in the EPM, HFD and
PIO also exerted no effects on the travel distances and the time spent in
the center, open arms, and closed arms (Fig. 7f–k).
To determine the effects of HFD and PIO on the performances in the
hippocampus-related learning and memory task, the ORT was carried
out. Results revealed that neither HFD nor PIO altered the formations of
short-term and long-term memory in the ORT (Fig. 7l and m). Taken
together, our results suggested that both HFD and PIO have no effect on
the exhibition of anxiety-like behaviors and the hippocampus-related
learning and memory in mice.
4. Discussion
This study highlights the therapeutic effects of PIO on both T2DM
and its concurrent depression. Our findings suggested that PIO rescues
the HFD-induced depressive-like phenotypes and ventral hippocampal
astrocytic deficits probably via improving the HFD-induced metabolic
dysregulations. This conclusion is supported by following evidence: (1)
PIO improved HFD-induced metabolic disorders; (2) PIO increased su­
crose preference in the SPT and reduced immobility time in the FST and
TST; (3) PIO decreased HFD-induced GFAP expression in the ventral
hippocampus; (4) PIO restored the process arborizations of the GFAP+
astrocytes in the ventral hippocampal CA1 of HFD mice.
An accumulating body of evidence links T2DM to the development of
MDD [33]. However, the mechanisms underlying the interactions be­
tween insulin resistance and neuropsychiatric disorders are still unclear.
PIO, a widely used insulin sensitizer, has been known to relieve
depressive symptoms. The improvements in insulin sensitivity and the
antidepressant-like effects were reported among patients with severe
depression receiving PIO therapy [35]. In current study, we showed that
long-term HFD feeding induced metabolic disorders accompanied with
depression-like phenotype in obese mice, which concurs with a previous
study [8]. We showed that a 12-week HFD feeding induced metabolic
disorders in mice, including obesity and systemic insulin resistance, and
dysregulated lipid metabolism. Our plasma biochemical examinations
showed elevated levels of plasma glucose, total cholesterol and tri­
glycerides in the HFD obese mice (Figs. 1f and 4). PIO reversed these
metabolic dysregulations through increasing the peripheral glucose
utilization (Figs. 2 and 4). We also found that HFD induced the devel­
opment of depression-like behaviors which was counteracted by PIO
treatment. It suggested that the PIO exerts antidepressant effect at least
in part through enhancing insulin sensitization and ameliorating lipid
distribution.
PPAR-γ is present in various brain regions, including the hippo­
campus, frontal cortex, and hypothalamus [36]. PIO is described to cross
the blood-brain barrier and mediate the depressive behaviors through
PPAR-γ [37]. The activation of PPAR-γ in the nervous system decreases
the expression of inflammatory cytokines, represses glutamate toxicity
and endoplasmic reticulum stress, and promotes neurogenesis and
neural plasticity [37]. Our results showed that PIO significantly
increased PPAR-γ expression in the hippocampus of mice (data not
shown), suggesting that PPAR-γ may be involved in the PIO-induced
antidepressant-like effects. Whether PPAR-γ antagonists abolish the
PIO-induced antidepressant effects in the HFD mice deserves further
investigations in the future.
Disrupted neuroplasticity is a major cause of hippocampus-related
behavioral impairments, including depression, anxiety and memory
loss. Astrocytes composed approximately 30% of all glial cells in the
central nervous system support the processes of neuroplasticity. In line
with our results, it has been reported that HFD induces astrocyte acti­
vation. Upregulated GFAP expression and increased area of GFAP+ as­
trocytes were observed in the cerebellum but not the cerebral cortex
after 16-week HFD feeding [38]. The number of astrocytic processes is
reduced in the hippocampal CA1 region of HFD fed rats [39]. Herein, we
demonstrated that PIO could reverse the altered astrocytic morphology
remodeling by increasing the total process length and the number of
branch points. Hence, it is possible that PIO counteracts the
HFD-induced depression-like phenotypes by correcting the
astrocyte-associated deficits in the ventral hippocampus of mice. A
previous study by Zhang et al. reported that shortened processes of
hypothalamic astrocyte in HFD-fed mice and by moderate-level
IKKβ/NFkB upregulation [40]. Zhang et al. revealed that IKKβ/NFkB
activation resulted in astrocytic process reduction while IKKβ/NFkB
inhibition reversed this process defect in obese mice by using genetic
models, suggesting IKKβ/NFkB pathway is very important for regulation
of astrocytic process [40]. In this study, we demonstrated that HFD led
to hippocampal astrocytic deficits while PIO reversed this process defect
in obese mice. Whether IKKβ/NFkB pathway participate in HFD-induced
hippocampal astrocytic deficits, and how? Whether PIO reverses the
altered astrocytic morphology remodeling by inhibition of IKKβ/NFkB
pathway? While the answers to these are waiting for further delineation.
It’s worth mentioning that the present study employs male mice
only, whereas in the clinic, T2DM-associated MDD is reported for both
males and females with some studies showing a higher prevalence in
women [41]. This obviously poses some limitations when concluding on
the present findings, but it is very interesting and worth of further
investigation.
5. Conclusions
In conclusion, herein, we demonstrated that chronic treatment of
PPARγ agonist, PIO, reverses the HFD-induced metabolic, behavioral,
and ventral hippocampal astrocytic impairments in mice. PIO could be a
promising therapeutic drug for the patients suffering from concurrent
metabolic disorders and depression.
CRediT authorship contribution statement
Y.Y.L. executed experiments and analyzed the results. S.F.T., P.C.C.
and Y.M.K. interpreted the results and edited manuscript. Y.W.C. su­
pervised this project, interpreted the results, and prepared the manu­
script. All authors have read and approved this manuscript.

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Y.-Y. Lam et al. Biomedicine & Pharmacotherapy 140 (2021) 111734

Fig. 7. Effects of PIO and HFD on exhibition anxiety-like behaviors and performances in the hippocampus-related memory test. (a) Quantitative results of total travel
distance in OFT. (b) Quantitative results of time fraction of immobility in OFT. (c) Quantitative results of time spent in center in OFT. (d) Quantitative results of
number of entries into center in OFT. (e) Quantitative results of travel distance within center in OFT. (f) Quantitative results of total travel distance in EPM. (g)
Quantitative results of time spent in open arm in EPM. (h) Quantitative results of time spent in center in EPM. (i) Quantitative results of travel distance in open arm in
EPM. (j) Quantitative results of travel distance in center in EPM. (k) Quantitative results of travel distance in closed arm in EPM. (l) Quantitative results of per­
formances of short-term memory in ORT. (m) Quantitative results of performances of long-term memory in ORT. Ordinary two-way ANOVA. OFT: open field test,
EPM: elevated plus maze, ORT: object recognition test. Values represent mean ± S.E.M. from three independent experiments.

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Y.-Y. Lam et al. Biomedicine & Pharmacotherapy 140 (2021) 111734

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