# Stress exposure alters brain mRNA expression of the genes involved in insulin signalling, an effect modified by a high fat/high fructose diet and cinnamon supplement

Frédéric Canini, Bolin Qin, Nathalie Arvy, Laurent Poulet, Cécile Batandier, Anne-Marie Roussel, Richard Anderson

► To cite this version:

Frédéric Canini, Bolin Qin, Nathalie Arvy, Laurent Poulet, Cécile Batandier, et al. Stress exposure alters brain mRNA expression of the genes involved in insulin signalling, an effect modified by a high fat/high fructose diet and cinnamon supplement. PLoS ONE, 2018, 13 (5), pp.1-15. 10.1371/journal.pone.0197094. hal-01930806

HAL Id: hal-01930806

https://hal.univ-grenoble-alpes.fr/hal-01930806

Submitted on 26 May 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH ARTICLE

# Stress exposure alters brain mRNA expression of the genes involved in insulin signalling, an effect modified by a high fat/high fructose diet and cinnamon supplement

Frédéric Canini¹,²*, Bolin Qin³,⁴, Nathalie Arvy⁵,⁶, Laurent Poulet⁷, Cécile Batandier⁷, Anne-Marie Roussel⁷, Richard A. Anderson⁴*

<figure><img src="image_2.png" /></figure>

1 Département Neurosciences & Contraintes Opérationnelles, Institut de Recherche Biomédicale des Armées (IRBA), Brétigny-sur-Orge Cedex, France, 2 École du Val de Grâce, 1 place Laveran, Paris, France, 3 In-ingredients.com, Columbia, TN, United States of America, 4 Diet, Genomics, and Immunology Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD, United States of America, 5 INRA, Laboratory of Nutrition and Integrative Neurobiology, UMR 1286, Bordeaux Cedex, France, 6 University of Bordeaux, Laboratory of Nutrition and Integrative Neurobiology, UMR 1286, Bordeaux Cedex, France, 7 LBFA/INSERM1055, Grenoble Alpes University, Grenoble, France

* Current address: PolyChrom Technology, LLC, Edgewater, MD, United States of America

* fredericcanini@gmail.com

<figure><img src="image_3.png" /></figure>

OPEN ACCESS

**Citation:** Canini F, Qin B, Arvy N, Poulet L, Batandier C, Roussel A-M, et al. (2018) Stress exposure alters brain mRNA expression of the genes involved in insulin signalling, an effect modified by a high fat/high fructose diet and cinnamon supplement. PLoS ONE 13(5): e0197094. https://doi.org/10.1371/journal.pone.0197094

**Editor:** Alexandra Kavushansky, Technion Israel Institute of Technology, ISRAEL

**Received:** June 11, 2017

**Accepted:** February 13, 2018

**Published:** May 29, 2018

**Copyright:** This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

**Data Availability Statement:** All data underlying the study are within the paper and its Supporting Information files.

**Funding:** The work was funded by USDA/ARS/USA CRADA: 58-3K95-7-1184 and French National Agency for Research (PNRA 007). The funders provided support in the form of salary for one of the authors [BQ], but did not have any additional role in the study design, data collection and

## Abstract

In occidental societies, high fat and high sugar diets often coincide with episodes of stress. The association is likely to modify brain energy control. Brain insulin signalling is rarely studied in stressed individuals consuming high fat diets. Furthermore the effects of cinnamon supplement are not known in these conditions. Therefore, we exposed rats, over a 12-week period, to a control (C) or a high fat/high fructose (HF/HFr) diet that induces peripheral insulin resistance. A cinnamon supplement (C+CN and HF/HFr +CN) was added or not. After diet exposure, one group of rats was exposed to a 30-min restraint followed by a 10-min open-field test, their combination featuring a moderate stressor, the other rats staying unstressed in their home cages. The insulin signalling in hippocampus and frontal cortex was studied through the mRNA expression of the following genes: insulin receptor (*Ir*), insulin receptor substrate (*Irs1*), glucose transporters (*Glut1* and *Glut3*), glycogen synthase (*Gys1*) and their modulators, *Akt1* and *Pten*. In C rats, stress enhanced the expression of *Ir*, *Irs1*, *Glut1*, *Gys1* and *Akt1* mRNA. In C+CN rats, stress induced an increase in *Pten* but a decrease in *Gys1* mRNA expression. In HF/HFr rats, stress was associated with an increase in *Pten* mRNA expression. In HF/HFr+CN rats, stress increased *Pten* mRNA expression but also decreased *Gys1* mRNA expression. This suggests that a single moderate stress favours energy refilling mechanisms, an effect blunted by a previous HF/HFr diet and cinnamon supplement.
analysis, decision to publish, or preparation of the
manuscript. The specific roles of the authors are
articulated in the 'author contributions' section.

**Competing interests:** BQ has a joint appointment with the USDA and IN-Ingredients Columbia, TN. The cinnamon powder used in the current study is not a product of IN-Ingredients and this study does not involve any products of IN-Ingredients. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

**Introduction**

People in western societies are chronically exposed to high fat/high sugar diets and repeatedly have to cope with life events that are moderate stressors. In rodents, chronic exposure to a high fat/high sugar diet induces peripheral insulin resistance [1] and changes brain insulin signalling [2–5]. However, the decrease in brain insulin sensitivity occurs independently from that of the periphery [6] and from adiposity [7], suggesting that a mechanism specific for the cerebral tissue may exist. When present, the brain insulin resistance is characterized by functional disturbances [3, 8] and structural abnormalities [4]. The relation between stress and brain insulin resistance is usually studied in animal models of depression and diabetes [9]. However, little is known in non pathological models. Prenatally stressed animals displaying depression-like behaviours in adulthood do not exhibit major alterations in brain insulin sensitivity [10]. Naive adult mice exposed repeatedly to foot shocks, a model of chronic high-level stress, exhibit insulin resistance in the periphery but not in the brain [11]. Repeated exposure to a moderate stress such as restraint is also followed by a peripheral insulin resistance [12]. However, the effects of single exposure to a moderate stressor on brain insulin signalling are not known.

Furthermore, the effects of a previous diet on stress-induced biological reactions have not been investigated. Evidence in humans and animals suggests that a high fat/high sugar diet and stress are interacting. Chronic stress exposure favours the emergence of both peripheral insulin resistance [13] and depression [14]. Furthermore, both are epidemiologically related [15]: patients with type 2 diabetes have a higher risk of depression than non diabetic subjects [16–19], while patients with depression also have an increased risk of diabetes [20–22], insulin resistance [23] and metabolic syndrome [24] compared to non depressed subjects. Brain inflammation and free-radical accumulation may be the crossroad for mechanisms as a high fat diet and stressor exposure enhance brain inflammation [25, 26] and free-radical levels [27–31]. In turn, free-radical accumulation favours the decrease in insulin sensitivity [32, 33], therefore triggering a vicious circle. Such evolution can be limited by polyphenols that counteract free radical consequences. For instance, cinnamon, a spice that is high in polyphenols, improves insulin sensitivity [34], alleviates peripheral insulin resistance [35], limits the stress-induced oxidation, especially in diabetic rats [36], and is neuroprotective of the cerebral tissue of rats exposed to high fat diet [5].

We hypothesized that a single moderate stress exposure would modify the brain mRNA expression of genes that are involved in insulin signalling in order to facilitate the energy supply. We also hypothesized that a previous high fat/high fructose diet would blunt this adaptive response. Lastly, we hypothesized that a cinnamon supplement would either limit the deleterious effects of a previous high fat/high fructose diet, or reduce the stress-induced adaptive responses. We designed the experiments accordingly: (i) to determine the effects of a single moderate stressor exposure on brain mRNA expression of genes involved in insulin sensitivity (ii) to evaluate the impact of a previous high fat/high sugar diet on mRNA reactions and (iii) to evaluate the effect of cinnamon on the brain mRNA expression in both normal chow and high fat/high sugar nourished rats.

**Materials and methods**

**Animals**

The 120 Wistar male rats (Charles River, L'Arbresle, France) were 5 weeks-old and weighed 64 ±1 g upon arrival at the laboratory. They were kept in a temperature-controlled room (ambient temperature: 22±1°C, relative humidity: 40-60%, 12 h light/12 h dark cycle with light at 8:00).
All experimental procedures were reviewed and approved by the Institutional Ethic Commit-
tee for Animal Care (Comité d'Ethique du CRSSA, Protocol N°2008/02.1). Rats were main-
tained and handled in agreement with the Guide for the Care and Use of Laboratory Rats
(NIH, 1985).

Diets

The diets were purchased from SAFE (Augis, France). The control diet contained 5% cellulose, 20% casein, 25% corn starch, 25% potato starch, 16% maltodextrin, 4% soybean oil, 3.5% AIN mineral mix, 1% AIN vitamin mix, 0.3% dl methionine and 0.2% choline bitartrate. The high fat/high fructose diet was similar except the corn starch, potato starch and maltodextrin were replaced by 46% fructose and 20% lard.

The cinnamon (CN) powder (*Cinnamomum burmannii*) was purchased from McCormick
Spice Co. (Baltimore, MD). The water extract of the CN contained more than 5% type A poly-
phenols with a tetramer, cassiatannin A, with a molecular weight of 1152 and two identified
trimers, cinnamtannin B-1 and cinnamtannin D-1, with a molecular weight of 864 plus two
unidentified type A trimers with a molecular weight of 864 [37–39]. The amount of CN used
was based upon our previous study showing a definite effect of 20 g of CN/kg of diet in sponta-
neously hypertensive rats [40].

**Experimental design**

After their arrival in the laboratory, the rats were familiarized with their housing environment (Fig 1). They were kept 3 per cage (26 x 40 x 15 cm) and fed a control diet for three weeks. They were then randomly divided into 4 groups and for 12 weeks they were fed *ad libitum* with one of the following four diets: control diet (C, n = 30), high fat/high fructose diet (HF/HFr, n = 30), or the respective diets containing 20 g cinnamon/kg of diet (C+CN and HF/HFr+CN, n = 30 each). During this time, they were housed individually in order to monitor their water and diet intakes. At the end of this phase, the rats from each diet group were randomly distributed into 2 sub-groups according to their exposure to stressor (Stress, n = 10) or not (Rest, n = 20). The Stress rats were submitted to a 30-min restraint immediately followed by a 10-min open field test [41]. This combination was assumed to mimic a life event stressor with the succession of movement limitation and novelty exposure. The unstressed Rest rats remained quiet in their housing cages. Stressed rats were sacrificed 1 hour following the beginning of the stress exposure. All investigations were carried out in the morning to take circadian rhythms into account.

Acute stressor exposure

The acute stressor used was the combination of a 30-min restraint followed by an open-field
test. The Rest rats were neither handled nor exposed to the combined stressor in order to keep
them free from stress.

The 30-min restraint is widely used as a moderate stressor [42]. To achieve it, the rats were placed gently into a net tube and their muzzle was left free. To ensure complete restraint without any body compression, the diameter of the net was adjusted to the rat's body size. The restraint was carried out in a separate room to avoid any interaction with Rest rats.

The open-field test consisted of a white plate (100x100 cm) surrounded by a 45-cm high wall. This test was performed under a 50-lux illumination in the centre of the test. The centre of the open-field was defined as the square 15-cm away from the wall. Behaviour assessment was carried out using video tracking (Viewpoint, Lyon). Just after restraint, the animals were transferred in their home cage to a room devoted to open field test. The animal was placed in the centre of the apparatus and left free for 10 min. Before and after each test, the open-field
<figure><img src="image_4.png" /><figcaption>**Fig 1.** Experimental design. Diets were the following: control (C), high fat/high fructose (HF/HFr), control+cinnamon (C+CN) and high fat/high fructose+cinnamon (HF/HFr+CN). The group sizes are indicated in brackets.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0197094.g001

was cleaned with 70% isopropylic alcohol. The behavioural variables used to evaluate the effect of diets on stress-induced reactivity were the following: (i) The defecation level during restraint and open-field; (ii) the locomotion in the open field as evaluated by the distance travelled and the duration of immobility periods; (iii) the propensity to go to the centre of the open field. Reduced time spent and distance travelled in the central area, long-lasting immobility in the open field and high defecation scores are indicative of anxiety [43, 44]. Locomotion speed under 5.7 cm/s was considered as immobility.

## Sacrifice and tissue sampling

After overnight fasting, the animals were transferred in their home cage to a particular room to avoid any added stress. They were quickly anesthetized with a mixture of isoflurane (3% in 100% O₂). Blood samples were taken by cardiac puncture in a tube coated with EDTA. The brain was quickly removed and dissected on an ice bed using a glass tool. The entire hippocampus and a sample of the frontal cortex were taken and immediately frozen in liquid nitrogen and stored at -80°C until analyses.
## Biochemical determination

The concentrations of plasma corticosterone were measured with an in-house RIA using a highly specific antibody provided by H. Vaudry (University of Rouen, France) as described [45].

## mRNA determinations

The gene expression both in the hippocampus and frontal cortex were evaluated in rats randomly taken from each experimental group: C (Rest: n = 8 and Stress: n = 8), C+CN (Rest: n = 9 and Stress: n = 7), HF/HFr (Rest: n = 8 and Stress: n = 8), HF/HFr+CN (Rest: n = 8 and Stress: n = 8). The assessed mRNA expressions were the following: insulin receptor (Ir), [46], insulin receptor substrate (Irs) type I and II [46], glucose transporter type 1 (Glut1) and 3 (Glut3) [47], glycogen synthase type 1 (Gys1) [48] and glycogen synthase kinase (Gsk3β), Akt1 protein (Akt1), phosphatase and tensin homolog (Pten), apoprotein E (ApoE), sterol regulatory element binding protein (Srebp1), tau and precursor amyloid protein (App).

Total RNA was isolated from hippocampus and cortex using Trizol reagent (Invitrogen, Carlsbad, CA). RNA concentrations and integrity were determined using RNA 6000 Nano Assay Kit and the Bioanalyzer 2100 according to the manufacturer's instructions (Agilent Technologies, Santa Clara, CA). The primers used for PCR were as follows: Ir primers, 5' - CAAAAGCAC AATCAGAGTGAGTATGAC-3' and 5' - ACCACGTTGTGCAGGTAATCC-3' ; Irs1 primers, 5' - GCCTGGAGTATTATGAGAACGAGAA-3' and 5' - GGGGATCGAGCGTTTGG-3' ; Irs2 primers, 5' - AAGATAGCGGGTACATGCGAAT-3' and 5' - GCAGCTTAGGGTCT GGGTTC T-3' ; Glut1-45kDa primers, 5' - GTGCTTATGGGTTTCTCCAAA-3' and 5' - GACACCTCC CCCACATACATG-3' ; Glut3 primers, 5' - TTTGCAGTAGGCGGAATGG-3' and 5' - GCCAA CATGGCTTTGATCCTT-3' ; Glut3 primers: 5' - TGGCTACAACACCGGAGTCATCAA-3' and 5' - CTGCCAAAGCGGTTGACAAAGAGT-3' ; Gys1 primers, 5' - TCCACTGTGCCTGTGTCTTC A-3' and 5' - AGAGAACTTCTTCACATTCAGTCCATT-3' ; Gsk3β primers, 5' - TT AAGGAA GGAAAAGGTGAATCGA-3' and 5' - CCAAAAGCTGAAGGCTGCTG-3' ; 18S primers, 5' - TAA GTCCCTGCCCTTTGTACACA-3' and 5' - ATCCGAGGGCCTCACTAAAC-3' ; Akt1 primers: 5' - GTGGCAAGATGTGTATGAG-3' and 5' - CTGGCTGAGTAGGAGAAC-3' ; Pten primers: 5' - ACACCGCCAAATTTAACTGC-3' and 5' - TACACCAGTCCGTCCTTTC-3' ; ApoE primers: 5' - TTGGTCCCATTGCTGACAG-3' and 5' - ACCGTCAGTTCCTGTGTGAC-3' ; Srebp1 primers: 5' - ACAAGATTGTGGAGCTCAAG-3' and TGCGCAAGACAGCAGAT TTA-3' ; Tau primers: 5' - CGGCGTAAGCAAAGACA-3' ; and 5' - TGTAGCCGCTTCGTTCT-3' ; App primers: 5' - GGATGCGGAGTTCGGACATG-3' , and 5' - GTTCTGCATCTGCTCAA AG-3' .

The mRNA levels were assessed using real-time quantitative RT-PCR. All PCR reactions were performed in a total volume of 25 µl and included the following components: cDNA derived from 25 ng of total RNA, 400 nM of each primer, RNase-free water, and 12.5 µl of SYBR Green PCR Master Mix (ABI), an optimised buffer system containing AmpliTaq Gold DNA polymerase and dNTPs. All PCR reactions were performed in duplicate and cycling parameters were as follows: after an initial denaturation step for 10 min at 95°C, 40 subsequent cycles were performed in which samples were denatured for 15 s at 95°C followed by primer annealing and elongation at 60°C for 1 min. The relative quantities of mRNA were normalised by 18S rRNA content.

## Statistical analyses

The statistical analyses were carried out using Statistica 7.1 software (Statsoft France, Maison-Alford). The effects of stress on C, HF/HFr, C+CN and HF/HFr+CN fed rats were evaluated using a three-may ANOVA (Stress, Diet, Cinnamon) followed, if necessary, by post-hoc Bonferroni tests for all couples. The impact of stress on C+CN, HF/HFr, HF/HFr+CN was studied using the interactions Stress x CN, Stress x HF/HFr and Stress x HF/HFr+CN, respectively.
The p value results are presented in Table 1. The effective stress-induced differences were evaluated for each mRNA gene expression using Bonferroni post-hoc test comparing stressed vs. unstressed animals in each diet. The results are reported in Fig 2. The specific effects of each diet in the absence of stress are reported elsewhere [5].

The relations among all the mRNA expressions were evaluated in C rats using factorial analysis with normalised Varimax to calculate the weight of each component for each factor. Factors were considered if their Eigenvalue exceeded 1. Components of the factors were considered if their weight exceeded 0.70. For this analysis, the hippocampus (h) and frontal cortex (fc) mRNA were assembled as they were submitted to the same history of diet and stress.

The relations between blood corticosterone and changes in brain mRNA expression were studied in each diet using multiple regression tests with corticosterone as the predictor. Resting (n = 8) and stressed (n = 8) animals were combined to take into account the high dynamic range of biological response. However, the analyses were done separately in the hippocampus and frontal cortex to respect their differences in glucocorticoid effects. To be conservative, only strongly adjusted multiple correlations (r²>0.40) were taken into account.

For all analyses, the threshold for statistical significance was set at 0.05. However, trends were considered at p<0.10. The values are given as mean ± SEM.

# Results

## Behavioural results

All stressed animals exhibited the same defecation level during the acute stress exposure and the same behaviour in the open field (Table 2, S2 Table).

<table><caption>Table 1. p-value results of the three-way ANOVA carried out on hippocampus and frontal cortex mRNA expressions.</caption><thead><tr><th></th><th>Ir</th><th>Irs1</th><th>Irs2</th><th>Glut1</th><th>Glut3</th><th>Gys1</th><th>Gsk3</th><th>Akt1</th><th>Pten</th><th>Srebp1</th><th>ApoE</th><th>Tau</th><th>App</th><th>Sirt1</th></tr></thead><tbody><tr><td colspan="15" style="text-align: center; font-weight: bold;">Hippocampus</td></tr><tr><td>Diet</td><td>&lt;0.0001</td><td>ns</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.05</td><td>ns</td><td>&lt;0.01</td><td>&lt;0.0001</td><td>&lt;0.001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>ns</td></tr><tr><td>CN</td><td>= 0.0878</td><td>&lt;0.001</td><td>ns</td><td>= 0.0606</td><td>ns</td><td>&lt;0.001</td><td>ns</td><td>= 0.0775</td><td>ns</td><td>ns</td><td>&lt;0.01</td><td>ns</td><td>ns</td><td>= 0.0797</td></tr><tr><td>Stress</td><td>&lt;0.001</td><td>&lt;0.01</td><td>&lt;0.05</td><td>&lt;0.05</td><td>ns</td><td>&lt;0.05</td><td>&lt;0.05</td><td>ns</td><td>&lt;0.0001</td><td>&lt;0.01</td><td>&lt;0.0001</td><td>&lt;0.001</td><td>&lt;0.0001</td><td>ns</td></tr><tr><td>Diet x CN</td><td>= 0.0952</td><td>ns</td><td>ns</td><td>ns</td><td>&lt;0.01</td><td>&lt;0.001</td><td>ns</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.05</td><td>&lt;0.0001</td><td>= 0.0642</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Stress x Diet</td><td>&lt;0.01</td><td>&lt;0.001</td><td>ns</td><td>&lt;0.0001</td><td>ns</td><td>= 0.0816</td><td>ns</td><td>&lt;0.01</td><td>ns</td><td>&lt;0.01</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>Stress x CN</td><td>ns</td><td>&lt;0.01</td><td>ns</td><td>&lt;0.05</td><td>= 0.0762</td><td>&lt;0.0001</td><td>ns</td><td>&lt;0.001</td><td>&lt;0.05</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>= 0.0782</td></tr><tr><td>Stress x Diet x CN</td><td>ns</td><td>&lt;0.01</td><td>ns</td><td>ns</td><td>= 0.0812</td><td>&lt;0.0001</td><td>ns</td><td>ns</td><td>&lt;0.01</td><td>&lt;0.01</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td colspan="15" style="text-align: center; font-weight: bold;">Frontal cortex</td></tr><tr><td>Diet</td><td>&lt;0.0001</td><td>ns</td><td>&lt;0.001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.01</td><td>&lt;0.05</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.001</td></tr><tr><td>CN</td><td>ns</td><td>&lt;0.0001</td><td>ns</td><td>ns</td><td>ns</td><td>&lt;0.01</td><td>ns</td><td>ns</td><td>ns</td><td>= 0.0535</td><td>ns</td><td>= 0.0818</td><td>ns</td><td>ns</td></tr><tr><td>Stress</td><td>&lt;0.001</td><td>&lt;0.01</td><td>&lt;0.05</td><td>&lt;0.05</td><td>ns</td><td>&lt;0.01</td><td>&lt;0.05</td><td>ns</td><td>&lt;0.0001</td><td>ns</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.001</td><td>ns</td></tr><tr><td>Diet x CN</td><td>&lt;0.05</td><td>ns</td><td>ns</td><td>ns</td><td>&lt;0.05</td><td>&lt;0.01</td><td>= 0.0510</td><td>&lt;0.01</td><td>&lt;0.0001</td><td>ns</td><td>&lt;0.0001</td><td>&lt;0.001</td><td>&lt;0.01</td><td>&lt;0.05</td></tr><tr><td>Stress x Diet</td><td>ns</td><td>ns</td><td>ns</td><td>&lt;0.01</td><td>= 0.0721</td><td>= 0.0799</td><td>ns</td><td>&lt;0.05</td><td>ns</td><td>ns</td><td>ns</td><td>&lt;0.01</td><td>ns</td><td>ns</td></tr><tr><td>Stress x CN</td><td>ns</td><td>&lt;0.001</td><td>ns</td><td>&lt;0.05</td><td>ns</td><td>&lt;0.0001</td><td>ns</td><td>= 0.0646</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>&lt;0.05</td><td>= 0.0667</td></tr><tr><td>Stress x Diet x CN</td><td>ns</td><td>&lt;0.01</td><td>ns</td><td>ns</td><td>&lt;0.05</td><td>&lt;0.0001</td><td>ns</td><td>&lt;0.05</td><td>&lt;0.05</td><td>&lt;0.001</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr></tbody></table>

The rats were fed for 12 weeks with control (C), high fat/high fructose (HF/HFr) diets with a supplement of cinnamon (CN) or not, then exposed (Stress) or not (Rest) to a 30-min restraint+10-min open-field before the sacrifice. There were 8 groups: C (Rest: n = 8, Stress: n = 8), HF/HFr (Rest: n = 8, Stress: n = 8), C+CN (Rest: n = 9, Stress: n = 7) and HF/HFr+CN (Rest: n = 8, Stress: n = 8). Comparisons are made using three-way factorial ANOVA with Stress, HF/HFr (Diet) and cinnamon (CN) effects and Interactions. The p values are given for ANOVA effects and interactions. The threshold for statistical significance was set at p<0.05 and trends were considered at p<0.10.
<figure><img src="image_5.png" /><figcaption>**Fig 2.** The expression of mRNA in the hippocampus and frontal cortex. The rats were fed with control (C) or high fat/high fructose (HF/HFr) diets supplemented or not with cinnamon (CN) for 12 weeks, then exposed (Stress) or not (Rest) before the sacrifice. The 8 groups were the following: C (Rest: n = 8, Stress: n = 8), HF/HFr (Rest: n = 8, Stress: n = 8), C+CN (Rest: n = 9, Stress: n = 7) and HF/HFr+CN (Rest: n = 8, Stress: n = 8). Comparisons are made using three-way ANOVA followed by Bonferroni *post-hoc* tests for all couples. Here are reported only those concerning the Rest vs. Stress conditions in each diet. Results are expressed as following: a: p<0.10, b: p<0.05, c: p<0.01, d: p<0.001 and e: p<0.0001. Values are expressed as mean ± SEM.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0197094.g002

## Stress-induced changes in mRNA expression in control animals

The factorial analysis (h: hippocampus and fc: frontal cortex) isolated 5 factors that explained 87% of the global variance. The factors (variables, percentage of explained variance) were the following: factor 1 (h-Irs1, fc-Irs1, h-Pten, fc-Pten, h-ApoE, fc-ApoE, fc-Sbrep1; 41%), factor 2 (h-Ir, h-Akt, fc-Akt, h-Gsk3, fc-Gsk3; 19%), factor 3 (h-Glut3, fc-Glut3, h-App, h-Sirt1, fc-Sirt1; 15%), factor 4 (h-Irs2, fc-Irs2; 7%) and factor 5 (no component weighing more than 0.70; 4%). Hippocampus and frontal cortex gene expressions were coordinated as both areas were found for the same gene in the same factor. The sole discordance are fc-Ir in the factor 2 (weight: 0.6823) and fc-App in the factor 3 (weight: 0.6913), both being near the threshold.

In the hippocampus (Table 1, S1 Table), stress induced a clear increase in mRNA gene expression of Ir (p<0.001), Irs1 (p<0.01), Ir2 (p<0.05), Glut1 (p<0.05), Gys1 (p<0.05), Gsk3 (p<0.05), Pten (p<0.0001), ApoE (p<0.0001), Tau (p<0.001) and App (p<0.0001), but a decrease in Sbrep1 (p<0.01). In the frontal cortex (Table 1, S1 Table), stressor exposure was
<table><caption>Table 2. Behaviour expressed by rats in open field immediately after a 30-min restraint.</caption><thead><tr><th></th><th>C</th><th>HF/HFr</th><th>C+CN</th><th>HF/HFr+CN</th></tr></thead><tbody><tr><td><strong>Defecation</strong></td><td>3.4±0.6</td><td>5.2±0.9</td><td>3.6±0.6</td><td>5.3±0.9</td></tr><tr><td colspan="5"><strong>Entire open field</strong></td></tr><tr><td><strong>Grooming Nb</strong></td><td>2.8±0.5</td><td>2.6±0.8</td><td>2.9±0.7</td><td>1.6±0.2</td></tr><tr><td><strong>Grooming duration</strong></td><td>60±8</td><td>58±13</td><td>58±15</td><td>29±5</td></tr><tr><td><strong>Rearing Nb</strong></td><td>22.6±3.8</td><td>18.2±3.4</td><td>21.6±2.2</td><td>18.8±3.8</td></tr><tr><td><strong>Rearing duration</strong></td><td>41±6</td><td>37±8</td><td>45±6</td><td>38±8</td></tr><tr><td><strong>Inactivity duration</strong></td><td>465±25</td><td>490±17</td><td>464±21</td><td>489±22</td></tr><tr><td><strong>Distance</strong></td><td>3329±618</td><td>2690±330</td><td>3140±356</td><td>2668±396</td></tr><tr><td colspan="5"><strong>Center of the open field</strong></td></tr><tr><td><strong>Entries Nb</strong></td><td>6.5±2.6</td><td>6.8±2.0</td><td>5.4±1.5</td><td>5.0±1.1</td></tr><tr><td><strong>Inactivity duration</strong></td><td>14±4</td><td>9±4</td><td>8±3</td><td>12±6</td></tr><tr><td><strong>Duration in the center</strong></td><td>34±8</td><td>26±7</td><td>28±8</td><td>25±9</td></tr><tr><td><strong>Distance</strong></td><td>461±177</td><td>363±85</td><td>375±125</td><td>268±58</td></tr></tbody></table>

The rats were fed with control (C, n = 10), high fat/high fructose (HF/HFr, n = 10), control+cinnamon (C+CN, n = 10), and high fat/high fructose+cinnamon (HF/HFr+CN, n = 10) diets for 12 weeks. Distance is given in cm and duration in s. Values are expressed as mean ± SEM.

https://doi.org/10.1371/journal.pone.0197094.t002

followed by an increase in mRNA gene expression of *Ir* (p<0.001), *Irs1* (p<0.01), *Ir2* (p<0.05), *Glut1* (p<0.05), *Gys1* (p<0.01), *Gsk3* (p<0.05), *Pten* (p<0.0001), *ApoE* (p<0.0001), *Tau* (p<0.0001) and *App* (p<0.001).

In Control animals (Fig 2), stressor exposure resulted in an increase in *Ir* (p<0.01), *Irs1* (p<0.0001), *Glut1* (p<0.001), *Gys1* (p<0.0001) and *Akt1* (p<0.01) in the hippocampus. In the frontal cortex (Fig 2), the increase in mRNA gene expression is observed for *Irs1* (p<0.0001), *Glut1* (0.01) and *Gys1* (p<0.0001).

## Stress-induced changes in mRNA expression in C+CN rats

Animals supplemented with CN did not exhibit the same stress-induced mRNA gene expression than rats without any supplement. In the hippocampus (Table 1), the interaction between Stress and CN effects was significant for *Irs1* (p<0.01), *Glut1* (p<0.05), *Glut3* (p<0.10), *Gys1* (p<0.0001), *Akt1* (p<0.001), *Pten* (p<0.05), and *Sirt1* (p<0.10). In the frontal cortex (Table 1), the interaction was observed for *Irs1* (p<0.01), *Glut1* (p<0.05), *Gys1* (p<0.0001), *Akt1* (p<0.10), *App* (p<0.05) and *Sirt1* (p<0.10). It resulted in an increase in hippocampus mRNA gene expression of *Pten* (p<0.0001), *ApoE* (p<0.05) and a decrease in *Gys1* (p<0.0001) and *Sbrep1* (p<0.0001) in Stress C+CN rats compared to Rest C+CN rats (Fig 2). In the frontal cortex (Fig 2), the stress-induced increase concerned *Pten* (p<0.01) and *App* (p<0.10) while a decrease was observed in *Gys1* (p<0.0001) (Fig 2).

## Stress-induced changes in mRNA expression in HF/HFr rats

When HF/HFr fed animals were exposed to stress, a clear interaction between their diet and the effect of stress was observed (Table 1). In the hippocampus, the interaction between Diet and Stress concerned the mRNA gene expression of *Ir* (p<0.01), *Irs1* (p<0.001), *Glut1* (p<0.0001), *Gys1* (p<0.10), *Akt1* (p<0.01) and *Srebp1* (p<0.01). In the frontal cortex (Table 1), the interaction was observed in *Glut1* (0.01), *Glut3* (p<0.10), *Gys1* (p<0.10), *Akt1* (p<0.05) and *Tau* (p<0.01). In the hippocampus (Fig 2), there was an increase in *Pten* (p<0.0001), *ApoE* (p<0.01) and *App* (p<0.05) in Stress HF/HFr rats as compared to Rest HF/HFr rats. In the frontal cortex
(Fig 2), Stress HF/HFr rats exhibited an increase in *Pten* (p<0.0001) and Tau (p<0.01) compared to Rest HF/HFr rats.

## Stress-induced changes in mRNA expression in HF/HFr+CN rats

The HF/HFr+CN diet also altered the stress-induced mRNA expression as suggested by the interaction between stress and diet and cinnamon supplement (Table 1). In the hippocampus, an interaction was observed for *Irs1* (p<0.01), *Glut3* (p<0.10), *Gys1* (p<0.0001), *Pten* (p<0.01) and *Srebp1* (p<0.01). In the frontal cortex, the HF/HFr+CN diet modified the stress induced mRNA expression for *Irs1* (p<0.01), *Glut3* (p<0.05), *Gys1* (p<0.0001), *Akt1* (p<0.05), *Pten* (p<0.05) and *Srebp1* (p<0.001). Finally (Fig 2), compared to non stressed HF/HFr+CN rats, the stressed HF/HFr+CN rats exhibited higher hippocampus mRNA levels of *Pten* (p<0.001) and *ApoE* (p<0.01) but lower levels of *Gys1* mRNA (p<0.10). In the frontal cortex (Fig 2), Stress HF/HFr+CN rats exhibited higher mRNA levels of *Pten* (p<0.05), *Tau* (p<0.01) and *App* (p<0.10) but lower levels of *Gys1* (p<0.01) mRNA than Rest HF/HFr+CN rats.

## Correlation between blood corticosterone level and brain mRNA expression

In C rats, stress exposure was followed by a large increase in corticosterone (Stress, p<0.001). This effect was blunted by prior HF/HFr diet consumption (Interaction: p<0.05; Stress, p<0.001). The previous cinnamon supplement did not modify the stress-induced increase in corticosterone (C vs. C+CN: Stress, p<0.001 and C vs. HF/HFr+CN: Stress, p<0.001) (Table 3, S3 Table).

In the C group, positive correlations were observed between blood corticosterone level and *Irs2* (hippocampus: r² = 0.420, p<0.01), *Glut1* (hippocampus: r² = 0.733, p<0.001; frontal cortex: r² = 0.593, p<0.001), *Glut3* (hippocampus: r² = 0.455, p<0.01; frontal cortex: r² = 0.478, p<0.01), *Gys1* (hippocampus: r² = 0.607, p<0.001), *Pten* (hippocampus: r² = 0.467, p<0.01) and *Sirt1* (hippocampus: r² = 0.424, p<0.01) mRNA expression levels. In the C+CN group, the sole correlation observed was between corticosterone and *Glut1* (hippocampus: r² = 0.538, p<0.001). No correlation was observed within the HF/HFr and HF/HFr+CN groups.

## Discussion

The main finding of the study is that a single moderate stressor exposure was followed by an enhanced mRNA expression of genes involved in insulin signalling and cell glucose control. This stress-induced mRNA expression was blunted by the previous HF/HFr diet, CN

<table><caption>Table 3. Plasma corticosterone values (ng/ml) assessed on 8 experimental groups.</caption><thead><tr><th></th><th>Rest</th><th>Stress</th></tr></thead><tbody><tr><td>C</td><td>59.10±12.35 (20)</td><td>555.37±72.14 (10) ***</td></tr><tr><td>HF/HFr</td><td>41.57±16.36 (20)</td><td>401.57±34.33 (10) *** #</td></tr><tr><td>C+CN</td><td>44.68±12.38 (20)</td><td>435.81±29.19 (10) ***</td></tr><tr><td>HF/HFr+CN</td><td>33.07±7.02 (20)</td><td>417.07±32.70 (9) ***</td></tr></tbody></table>

The group sizes are indicated in brackets. Comparisons were made using the two-way factorial ANOVA (diet, stress).

The results are given as: Stress effect

***: p<0.001 and Interaction

#: p<0.05. Values are given as mean ± SEM.

https://doi.org/10.1371/journal.pone.0197094.t003
supplement or a combination of both. However, no difference in behaviour was observed among acutely stressed rats according to diets.

## The effect of stress on mRNA expression of genes involved in insulin signalling

Restraint is considered as a moderate stressor that activates the brain [42]. It induced changes in brain mRNA expression of genes involved in energy management. These modifications occurred in less than one hour. This time course is similar to that of early genes [49] and is in accordance with studies showing that *Glut1* transcription behaves like early genes [47]. The hippocampus and frontal cortex, 2 brain areas known for their role in emotion regulation, expressed *Ir* [50] and *Glut1* [51] proteins and have similar stress-induced modification in mRNA expression (our study). The concomitant increase in *Gys1* [48, 52] and *Glut1* [51] under stress-induced brain activation suggests a participation of astrocytes [53, 54]. We also observed the lack of increase in *Glut3* mRNA expression reported under stress [55]. Lastly, the stress-induced mRNA expression we observed is likely to be coordinated as the factor analysis isolates 4 factors with a biological consistency: factor 1 is related to the transduction of insulin signals, factor 2 and 3 are related to insulin signalling linked to oxidative signalling control [56] and factor 4 is related to nutrient homeostasis [57].

The value of the biological value of the stress-induced reaction is likely to be adaptive. The mRNA expression can be considered as the early phase of a coordinated diachronic reaction triggered by stress and aimed at facilitating the glucose entrance in the cerebral tissue and refilling brain with energy after the large expenditure driven by stress [58]. Although the increase in *Irs1* mRNA expression can reflect either a decreased [59], or enhanced [60, 61] insulin sensitivity, the latter is more probable as the *Akt1* mRNA expression also increased [61–63]. The increase in *Ir* concomitantly to *Glut1*, at least in the hippocampus, is in agreement with this interpretation. The increased insulin sensitivity favours therefore large brain glucose entry [58]. In line with this hypothesis, a similar reaction is triggered by other stressors: (i) a pulse of catecholamines trigger *in vitro* spontaneous synthesis of glycogen 9h later [64] and (ii) hypoglycemia is followed by an over refilling of brain glycogen [52].

## The effect of a previous HF/HFr diet on stress-induced mRNA expression

This 12-week HF/HFr diet was shown to induce peripheral [1] and brain [5] insulin resistance. The stress-induced increase in *Irs1*, *Glut1* and *Gys1* was blunted in animals fed with HF/HFr diet. This effect suggests an impairment of the stress-induced adaptive reaction. This is supported by the contrast of *Akt1* and *Pten* mRNA expression between C and HF/HFr rats ([C: ↗ *Akt1*→ *Pten*] vs. [HF/HFr:→*Akt1*-/'*Pten*]) [65]. This effect observed after single exposure to a moderate stressor remained restricted to the biological level and did not reach a behavioural level as no difference in behaviour was observed in the open field test carried out shortly after stressor exposure.

Furthermore, the increase in *Akt1* mRNA expression in C group supports a neuroprotective effect as the insulin is neuroprotective through the *Akt1* and *Gsk3β* pathway [56, 66], the Akt target being generic for neuroprotection [67]. Conversely, the blockade of the stress-induced increase in *Akt1* mRNA expression in HF/HFr group suggested a decrease in a neuroprotective effect.

## The effect of a previous CN supplement on the stress-induced mRNA expression

The cinnamon supplement also modified the changes observed in mRNA expression after stress exposure. The stress-induced enhancement in *Glut1* mRNA expression observed in C
rats was limited after CN supplement. However, the stress-induced increase in *Gys1* mRNA expression observed in C animals was not seen in C+CN animals that exhibited rather a decrease in *Gys1* expression as compared to their resting condition. The high *Gys1* expression observed in Rest C+CN animals is congruent to the increase in glycogen observed in the liver of resting animals after the CN supplement [68]. All that suggests that the stress-induced increase in need of glucose was less important in C+CN rats.

The CN supplement has been shown to produce a neuroprotective effect [34]. In our case, the pattern of stress-induced change in *Akt1*, *Gsk3β* and *Pten* was similar in C+CN and HF/HFr diets. The lack of *Akt1* increase associated with the *Pten* increase favours a deleterious effect [65]. However, the HF/HFr rats differed from the C+CN rats by their *Srebp1* mRNA expression. *Srebp1* participates in fatty acid biosynthesis regulation [69] and favours membrane fluidity [70]. A decrease in *Srebp1* after stress in C+CN rats suggests that there is no assistance in maintaining membrane fluidity, a situation of low allostatic load [70]. The specific decrease in *Srebp1* mRNA expression after stress indicates a neuroprotective effect. Accordingly, brain *Srebp1* mRNA expression is increased after a high fat diet [5] and aging [71], both situations related to a high allostatic load.

## Role of glucocorticoid on stress-induced mRNA expression

During this early stress phase, the high concentration of glucocorticoids may have an important modulator effect on the brain, especially on the insulin pathway [72]. In C rats, blood corticosterone levels were positively correlated with *Irs2*, *Glut1*, *Glut3*, *Gys1*, *Pten* and *Sirt1*. Thus suggests that glucocorticoid favours glucose use by neurons and astrocytes, either immediately or directly afterwards. This effect was only partly observed in C+CN rats in accordance with the effect of cinnamon on the stress-induced changes in mRNA expression. However, this effect of glucocorticoids was blunted in HF/HFr and HF/HFr+CN rats, suggesting that their brain energy regulation becomes partly independent from blood glucocorticoid levels.

## Conclusion

In conclusion, a single exposure to a moderate stressor is followed by genomic activation favouring replenishment of cell energy, an effect congruent to an adaptive response. This suggests that a moderate stress favours energy refilling mechanisms, an effect blunted by a previous HF/HFr diet and cinnamon supplement.

## Supporting information

*S1 Table. Data related to molecular biology analysis.*
(XLSX)

*S2 Table. Data related to behavioral test.*
(XLSX)

*S3 Table. Data related to plasma corticosterone values.*
(XLSX)

## Acknowledgments

We are indebted to Renaud Maury and Nadine Fidier who participated in the conditioning of the animals.
## Author Contributions

**Conceptualization:** Frédéric Canini, Bolin Qin, Cécile Batandier, Anne-Marie Roussel, Richard A. Anderson.

**Formal analysis:** Frédéric Canini.

**Funding acquisition:** Frédéric Canini, Anne-Marie Roussel, Richard A. Anderson.

**Investigation:** Frédéric Canini, Bolin Qin, Nathalie Arvy, Laurent Poulet, Cécile Batandier.

**Methodology:** Frédéric Canini, Anne-Marie Roussel, Richard A. Anderson.

**Project administration:** Frédéric Canini, Anne-Marie Roussel, Richard A. Anderson.

**Supervision:** Anne-Marie Roussel.

**Validation:** Bolin Qin, Nathalie Arvy.

**Writing – original draft:** Frédéric Canini.

**Writing – review & editing:** Frédéric Canini, Cécile Batandier, Anne-Marie Roussel, Richard A. Anderson.

## References

1. Couturier K, Batandier C, Awada M, Hininger-Favier I, Canini F, Anderson RA, et al. Cinnamon improves insulin sensitivity and alters the body composition in an animal model of the metabolic syndrome. Arch Biochem Biophys. 2010; 501:158–61. https://doi.org/10.1016/j.abb.2010.05.032 PMID: 20515642

2. Arnold SE, Lucki I, Brookshire BR, Carlson GC, Browne CA, Kazi H, et al. High fat diet produces brain insulin resistance, synaptodendritic abnormalities and altered behavior in mice. Neurobiol Dis. 2014; 67:79–87. https://doi.org/10.1016/j.nbd.2014.03.011 PMID: 24686304

3. Stranahan AM, Norman ED, Lee K, Cutler RG, Telljohann R, Egan JM, et al. Diet-induced insulin resistance impairs hippocampal synaptic plasticity and cognition in middle-aged rats. Hippocampus. 2008; 18(11):1085–8. https://doi.org/10.1002/hipo.20470 PMID: 18651634

4. Calvo-Ochoa E, Hernandez-Ortega K, Ferrera P, Morimoto S, Arias C. Short-term high-fat-and-fructose feeding produces insulin signaling alterations accompanied by neurite and synaptic reduction and astroglial activation in the rat hippocampus. J Cereb Blood Flow Metab. 2014; 34:1001–8. https://doi.org/10.1038/jcbfm.2014.48 PMID: 24667917

5. Anderson RA, Qin B, Canini F, Poulet L, Roussel AM. Cinnamon counteracts the negative effects of a high fat/high fructose diet on behavior, brain insulin signaling and Alzheimer-associated changes. PLoS ONE. 2013; 8(12):e83243. https://doi.org/10.1371/journal.pone.0083243 PMID: 24349472

6. Pratchayasakul W, Kerdphoo S, Petsophonsakul P, Pongchaidecha A, Chattipakorn N, Chattipakorn SC. Effects of high-fat diet on insulin receptor function in rat hippocampus and the level of neuronal corticosterone. Life Sci. 2011; 88:619–27. https://doi.org/10.1016/j.lfs.2011.02.003 PMID: 21315737

7. Clegg DJ, Gotoh K, Kemp C, Wortman MD, Benoit SC, Brown LM, et al. Consumption of a high-fat diet induces central insulin resistance independent of adiposity. Physiol Behav. 2011; 103:10–6. https://doi.org/10.1016/j.physbeh.2011.01.010 PMID: 21241723

8. McNay EC, Ong CT, McCrimmon RJ, Cresswell J, Bogan JS, Sherwin RS. Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance. Neurobiol Learn Mem. 2010; 93(4):546–53. https://doi.org/10.1016/j.nlm.2010.02.002 PMID: 20176121

9. Detka J, Kurek A, Basta-Kaim A, Kubera M, Lasoń W, Budziszewska B. Neuroendocrine link between stress, depression and diabetes. Pharmacol Reports. 2013; 65:1591–600.

10. Głombik K, Ślusarczyk J, Trojan E, Chamera K, Budziszewska B, Lasoń W, et al. Regulation of insulin receptor phosphorylation in the brains of prenatally stressed rats: new insight into the benefits of antidepressant drug treatment. Eur Neuropsychopharmacol. 2017; 27:120–31. https://doi.org/10.1016/j.euroneuro.2016.12.005 PMID: 28063625

11. Li L, Li X, Zhou W, Messina JL. Acute psychological stress results in the rapid development of insulin resistance. J Endocrinol. 2013; 217(2):175–84. https://doi.org/10.1530/JOE-12-0559 PMID: 23444388
12. Depke M, Fusch G, Domanska G, Geffers R, Völker U, Schuett C, et al. Hypermetabolic syndrome as a consequence of repeated psychological stress in mice. Endocrinology. 2008; 149:2714-23. https://doi.org/10.1210/en.2008-0038 PMID: 18325986

13. Tamashiro KL, Sakai RR, Shively CA, Karatsoreos IN, Reagan LP. Chronic stress, metabolism, and metabolic syndrome. Stress. 2011; 14(5):468-74. https://doi.org/10.3109/10253890.2011.606341 PMID: 21848434

14. Kendler KS, Karkowski LM, Prescott CA. Causal relationship between stressful life events and the onset of major depression. Am J Psychiatry. 1999; 156:837-41. https://doi.org/10.1176/ajp.156.6.837 PMID: 10360120

15. Rustad JK, Musselman DL, Nemeroff CB. The relationship of depression and diabetes: pathophysiological and treatment implications. Psychoneuroendocrinology. 2011; 36:1276-86. https://doi.org/10.1016/j.psyneuen.2011.03.005 PMID: 21474250

16. Ali S, Stone MA, Peters JL, Davies MJ, Khunti K. The prevalence of co-morbid depression in adults with type 2 diabetes: a systematic review and meta-analysis. Diabet Med. 2006; 23:1165-73. https://doi.org/10.1111/j.1464-5491.2006.01943.x PMID: 17054590

17. Andreoulakis E, Hyphantis T, Kandylis D, Iacovides A. Depression in diabetes mellitus: a comprehensive review. Hippocratia. 2012; 16(3):205-14.

18. Niraula K, Kohrt BA, Flora MS, Thapa N, Mumu SJ, Pathak R, et al. Prevalence of depression and associated risk factors among persons with type-2 diabetes mellitus without a prior psychiatric history: a cross-sectional study in clinical settings in urban Nepal. BMC Psychiatry. 2013; 13(309):1-12.

19. Nouwen A, Winkley K, Twisk J, Lloyd CE, Peyrot M, Ismail K, et al. Type 2 diabetes mellitus as a risk factor for the onset of depression: a systematic review and meta-analysis. Diabetologia. 2010; 53:2480-6. https://doi.org/10.1007/s00125-010-1874-x PMID: 20711716

20. Knol MJ, Twisk JWR, Beekman ATF, Heine RJ, Snoek FJ, Pouwer F. Depression as a risk factor for the onset of type 2 diabetes mellitus. A meta-analysis. Diabetologia. 2006; 49:837-45. https://doi.org/10.1007/s00125-006-0159-x PMID: 16520921

21. Kokoszka A, Pouwer F, Jodko A, Radzio R, Mucko P, Bienkowska J, et al. Serious diabetes-specific emotional problems in patients with type 2 diabetes who have different levels of comorbid depression: a polish study from the European Depression in Diabetes (EDID) Research consortium. Eur Psychiatry. 2009; 24:425-30. https://doi.org/10.1016/j.eurpsy.2009.04.002 PMID: 19541457

22. Mezuk B, Eaton WW, Albrecht S, Golden SH. Depression and type 2 diabetes over the lifespan. A meta-analysis. Diabetes Care. 2008; 31:2383-90. https://doi.org/10.2337/dc08-0985 PMID: 19033418

23. Winokur A, Maislin G, Phillips JL, Amsterdam JD. Insulin resistance after oral glucose tolerance testing in patients with major depression. Am J Psychiatry. 1988; 145:325-30. https://doi.org/10.1176/ajp.145.3.325 PMID: 2894176

24. McIntyre RS, Soczynska JK, Konarski JZ, Woldeyohannes HO, Law CWY, Miranda A. Should depressive syndromes be reclassified as "metabolic syndrome type II"? Ann Clin Psychiat. 2007; 19(4):257-64.

25. Zhang X, Dong F, Ren J, Driscoll MJ, Culver B. High dietary fat induces NADPH oxidase-associated oxidative stress and inflammation in rat cerebral cortex. Exp Neurol. 2005; 191:318-25. https://doi.org/10.1016/j.expneurol.2004.10.011 PMID: 15649487

26. Munhoz CD, García-Bueno B, Madrigal JLM, Lepsch LB, Scavone C, Leza JC. Stress-induced neuroinflammation: mechanisms and new pharmacological targets. Braz J Med Biol Res. 2008; 41:1037-46. PMID: 19148364

27. Park HR, Park M, Choi J, Park K-Y, Chung HY, Lee J. A high-fat diet impairs neurogenesis: involvement of lipid peroxidation and brain-derived neurotrophic factor. Neurosci Lett. 2010; 482:235-9. https://doi.org/10.1016/j.neulet.2010.07.046 PMID: 20670674

28. Fachinetto R, Burger ME, Wagner C, Wondracek DC, Brito VB, Nogueira CW, et al. High fat diet increases the incidence of orofacial dyskinesia and oxidative stress in specific brain regions of rats. Pharmacol Biochem Behav. 2005; 81:585-92. https://doi.org/10.1016/j.pbb.2005.05.001 PMID: 15936064

29. Pereira Ribeiro MC, de Vargas Barbosa NB, de Almeida TM, Parcianello LM, Perottoni J, de Avila DS, et al. High-fat diet and hydrochlorothiazide increase oxidative stress in brain of rats. Cell Biochem Funct. 2009; 27:473-8. https://doi.org/10.1002/cbf.1599 PMID: 19784960

30. Atif F, Yousuf S, Agrawal SK. Restraint stress-induced oxidative damage and its amelioration with selenium. Eur J Pharmacol. 2008; 600:59-63. https://doi.org/10.1016/j.ejphar.2008.09.029 PMID: 18854182

31. Lee YJ, Choi B, Lee EH, Choi KS, Sohn S. Immobilization stress induces cell death through production of reactive oxygen species in the mouse cerebral cortex. Neurosci Lett. 2006; 392:27-31. https://doi.org/10.1016/j.neulet.2005.08.065 PMID: 16203091
32. Archuleta TL, Lemieux AM, Saengirisuwan V, Teachey MK, Lindborg KA, Kim JS, et al. Oxidant stress-induced loss of IRS-1 and IRS-2 proteins in rat skeletal muscle: role of p38 MAPK. Free Radic Biol Med. 2009; 47:1486-93. https://doi.org/10.1016/j.freeradbiomed.2009.08.014 PMID: 19703555

33. Henriksen EJ. Effects of H2O2 on insulin signaling the glucose transport system in mammalian skeletal muscle. In: Cadenas E, Packer L, editors. Methods in Enzymology. 528: Elsevier Inc.; 2013. p. 269-78. https://doi.org/10.1016/B978-0-12-405881-1.00016-1 PMID: 23849871

34. Sartorius T, Peter A, Schulz N, Drescher A, Bergheim I, Machann J, et al. Cinnamon extract improves insulin sensitivity in the brain and lowers liver fat in mouse models of obesity. PLoS ONE. 2014; 9(3): e92358. https://doi.org/10.1371/journal.pone.0092358 PMID: 24643026

35. Yan J, Zhao Y, Suo S, Liu Y, Zhao B. Green tea catechins ameliorate adipose insulin resistance by improving oxidative stress. Free Radic Biol Med. 2012; 52:1648-57. https://doi.org/10.1016/j.freeradbiomed.2012.01.033 PMID: 22330066

36. Reagan LP, Magarinos AM, Yee DK, Swzeda LI, Van Bueren Antonia, McCall AL, et al. Oxidative stress and HNE conjugation of GLUT3 are increased in the hippocampus of diabetic rats subjected to stress. Brain Res. 2000; 862:292-300. PMID: 10799703

37. Lu J, Zhang K, Nam S, Anderson RA, Jove R, Wen W. Novel angiogenesis inhibitory activity in cinnamon extract blocks VEGFR2 kinase and downstream signaling. Carcinogenesis. 2010; 31(3):481-8. https://doi.org/10.1093/carcin/bgp292 PMID: 19969552

38. Anderson RA, Broadhurst CL, Polansky MM, Schmidt WF, Khan A, Flanagan VP, et al. Isolation and characterization of polyphenol type-A Polymers from cinnamon with insulin-like biological activity. J Agric Food Chem. 2004; 52:65-70. https://doi.org/10.1021/jf034916b PMID: 14709014

39. Killday KB, Davey MH, Glinski JA, Duan P, Veluri R, Proni G, et al. Bioactive A-type proanthocyanidins from cinnamomum cassia. J Nat Prod. 2011; 74:1833-41. https://doi.org/10.1021/np1007944 PMID: 21875098

40. Preuss HG, Echard B, Polansky MM, Anderson R. Whole cinnamon and aqueous extracts ameliorate sucrose-induced blood pressure elevations in spontaneously hypertensive rats. J Am Coll Nutr. 2006; 25(2):144-50. PMID: 16582031

41. Drouet J-B, Fauvelle F, Maunoir-Regimbal S, Fidier N, Maury R, Peinnequin A, et al. Differences in prefrontal cortex GABA/glutamate ratio after acute restraint stress in rats are associated with specific behavioral and neurobiological patterns. Neuroscience. 2015; 285:155-65. https://doi.org/10.1016/j.neuroscience.2014.10.058 PMID: 25451275

42. Buynitsky T, Mostofsky DI. Restraint stress in biobehavioral research: recent developments. Neurosci Biobehav Rev. 2009; 33:1089-98. https://doi.org/10.1016/j.neubiorev.2009.05.004 PMID: 19463853

43. Denenberg VH. Open-field behavior in the rat: what does it mean? Ann NY Acad Sci. 1969; 59:852-9.

44. Ramos A, Mormède P. Stress and emotionality: a multidimensional and genetic approach. Neurosci Biobehav Rev. 1998; 22(1):33-57. PMID: 9491939

45. Marissal-Arvy N, Batandier C, Dallennes J, Canini F, Poulet L, Couturier K, et al. Effect of a high-fat-high-fructose diet, stress and cinnamon on central expression of genes related to immune system, hypothalamic-pituitary-adrenocortical axis function and cerebral plasticity in rats. Br J Nutr. 2013:1-12. https://doi.org/10.1017/S0007114513003577 PMID: 24252462

46. Ghasemi R, Haeri A, Dargahi L, Mohamed Z, Ahmadiani A. Insulin in the brain: sources, localization and functions. Mol Neurobiol. 2013; 47:145-71. https://doi.org/10.1007/s12035-012-8339-9 PMID: 22956272

47. McGowan KM, Long SD, Pekala PH. Glucose transporter gene expression: regulation of transcription and mRNA stability. Pharmac Ther. 1995; 66(3):465-505.

48. Pellerin L, Magistretti PJ. Sweet sixteen for ANLS. J Cereb Blood Flow Metab. 2012; 32:1152-66. https://doi.org/10.1038/jcbfm.2011.149 PMID: 22027938

49. Cullinan WE, Herman JP, Battaglia DF, Akil H, Watson SJ. Pattern and time course of immediate early gene expression in rat following acute stress. Neuroscience. 1995; 64(2):477-505. PMID: 7700534

50. Havrankova J, Roth J, Brownstein M. insulin receptors are widely distributed in the central nervous system of the rat. Nature. 1978; 272:827-9. PMID: 205798

51. McEwen BS, Reagan LP. Glucose transporter expression in the central nervous system: relationship to synaptic function. Eur J Pharmacol. 2004; 490:13-24. https://doi.org/10.1016/j.ejphar.2004.02.041 PMID: 15094070

52. Gruetter R. Glycogen: the forgotten cerebral energy store. J Neurosci Res. 2003; 74:179-83. https://doi.org/10.1002/jnr.10785 PMID: 14515346

53. Brown RC, Nugent NR, Hawn SE, Koenen KC, Miller A, Amstadter AB, et al. Predicting the transition from acute stress disorder to posttraumatic stress disorder in children with severe injuries. J Pediatr Health Care. 2016; 30(6):558-68. https://doi.org/10.1016/j.pedhc.2015.11.015 PMID: 26776839
54. Falkowska A, Gutowska I, Goschorska M, Nowacki P, Chlubek D, Baranowska-Bosiacka I. Energy metabolism of the brain, including the cooperation between astrocytes and neurons, especially in the context of glycogen metabolism. Int J Mol Sci. 2015; 16:25959–81. https://doi.org/10.3390/ijms161125939 PMID: 26528968

55. Reagan LP, Margarinos AM, McEwen BS. Neurological changes induced by stress in streptozotocin diabetic rats. Ann NY Acad Sci. 1999; 893:126–37. PMID: 10672234

56. Duarte AI, Santos P, Oliveira CR, Santos MS, Rego AC. Insulin neuroprotection against oxidative stress is mediated by Akt and GSK-3β signaling pathways and changes in protein expression. Biochim Bio-phys Acta. 2008; 1783:994–1002. https://doi.org/10.1016/j.bbamcr.2008.02.016 PMID: 18348871

57. Taguchi A, Wartschow LM, White MF. Brain IRS2 signaling coordinates life span and nutrient homeostasis. Science. 2007; 317(20 july):369–72.

58. Fernandez AM, Hernandez E, Guerrero-Gomez D, Miranda-Vizuete A, Torres Aleman I. A network of insulin peptides regulate glucose uptake by astrocytes: potential new druggable targets for brain hypometabolism. Neuropharmacol. 2017:1–7.

59. Moroz N, Tong M, Longato L, Xu H, de la Monte SM. Limited Alzheimer-type neurodegeneration in experimental obesity and type 2 diabetes mellitus. J Alzheimer's Dis. 2008; 15:29–44.

60. Sun XJ, Miralpeix M, Myers Jr. MG, Glasheen EM, Backer JM, Kahn CR, et al. Expression and function of IRS-1 in insulin signal transmission. J Biol Chem. 1992; 267(31):22662–72. PMID: 1385403

61. Bhat NR, Thirumangalakudi L. Increased Tau phosphorylation and impaired brain insulin/IGF signaling in mice fed a high fat/high cholesterol diet. J Alzheimer's Dis. 2013; 36:781–9. https://doi.org/10.3233/JAD-2012-121030 PMID: 23703152

62. Harmon BT, Devaney SA, Gordish-Dressman H, Reeves EK, Zhao P, Devaney JM, et al. Functional characterization of a haplotype in the AKT1 gene associated with glucose homeostasis and metabolic syndrome. Hum Genet. 2010; 128:635–45. https://doi.org/10.1007/s00439-010-0891-7 PMID: 20872231

63. Devaney JM, Gordish-Dressman H, Harmon BT, Bradbury MK, Devaney SA, Harris TB, et al. AKT1 polymorphisms are associated with risk for metabolic syndrome. Hum Genet. 2011; 129:129–39. https://doi.org/10.1007/s00439-010-0910-8 PMID: 21061022

64. Sorg O, Magistretti PJ. Vasoactive intestinal peptide and noradrenaline exert long-term control on glycogen levels in astrocytes: blockade by protein synthesis inhibition. J Neurosci. 1992; 12:4923–31. PMID: 1334506

65. Lai TW, Zhang S, Wang YT. Excitotoxicity and stroke: Identifying novel targets for neuroprotection. Prog Neurobiol. 2014; 115:157–88. https://doi.org/10.1016/j.pneurobio.2013.11.006 PMID: 24361499

66. Ramalingam M, Kim S-J. Insulin exerts neuroprotective effects via Akt/Bcl-2 signaling pathways in differentiated SH-SY5Y cells. J Receptor Signal Transduction Res. 2015; 35(1):1–7. https://doi.org/10.3109/10799893.2014.922576 PMID: 24849496

67. Fukunaga K, Kawano T. Akt is a molecular target for signal transduction therapy in brain ischemic insult. J Pharmacol Sci. 2003; 92:317–27. PMID: 12939516

68. Couturier K, Qin B, Batandier C, Awada M, Hininger-Favier I, Canini F, et al. Cinnamon increases liver glycogen in an animal model of insulin resistance. Metab Clin Exp. 2011; 60:1590–7. https://doi.org/10.1016/j.metabol.2011.03.016 PMID: 21550075

69. Brown MS, Goldstein JL. The SREBP pathway: regulation review of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell. 1997; 89:331–40. PMID: 9150132

70. Hagen RM, Rodriguez-Cuenca S, Vidal-Puig A. An allostatic control of membrane lipid composition by SREBP1. FEBS Lett. 2010; 584:2689–98. https://doi.org/10.1016/j.febslet.2010.04.004 PMID: 20385130

71. Okamoto K, Kakuma T, Fukuchi S, Masaki T, Sakata T, Yoshimatsu H. Sterol regulatory element binding protein (SREBP)-1 expression in brain is affected by age but not by hormones or metabolic changes. Brain Res. 2006; 1081:19–27. https://doi.org/10.1016/j.brainres.2006.01.081 PMID: 16554040

72. Sapolsky RM, Romero LM, Munck AU. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocrin Rev. 2000; 21(1):55–89.