# Resveratrol protection against cardiac remodeling and ischemia-reperfusion injury associated with hepatic lipid metabolism ameliorations and adipose tissue autotaxin pathway inhibition in a diet induced prediabetic female rat model

Alexis Jouenne, Joevin Sourdon, Isabelle Varlet, Christophe Vilmen, Frank

Kober, Jean-François Landrier, Monique Bernard, Martine Desrois

► To cite this version:

Alexis Jouenne, Joevin Sourdon, Isabelle Varlet, Christophe Vilmen, Frank Kober, et al. Resveratrol protection against cardiac remodeling and ischemia-reperfusion injury associated with hepatic lipid metabolism ameliorations and adipose tissue autotaxin pathway inhibition in a diet induced prediabetic female rat model. Journal of Nutritional Biochemistry, 2025, 144, pp.109992. 10.1016/j.jnutbio.2025.109992. hal-05140849

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RESEARCH PAPER

# Resveratrol protection against cardiac remodeling and ischemia-reperfusion injury associated with hepatic lipid metabolism ameliorations and adipose tissue autotaxin pathway inhibition in a diet induced prediabetic female rat model

Alexis Jouenne<sup>a,*</sup>, Joevin Sourdon<sup>a</sup>, Isabelle Varlet<sup>a</sup>, Christophe Vilmen<sup>a</sup>, Frank Kober<sup>a</sup>, Jean-François Landrier<sup>b</sup>, Monique Bernard<sup>a</sup>, Martine Desrois<sup>a</sup>

Received 29 July 2024; received in revised form 13 March 2025; accepted 5 June 2025

## Abstract

Prediabetes is associated with an increased CV risk, especially for women. Recent evidence highlights the importance of liver and adipose tissue (AT) in its stratification. Current therapeutic strategies lack cardioprotective effects in this context. We evaluated the cardioprotective effects of resveratrol supplementation versus diet intervention and their association with hepatic and AT alterations in prediabetic female rats submitted to a high-fat-high-sucrose diet (HFS). Female Wistar rats were divided into 4 groups fed for 5 months with: standard diet (CTRL), HFS diet (HFS), HFS diet supplemented for the last 2 months with resveratrol (RSV) or 2 months of standard diet after 3 months of HFS diet (RSD). *In vivo* MRI was performed to study cardiac morphology, function and perfusion. Tolerance to ischemia-reperfusion (IR) injury was investigated *ex vivo* by a simultaneous measurement of cardiac function and energy metabolism with ³¹P MRS. Hepatic steatosis, along with hepatic and AT lipid metabolism and inflammation gene expression, was studied. HFS induced glucose intolerance, hepatic steatosis, higher diastolic LV volume, wall thickness, heart weight to tibia length ratio and altered myocardial tolerance to IR, with reduced function and energy metabolism. Both approaches improved glucose tolerance, reduced hepatic steatosis with lipid oxidation genes expression and ameliorated myocardial tolerance to IR. However, only RSV inhibited deleterious cardiac remodeling and autotaxin AT expression and plasma levels induced by HFS diet. RSV exhibited better cardioprotection than RSD, along with decreased hepatic steatosis and AT derived ATX, suggesting its protective potential against early cardiac alterations associated with prediabetes and NAFLD.

© 2025 The Authors. Published by Elsevier Inc.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Keywords: Prediabetes; NAFLD; Heart; Female sex; NMR; Resveratrol.

## 1. Introduction

Prediabetes is an asymptomatic abnormal stage that precedes the development of overt diabetes. It is defined by impaired fasting

glucose (100–125 mg/dL), impaired glucose tolerance (2 h plasma glucose during 75 g OGTT 140 to 199 mg/dL for humans), or HbA1c in the range of 39–47 mmol/mol [1]. Its prevalence is in constant rising; the International Diabetes Federation estimated the prevalence

Prediabetes is an asymptomatic abnormal stage that precedes the development of overt diabetes. It is defined by impaired fasting

<footnote><sup>a</sup>Aix-Marseille Univ, CNRS, CRMBM, Marseille, France</footnote>

<footnote><sup>b</sup>Aix-Marseille Univ, INSERM, INRAE, C2VN, Marseille, France</footnote>

<footnote>Abbreviations: ACO, Acyl-CoA Oxidase; AMPK, AMP-activated protein kinase; AT, Adipose tissue; ATP, Adenosine Tri Phosphate; ATX, Autotaxin; AUC, Area Under Curve; CPT1a, Carnitine Palmitoyl Transferase 1A; CTRL, Control; CV, Cardiovascular; CVD, Cardiovascular Diseases; DP, Developed Pressure; ECG, Electrocardiogram; EDP, End-Diastolic Pressure; EDV, End-Diastolic Volume; FAS, Fatty Acid Synthase; FFA, Free Fatty Acid; HFS, High-Fat-high-Sucrose; HR, Heart Rate; HTLR, Heart weight to Tibia Length Ratio; IPGTT, Intraperitoneal Glucose Tolerance Test; IR, Ischemia-Reperfusion; LCAD, Long Chain Acyl-CoA Dehydrogenase; LV, Left Ventricular; MASLD, Metabolic dysfunction-associated Steatotic Liver Disease; MCAD, Medium Chain Acyl-CoA Dehydrogenase; MRI, Magnetic Resonance Imaging; MRS, Magnetic Resonance Spectroscopy; NAFLD, Non-Alcoholic Fatty Liver Disease; PCr, Phosphocreatine; PCR, Polymerase Chain Reaction; PGC-1α, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Pi, Inorganic Phosphate; PME, Phosphomonoesters; PPARγ, peroxisome proliferator-activated receptor γ; ROS, Reactive Oxygen Species; RPP, Rate-Pressure-Product; RSD, Return to Standard Diet; RSV, Resveratrol; SIRT1, NAD-dependent protein deacetylase sirtuin-1; STZ, Streptozotocin; T2D, Type 2 Diabetes; TG, Triglycerides.</footnote>

<footnote>* Corresponding author at: Tel.: +33(0)4 91 32 48 08.</footnote>

<footnote>E-mail address: alexis.jouenne@univ-amu.fr (A. Jouenne).</footnote>
lence in the adult population to be 8.6% (548.4 million) in 2045 [2]. This stage is already associated with increased risks of cardiovascular disease (CVD) such as coronary artery disease, stroke or composite cardiovascular diseases [3–5] with a sexual dimorphism implying a greater cardiovascular disease risk for prediabetic women [6,7].

Moreover, growing evidence suggests that multi organ crosstalk, involving in parts adipose tissue (AT) or liver, should be considered in the CV risk and the stratification of complications in a prediabetic context [8–10]. Indeed, AT is known to be involved in cardiac alterations as deleterious remodeling through, for example, inflammation signaling, insulin resistance [8], and through secreted proteins such as the Adipocyte Fatty Acid Binding Protein (AFABP), also known as aP2 [11] or autotaxin [12,13]. Interestingly, autotaxin (ATX) secreted by AT was found to be involved in deleterious cardiac remodeling associated with a high fat diet in a rodent model [13]. In addition, the main liver pathology in this dysmetabolic state is MASLD, formerly called Non-Alcoholic Fatty Liver Disease (NAFLD) [14], which is the most common chronic liver disease in developed countries affecting approximately 25% of the adult population [15]. NAFLD definition is based on the presence of hepatic steatosis (>5% of hepatocytes) in association with metabolic risk factors (particularly, obesity and type 2 diabetes) and in the absence of excessive alcohol consumption [16]. NAFLD has been recently shown to be independently associated with CVD and should therefore be considered into the CV risk spectrum due to a potential lipotoxic effect [10,17–21]. Interestingly, the presence of NAFLD in association with prediabetes or T2D is linked with a worse prognosis and an increased CV risk compared to prediabetes or diabetes alone [22,23]. However, little is still known about the relationship between NAFLD, prediabetes and CVD.

Prediabetes has the benefit of being a reversible and long stage and represents an interesting therapeutic window [24]. By now, first line treatments of NAFLD and prediabetes, focused on restoring euglycemia, are diet and lifestyle interventions. However, they are notoriously difficult to maintain [25], suggesting that some patients may benefit from pharmacological therapy. In addition, a recent study on the outcomes of the Diabetes Prevention Program suggested that neither lifestyle intervention nor metformin reduced major cardiovascular events over 20 years despite the long-term diabetes prevention, suggesting that relevant short and long term cardioprotective therapeutic strategies are still warranted [26]. Resveratrol, a well-known antioxidant polyphenol, has already demonstrated cardioprotective effects in T2D rodent models [27–30] and prevented deleterious cardiac remodeling in a rodent model of dilated cardiomyopathy [31], but its effects in a prediabetic context and NAFLD remain to be elucidated.

In this context, we aimed to evaluate the cardioprotective effects of RSV supplementation versus diet intervention, and its association with hepatic and AT alterations in a HFS-induced female rat prediabetic model. Cardiac morphology, function, perfusion, and fibrosis were explored with multimodal longitudinal in vivo magnetic resonance imaging (MRI) at the 3rd and 5th months of diet, before and after intervention, respectively. At the end of the 5th month, rats underwent an IPGTT to evaluate the prediabetic state. Ex vivo isolated-heart experiments were carried out to evaluate tolerance to ischemia-reperfusion injury with simultaneous exploration of cardiac function and energy metabolism by ³¹P MRS. We then assessed hepatic steatosis along with hepatic lipid metabolism and inflammation gene expression. To assess lipid metabolism and autotaxin expression levels, AT gene expression was evaluated through biomolecular analyses.

<table><caption>Table 1
Nutritional composition and energy contents of control (SAFE, A04) and HFS (SAFE, U8978 v19) diets.</caption><thead><tr><th>Nutritional composition (%)</th><th>Control</th><th>HFS</th></tr></thead><tbody><tr><td>Nitrogen Free Extract</td><td>60.4</td><td>37.23</td></tr><tr><td>of which Starch</td><td>43.5</td><td>14.52</td></tr><tr><td>of which Sugars</td><td>3.2</td><td>20.25</td></tr><tr><td>Crude proteins</td><td>16.1</td><td>19.84</td></tr><tr><td>Crude Fat</td><td>3.1</td><td>35.92</td></tr><tr><td>Crude Ash</td><td>4.6</td><td>4.2</td></tr><tr><td>Crude Fiber</td><td>3.9</td><td>&lt;0.5</td></tr><tr><td>Moisture</td><td>11.9</td><td>2.8</td></tr></tbody><thead><tr><th>Energy content (%)</th><th>Control</th><th>HFS</th></tr></thead><tbody><tr><td>Energy from proteins</td><td>19.3</td><td>14.4</td></tr><tr><td>Energy from lipids</td><td>8.4</td><td>58.6</td></tr><tr><td>Energy from NFE</td><td>72.4</td><td>27</td></tr></tbody></table>

## 2. Materials and methods

### 2.1. Animal model and diets

Seven-week-old female Wistar rats (n=40) (Charles River, France) were housed in a ventilated rack cabinet with monitored temperature (22–24°C), light-dark cycle of 12:12 h and environmental enrichments. All procedures were approved by the animal experiment ethic committee of the University (APAFIS #10547-2017071009112930) and were carried out in accordance with the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Animals had access to water and food ad libitum. The study design is shown in Fig 1. Rats were randomly assigned into the different groups. The control group was fed a standard diet (A04C-10, SAFE diets, Augy, France) for 5 months, the HFS group was fed a High Fat High Sucrose diet (U8978 v19, SAFE diets, Augy, France) for 5 months, as previously described [32–34]. The RSV supplemented group was fed HFS diet for 5 months and supplemented with RSV [35] in ad libitum water (1 mg/kg/d, based on Rocha et al. and Lin et al. studies [36,37]) during the last 2 months. RSV was added to ad libitum water in light-protected feeding bottles, which were changed every 2 days. RSV amount was adjusted to the average water consumption of the cage. The diet intervention group (Return to Standard Diet, RSD), which is one of the most efficient therapeutic approaches against prediabetes [38], was fed the HFS diet for 3 months and then standard chow (A04C-10, SAFE diets, Augy, France) for 2 months. The nutritional composition and energy contents of both diets are listed in Table 1. Food intake and animal weight were monitored weekly throughout the 5 months. Food efficiency was estimated as the ratio of weight gain and food intake over the 5 months of diet.

### 2.2. In vivo Magnetic Resonance Imaging

In order to evaluate cardiac morphology, function, fibrosis and perfusion, magnetic resonance imaging experiments were carried out at the 3rd and 5th month. All acquisitions were performed using a Bruker Pharmascan Magnetic Resonance system equipped with a 7 Tesla superconducting magnet (Bruker Biospin, Ettlingen, Germany) and an actively decoupled Bruker 4 channel receive surface coil array. Animals were sedated with 3.5% isoflurane and then placed prone into a homemade cradle designed for rats. During the MRI investigations, anesthesia was maintained at 1.5–2.5% isoflurane in 1 L/min pure oxygen-flow to obtain regular breathing at a rate of approximately 70 breaths per minute. Respiration
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Experimental protocol. CTRL, control group; HFS, high fat high sucrose fed group; HFS+RSV, RSV supplemented group; RSD, return to standard diet group. IPGTT, intra peritoneal glucose tolerance test; MRS, magnetic resonance spectroscopy.</figcaption></figure>

rate, ECG signal and body temperature were monitored through-
out the whole exploration. An ECG-gated gradient-echo cine MRI
sequence (cine FLASH) was used to assess myocardial morphology,
mass and systolic function [39]. To evaluate myocardial fibrosis, a
standard ECG- and respiration-gated spin-echo pulse sequence was
used with two echo times in order to create relaxation time maps
throughout the slice. The trigger timing was set such as to ob-
tain images in the same cardiac phase for both echo times [40]. In
vivo myocardial perfusion was measured using a Look-Locker Flow-
sensitive Alternating Inversion Recovery Gradient-Echo (LLFAIRGE)
Arterial Spin Labelling (ASL) technique [41]. All imaging data were
post processed using homemade software developed in Python.

2.3. *Intraperitoneal Glucose Tolerance Test*

At the end of the 5-months diet, an intraperitoneal glucose tol-
erance test (IPGTT) was performed to evaluate glucose homeosta-
sis. Animals were fasted for 6 h prior to IPGTT but had free access
to water during the entire experiment. The extremity of the tail
was incised in order to collect small drops of blood for glycemia
assessment using test strips of the blood nano-glucometer (ACCU-
CHEK Performa, Roche, Basel, Switzerland). The first value corre-
sponded to fasting glycemia. Rats were then weighed, and a bo-
lus of glucose was injected into the intraperitoneal cavity at a
dose of 1 g/kg. Glycemia was measured again at 15, 30, 60, 90,
and 120 min post glucose injection. Glucose intolerance was estab-
lished by significantly higher IPGTT's AUC than the control group
[42,43].

2.4. Isolated perfused rat heart preparation and tolerance to ischemia-reperfusion injury

Animals were sedated by inhalation of 3.5% isoflurane then
weighted and anesthetized by intraperitoneal injection of pen-
tobarbital sodium (132 mg/kg Euthasol Vet, Dechra Veterinary
Products SAS, Montigny-le-bretonneux, France). Hearts were then
quickly collected by thoracotomy and stabilized in ice-cold Krebs-
Henseleit buffer (containing (mM): NaCl (118), KCl (4.7), MgSO4
(1.2), CaCl2 (1.75), NaHCO3 (25), KH2PO4 (1.2), EDTA (0.5) and d-
glucose (11)). Hearts were then weighed and cannulated via the
ascending aorta for retrograde Langendorff perfusion of the coro-
nary arterial network, at a constant pressure of 100 mmHg, as pre-
viously described [33].

2.4.1. *Experimental protocol*

After stabilization, hearts were perfused for 4 min with Krebs-
Henseleit buffer and then for 24 min with a physiological re-
circulating Krebs-Henseleit buffer containing 0.4 mM palmitate,
3% albumin, 11 mM glucose, 3 U/L insulin, 0.8 mM lactate, and
0.2 mM pyruvate. Four minutes before low-flow ischemia, hearts
were perfused with a physiological Krebs-Henseleit buffer contain-
ing 1.2 mM palmitate. Then the hearts underwent a low-flow is-
chemia (0.5 mL/min/g wet weight) during 32 min with the same
buffer. Finally, flow was restored entirely for 32 min with the phys-
iological recirculating Krebs-Henseleit buffer containing 0.4 mM
palmitate. The perfusates were continually gassed with a mixture
of 95% O2 and 5% CO2 to maintain pH at 7.4. The buffer temper-
ature was maintained and monitored with a thermal probe (SA
Instruments, Stony Brook, USA) placed next to the heart at 37°C
throughout the protocol.

2.4.2. Myocardial function

During the cannulation process, a water-filled latex balloon con-
nected to a pressure sensor was inserted into the left ventricle to
record left ventricular developed pressure (DP) and heart rate (HR).
The water filled latex balloon was then inflated in order to pro-
duce an end diastolic pressure of 10 mm Hg at the start of perfu-
sion. The rate pressure product (RPP), the product of DP and HR
was used as an index of cardiac function. Coronary flow was mea-
sured by collection of coronary effluent before and after ischemia
(at 20 min and 80 min) and expressed in mL/min/g wet weight, as
previously described [33].

2.4.3. Myocardial energy metabolism

To evaluate cardiac high-energy compound concentrations, per-
fused rat hearts were placed in a 20-mm magnetic resonance sam-
ple tube, inserted in a ³¹P probe seated in the bore of a super-
conducting 9.4 Tesla Bruker magnet (Avance 400 MHz spectrome-
ter [Bruker, Ettlingen, Germany]). A series of eight ³¹P NMR spec-
tra were recorded during each period of the experimental proto-
col to quantify phosphorus metabolites (Adenosine triphosphate:
ATP, phosphocreatine: PCr and inorganic phosphate: Pi). Intracellu-
lar pH (pHi) was calculated from the chemical shift of the Pi reso-
nance, accounting for temperature changes. Spectra were obtained
by signal averaging 269 free induction decay signals acquired dur-
ing 4 min. Prior to Fourier transformation, the free induction de-
cay was multiplied by an exponential filter function that gener-
ated a 20 Hz line broadening. The positions and areas of the reso-
nances were determined using the AMARES software package (jM-
RUI; http://www.jmrui.eu/; accessed on February 26, 2018) [44], in-
cluded in a homemade software developed in Python. Phenylphos-
phonic acid (0.6 M) in a glass capillary placed along the heart was
used as an external reference for quantification. Cytosolic concen-
trations (mmol/L) were calculated using the intracellular volume,
tissue weight and saturation factors.

2.4.4. Sample collection

At the time of heart collection, blood was withdrawn for further
analyses. Liver and perirenal AT were then weighed and collected
for histology (liver) and biochemical analyses. At the end of the
experiments, hearts were “freeze-clamped” in liquid nitrogen and
kept at –80°C for further analysis.

2.5. Biochemical analyses

2.5.1. Nonfasting plasma and heart analyses

Glycemia was measured using the Randox GLUC-PAP kit (GL364, Randox, Crumlin, United Kingdom), according to the manufacturer's instructions. The colorimetric Free Fatty Acid (FFA) Quantification Kit (MAK044-1KT, Sigma Aldrich, Saint-Louis, MO, USA) was used to determine FFA plasma concentrations. Plasma total antioxidant power was assessed using the Total Antioxidant Power Kit (OX-TA02, Oxford biomedical research, Rochester Hills, USA) and was also used to evaluate cardiac total antioxidant power in freeze clamped hearts, according to the manufacturer's instruction. Plasma ATX was measured using the Rat Autotaxin/ENPP2 ELISA Kit (SK00526-01, Aviscera Bioscience, Santa Clara, CA, USA), according to the manufacturer's instructions.

2.5.2. Liver analyses

2.5.2.1. *Total lipid mass and triglycerides concentration.* Total lipid mass was determined using the Folch lipid extraction method. Briefly, weighed liver samples were placed and ground in a chloroform/methanol (2/1) solution. After centrifugation, the chloroform phase was collected and underwent another centrifugation. At the end of the extraction, the lipidic phase (chloroform) was placed into preweighted glass tubes and evaporated in order to have the total lipid content of the sample in the tube. Total lipid mass was then normalized by the sample weight. Liver triglyceride concentration was determined using the colorimetric GPO method Triglyceride assay kit (80019, Biolabo, Maizy, France).

2.5.2.2. *Liver histology.* Liver tissue samples were fixed in 10% buffered formalin, embedded in paraffin and sliced to prepare 5 µm tissue sections whose were stained with hematoxylin and eosin (H&E) [45]. The images were captured by a light microscope (EZAD, Leica, Germany).

2.6. Liver and perirenal AT genes expression by RT-qPCR

Total RNA from liver and perirenal AT was extracted using TRIzol reagent according to the manufacturer's instructions (Thermo Fisher, Courtaboeuf, France). cDNAs were then synthesized from 1 µg of total RNA in 20 µL using random primers and Moloney Murine Leukemia Virus reverse transcriptase (Thermo Fisher, Courtaboeuf, France). Real-time quantitative RT-PCR analyses were performed using the AriaMX real time PCR system (Agilent Technologies France, Les Ullis) as previously described [46]. Data were analyzed using the Aria Agilent software 1.8 (Agilent, Santa Clara, CA, USA). All PCR reactions were using a SYBR Green Master mix (PowerUp SYBR, Thermo Fisher, Courtaboeuf, France)

using 10 ng of cDNA in 10 µL. The PCR cycles were composed
of one step of UDG activation (50 °C for 2 min), then DNA Poly-
merase activation step (95 °C for 2 min) followed by 40 cycles
composed of a denaturation step (95 °C for 15 s) and an extend
step (60 °C for 30 s), according to the manufacturer's instructions.
The expression was quantified in duplicate, and the ribosomal pro-
tein 18S rRNA was used as the endogenous control in the compar-
ative cycle threshold (CT) method [47] in order to express data as
a relative expression ratio. Primers used for this study are listed in
Table 2.

2.7. Statistical analysis

Data are graphically provided as mean ± SEM of absolute val-
ues. Graph Pad Prism software 9.0 (La Jolla, CA, USA) was used
for all statistical processing. Significant differences between groups
were determined using two-way analysis of variance (ANOVA) with
repeated measures over time for the time-dependent variables fol-
lowed by Bonferroni post-hoc test. One-way analysis of variance
(ANOVA) followed by Bonferroni post-hoc test was used for the
other parameters. Pearson test was realized to evaluate correla-
tions between observed cardiac ex vivo, in vivo parameters, phys-
iological, hepatic and AT parameters where only r>0.5 were in-
cluded. A P value of less than or equal to .05 was considered to
indicate significant difference.

3. Results

3.1. Therapeutic approaches effects on physiological parameters

The effects of therapeutic approaches on physiological parame-
ters are shown in Table 3 and Figure S1. Although a tendency for
a higher body weight for the HFS fed groups was observed, it was
not significant (Fig. S1A). Both therapeutic approaches significantly
restored the elevated fasting glycemia induced by HFS diet to con-
trol levels (P<.05 CTRL, RSV, RSD vs HFS). The IPGTT emphasized
a significant glucose intolerance associated with an increased area
under the curve (AUC) for HFS (P<.05 vs CTRL), as shown in Figure
S1B and Table 3. RSV supplementation decreased glucose intoler-
ance at an intermediary level (P<.05 vs CTRL, HFS), while return
to standard diet significantly reduced it to values comparable to
the CTRL level, as shown by the area under the curve (AUC). Food
efficiency was significantly higher in the three HFS groups versus
CTRL at 3 months (P<.05). After 2 months of treatment, food effi-
ciency was found unchanged for HFS and RSV groups but returned
to CTRL levels for RSD (P<.05). HFS diet induced elevated non-
fasting glycemia, without an effect of RSV supplementation, while
RSD induced a return to CTRL level (P<.05 CTRL, RSD vs HFS, RSV).
FFA levels were significantly higher for HFS (P<.05 vs CTRL, RSV,
and RSD). RSV supplementation significantly reduced FFA, but the
levels were still higher than in CTRL (P<.05 vs CTRL, HFS, and RSD).
RSD returned FFA level to that of CTRL (P<.05 vs HFS and RSV).
Perirenal AT weight normalized to body weight was found signif-
icantly higher in HFS and RSV groups compared to CTRL (P<.05),
while diet intervention partially reduced it (P<.05 vs HFS, RSV,
and CTRL). Both therapeutic approaches significantly reduced liver
weight normalized to body weight ratio compared to HFS (P<.05).
Interestingly, heart weight normalized to tibia length was signifi-
cantly higher in HFS and RSD groups compared to CTRL and RSV
groups (P<.05). Plasma total antioxidant power, estimated as µM
Trolox equivalent, was higher in HFS and RSV groups, compared to
CTRL and RSD groups (P<.05). However, no significant difference
was found in cardiac total antioxidant power among groups (data
not shown).
<table><caption>Table 2
Primer sequences used for real-time-PCR experiments.</caption><thead><tr><th>Gene</th><th>Forward 3'–5'</th><th>Reverse 3'–5'</th></tr></thead><tbody><tr><td>18S</td><td>GTGGGCCTGCGGCTTAAT</td><td>GCCAGAGTCTCGTTCGTTATC</td></tr><tr><td>Enpp2/Atx</td><td>TCTACGAGGGCGAGAGAAGT</td><td>GCTTGGTGGCTGTAATCCAT</td></tr><tr><td>Lipid metabolism</td><td>Forward 3'–5'</td><td>Reverse 3'–5'</td></tr><tr><td>Fas</td><td>GGCCACCTCAGTCCTGTTAT</td><td>AGGGTCCAGCTAGAGGGTACA</td></tr><tr><td>Aco</td><td>CACCTTCGAGGGAGAGAACA</td><td>CGCACCTGGTCGTAGATTTT</td></tr><tr><td>Cpt1a</td><td>ACAATGGGACATTCCAGGAG</td><td>AAAGACTGGCGCTGCTCA</td></tr><tr><td>Mcad</td><td>CTTAGCTTCTGCCCTGTGGT</td><td>TGAGAGACACTTCTCAGGACCTT</td></tr><tr><td>Lcad</td><td>GCAGTTACTTGGGAAGAGCAA</td><td>GGCATGACAATATCTGAATGGA</td></tr><tr><td>Pparγ</td><td>GGTGAAACTCTGGGAGATCCT</td><td>AATGGCATCTCTGTGTCAACC</td></tr><tr><td>Inflammation</td><td>Forward 3'–5'</td><td>Reverse 3'–5'</td></tr><tr><td>Tnf-α</td><td>GCCCAGACCCTCACACTC</td><td>CCACTCCAGCTGCTCCTCT</td></tr><tr><td>Il6</td><td>CCCTTCAGGAACAGCTATGAA</td><td>ACAACATCAGTCCCAAGAAGG</td></tr><tr><td>Mcp1</td><td>AGCATCCACGTGCTGTCTC</td><td>GATCATCTTGCCAGTGAATGAGT</td></tr></tbody></table>

18S, ribosomal protein S18; FAS, fatty acid synthase; ACO, Acyl CoA oxidase; CPT1 a, Carnitine palmitoyltransferase 1a (Liver); ENPP2/ATX, ectonucleotide pyrophosphatase/phosphodiesterase 2; MCAD, Medium-chain acyl-CoA dehydrogenase; LCAD, Long-chain acyl-CoA dehydrogenase; PPARγ, peroxisome proliferator-activated receptor γ; TNF-α, Tumor necrosis factor α; IL-6, Interleukin 6; MCP1, Monocyte chemoattractant protein 1.

<table><caption>Table 3
Therapeutic approach effects on physiological parameters.</caption><thead><tr><th>Physiological parameters</th><th>CTRL</th><th>HFS</th><th>RSV</th><th>RSD</th></tr></thead><tbody><tr><td>Fasting glycemia (mg/dL)</td><td>101.3±2.452 b</td><td>119.9±2.347 a</td><td>108.8±2.55 b</td><td>102.9±2.013 b</td></tr><tr><td>IPGTT AUC</td><td>641.3±15.11 b,c</td><td>852.8±18.28 a,c,d</td><td>767.5±22.92 a,b</td><td>716±19.71 b</td></tr><tr><td>Food efficiency (3rd month)</td><td>0.013±0.004 b,c,d</td><td>0.0608±0.012 a</td><td>0.064±0.007 a</td><td>0.061±0.013 a</td></tr><tr><td>Food efficiency (5th month)</td><td>0.011±0.002 b,c</td><td>0.065±0.014 a,d</td><td>0.067±0.007 a,d</td><td>0.008±0.004 b,c</td></tr><tr><td>Non-fasting glycemia (mg/dL)</td><td>144.3±9.164 b,c</td><td>206.5±12.13 a,d</td><td>193.3±5.399 a,d</td><td>149.3±7.792 b,c</td></tr><tr><td>Plasma FFA (mmol/L)</td><td>0.096±0.019 b,c</td><td>0.389±0.047 a,c,d</td><td>0.272±0.018 a,b,d</td><td>0.151±0.029 b,c</td></tr><tr><td>Perirenal AT weight/ Animal weight</td><td>0.008±0.0005 b,c,d</td><td>0.019±0.0012 a,d</td><td>0.018±0.0008 a,d</td><td>0.011±0.0008 a,b,c</td></tr><tr><td>Liver weight/ Animal weight</td><td>0.026±0.0006 b</td><td>0.029±0.0007 a,c,d</td><td>0.025±0.0004 b</td><td>0.026±0.0008 b</td></tr><tr><td>Heart weight/ Tibia length (g/cm)</td><td>0.207±0.003 b,d</td><td>0.231±0.003 a,c</td><td>0.206±0.005 b,d</td><td>0.228±0.005 a,c</td></tr><tr><td>Plasma ATX (ng/L)</td><td>0.787±0.0789</td><td>5.115±2.429 c</td><td>0.448±0.069 b</td><td>1.158±0.249</td></tr><tr><td>Plasma Total Antioxidant Power (μM Trolox equivalent)</td><td>13.48±0.75 b,c</td><td>18.23±1.09 a,d</td><td>18.92±1.6 a,d</td><td>12.62±1.21 b,c</td></tr></tbody></table>

CTRL, female control group; HFS, female high-fat high-sucrose diet group; HFS+RSV, RSV supplemented female high-fat high-sucrose group; RSD, diet intervention female group.

Physiological parameters: IPGTT AUC: IPGTT's area under curve, FFA, free fatty acid, ATX, autotaxin.

Data are means ± SEM. One-way ANOVA test followed by Bonferroni post-hoc test was performed on all parameters.

## 3.2. Therapeutic approaches effects on in vivo cardiac morphology, function, and perfusion

The effects of the therapeutic approaches on *in vivo* cardiac parameters before and after treatment (after 3 and 5 months, respectively), are shown in Figure 2. After 3 months of diet, no differences in left-ventricular end-diastolic volume (LVEDV) were found among the different groups (Fig. 2A). Interestingly, LVEDV was found higher in HFS and RSD groups compared to CTRL and RSV after 5 months (P<.05, Fig. 2A). Increased myocardial wall thickness was found in HFS, RSV and RSD groups after 3 months compared to CTRL (P<0.01, Fig. 2A). After 5 months, only HFS and RSD myocardial wall thickness were found significantly higher compared to CTRL and RSV (P<.05). Only a trend towards higher left ventricular mass for the three HFS fed groups compared to CTRL was observed after 3 months. After 5 months, a significant increase of left ventricular mass was observed for HFS and RSD compared to CTRL and RSV (P<.05). Significantly higher myocardial perfu-

sion was observed for the three HFS fed groups compared to CTRL after 3 months (P<.0001) and 5 months (P<.0001), as shown in Figure 2B (Myocardial Blood Flow maps shown). There were no differences among the groups in Left Ventricular Ejection Fraction (Figure S2A), cardiac output (Figure S2B), cardiac index (Fig S2C) or cardiac fibrosis (Fig S2D) at any timepoint.

## 3.3. Therapeutic approaches effects on hepatic steatosis state

Therapeutic approaches effects on hepatic lipid content are shown in Figure 3A. Increased total lipid mass was found for HFS compared to CTRL (P<.0001), while both therapeutic approaches significantly reduced total lipid mass compared to HFS (P<.001 vs HFS). HFS diet increased triglyceride concentration as well compared with CTRL (P<.05). RSV supplementation significantly reduced the increased TG concentration (P<.05 vs HFS) while only a trend was observed for RSD. Increased triglyceride concentration was associated with histology assessment of lipid droplets in liver

<footnote>a P<.05 vs. CTRL.</footnote>

<footnote>b P<.05 vs. HFS.</footnote>

<footnote>c P<.05 vs. RSV.</footnote>

<footnote>d P<.05 vs. RSD.</footnote>
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** *In vivo* cardiac parameters. (A): left ventricular end-diastolic volume (LVEDV). Myocardial wall thickness and left ventricular mass; (B) myocardial perfusion and myocardial blood flow maps at the 3rd and 5th months. Data are means ± SEM. Two-way ANOVA followed by Bonferroni post-hoc test were performed on all parameters. \*: P<.05; \*\*: P<.01; \*\*\*: P<.001; \*\*\*\*: P<.0001.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Hepatic lipid content and lipid metabolism gene expression. (A) Total lipid mass and Triglyceride concentration; (B) Hepatic histological slices (HE staining), arrows showing lipid vacuoles Lipid; (C) β-oxidation gene relative expression; (D) Fatty-Acid-Synthase relative expression. Data are means ± SEM. One-way ANOVA followed by Bonferroni post-hoc test were performed on all parameters. \*: P<.05; \*\*: P<.01; \*\*\*: P<.001; \*\*\*\*: P<.0001.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Adipose tissue lipid metabolism, ATX gene expression and plasma ATX levels. (A) Perirenal adipose tissue lipid metabolism and ATX relative expression. (B) ATX Plama levels. Data are means ± SEM. One-way ANOVA followed by Bonferroni post-hoc test were performed on all parameters. \*: P<.05; \*\*: P<.01; \*\*\*: P<.001; \*\*\*\*: P<.0001.</figcaption></figure>

histological samples in HFS, as shown in **Figure 3B**. In addition, reduced TG hepatic concentration was associated with reduced number of lipid droplets on histology assessment for both therapeutic approaches, as shown in **Figure 3B**.

### 3.4. Therapeutic approaches effects on hepatic metabolism and inflammation gene relative expression

The effects of the two therapeutic approaches on the relative expression of hepatic lipid metabolism are shown in **Figure 3C**. Both therapeutic approaches decreased relative hepatic *Cpt1a* and *Lcad* expression compared to HFS (*P*<.05), but diet intervention induced a higher reduction compared to RSV supplementation (*P*<.01). In addition, relative *Lcad* expression was significantly higher for RSV than CTRL (*P*<.01). Both therapeutic approaches significantly reduced in a similar way the increase in mRNA relative expression of *Aco* and *Mcad* induced by HFS diet (*P*<.05). No differences among groups were observed on the relative mRNA level of hepatic fatty acid synthase (*Fas*) as shown in **Figure 3D**, nor on the relative gene expressions of Interleukin 6 (Il-6), Tumor Necrosis Factor α (*Tnf-α*), and hepatic Monocyte Chemoattractant Protein 1 (*Mcp1*, also known as *Ccl2*), (Fig S3).

### 3.5. Therapeutic approaches effects on AT metabolism and inflammation gene relative expression

The effects of the therapeutic approaches on the relative expression of AT genes are shown in **Figure 4A**. Increased *Aco* mRNA level was observed for HFS compared to CTRL (*P*<.01). RSV supplementation induced increased *Aco* mRNA level compared to CTRL and RSD (*P*<.05). A significantly decreased *Pparγ* relative gene expression was found only for RSV (*P*<.05 vs CTRL, HFS, and RSD).

No significant differences were found in *Il-6* mRNA gene relative expression among groups (Fig S3). HFS diet induced a significant increased ATX/Enpp2 mRNA relative expression compared to CTRL (*P*<.05). RSV supplementation restored ATX/Enpp2 mRNA levels to CTRL levels (*P*<.05 vs HFS, RSD) while RSD had no effect on this elevated mRNA expression (*P*<.001 vs CTRL). Elevated ATX/Enpp2 mRNA relative expression was associated with higher ATX plasma concentration for HFS compared to CTRL (*P*<.05), as shown in **Figure 4B**. Nonetheless, both therapeutic approaches led to significantly lower ATX plasma levels compared to HFS (*P*<.01 RSV vs HFS, *P*<.05 RSD vs HFS), as shown in **Figure 4B**.

## 3.6. Therapeutic approaches effects on ex vivo myocardial ischemia-reperfusion injury tolerance

### 3.6.1. Therapeutic approaches effects on cardiac function

The effects of therapeutic approaches on cardiac function are shown in **Figure 5** and Supplementary Figure S4. As shown in **Figure 5A** and S4, HFS diet induced a significant decrease in basal RPP (*P*<.001) due to decreased basal developed pressure (S4, *P*<.01), compared to CTRL. RSV supplementation significantly improved basal RPP via increased DP compared to HFS (S4, *P*<.05). RSD induced higher basal RPP than HFS, but a significantly lower basal RPP compared to CTRL (*P*<.05). No significant differences were observed for basal HR between the groups, as shown in Figure S4. During ischemia, cardiac function was the same across all groups. As shown in **Figure 5A**, HFS diet led to reduced reperfusion RPP, due to lower DP and HR compared to CTRL (Fig. S4, *P*<.001). However, both therapeutic approaches significantly improved RPP compared to HFS (*P*<.05), due to higher reperfusion developed pressure compared to HFS (*P*<.01), as shown in **Figure 5A** and S4. Interestingly, HFS diet also induced higher end-diastolic pressure compared to CTRL during all the experimental time course, and the return to standard diet for 2 months had no measurable effect (Fig S5, *P*<.05 HFS, RSD vs CTRL). RSV supplementation restored the end diastolic pressure compared to HFS and RSD (*P*<.05) throughout the IR injury experiment, as shown in **Figure 5B**. No significant differences in coronary flow were found among groups throughout the protocol (Fig S4)

### 3.6.2. Therapeutic approaches effects on cardiac energy metabolism

The effects of therapeutic approaches on cardiac energy metabolism are shown in **Figure 6**. Basal ATP and PCr levels were significantly lower in HFS compared to CTRL rats (*P*<.01), while only a tendency was observed for both therapeutic approaches (Fig. 6A and B). No significant differences were observed for ATP and PCr levels during ischemia among groups. Significantly higher reperfusion ATP and PCr levels were found for both therapeutic approaches compared to HFS (*P*<.05). No significant differences were found for PCr levels between RSV, RSD and CTRL groups during reperfusion. However, RSV and RSD reperfusion ATP level were significantly lower compared to CTRL (*P*<.001). As shown in **Figure 6C**, a trend to a higher basal Pi level for HFS was observed compared to CTRL, RSV and RSD. RSV supplementation lowered the Pi levels during ischemia compared to CTRL, HFS and RSD (*P*<.01). A tendency to lower ischemia Pi levels in the RSD group compared to CTRL and HFS was observed but not statistically significant. Both therapeutic approaches restored Pi levels compared to HFS at reperfusion (*P*<.001). As shown in **Figure 6D**, HFS diet induced elevated basal and reperfusion levels of phosphomonoesters compared to CTRL (*P*<.05). Neither therapeutic approach had an effect on the HFS-induced basal and reperfusion PME increase compared to CTRL (*P*<.05 RSV, RSD vs CTRL), and no significant differences were found between groups during ischemia. No difference
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Ex vivo cardiac function parameters. (A) Rate Pressure Product (RPP); (B) End diastolic pressure. Data are means ± SEM. Two-way ANOVA followed by Bonferroni post-hoc test were performed on all parameters.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Ex vivo cardiac metabolism parameters. (A) ATP levels; (B) Phosphocreatine; (C) Inorganic Phosphate; (D) Phosphomonoesters levels during ischemia reperfusion protocol on isolated perfused heart ex vivo. Data are means ± SEM. Two-way ANOVA followed by Bonferroni post-hoc test were performed on all parameters.</figcaption></figure>
<table><caption>Table 4
Correlation analysis.</caption><thead><tr><th>Correlations</th><th></th><th>P</th><th>r</th></tr></thead><tbody><tr><td rowspan="2">Hepatic total lipids</td><td>Adipose tissue/ body weight</td><td>.0019</td><td>0.64</td></tr><tr><td>Heart weight/ Tibia length</td><td>.0001</td><td>0.69</td></tr><tr><td rowspan="5">Basal RPP</td><td>Left Ventricular Mass</td><td>.0206</td><td>-0.57</td></tr><tr><td>Left Ventricular End-Diastolic Volume</td><td>.0019</td><td>-0.71</td></tr><tr><td>IPGTT AUC</td><td>.0114</td><td>-0.60</td></tr><tr><td>Plasma FFA</td><td>.0313</td><td>-0.58</td></tr><tr><td>Hepatic total lipids</td><td>.0249</td><td>-0.59</td></tr><tr><td rowspan="4">Reperfusion RPP</td><td>Left Ventricular End-Diastolic Volume</td><td>.0060</td><td>-0.62</td></tr><tr><td>IPGTT AUC</td><td>.0004</td><td>-0.73</td></tr><tr><td>Plasma FFA</td><td>.0083</td><td>-0.62</td></tr><tr><td>Hepatic total lipids</td><td>.0041</td><td>-0.67</td></tr><tr><td rowspan="3">Myocardial Perfusion</td><td>Non-fasting glycemia</td><td>.0050</td><td>0.63</td></tr><tr><td>Plasma FFA</td><td>4E-05</td><td>0.65</td></tr><tr><td>Adipose tissue weight/ body weight</td><td>1E-03</td><td>0.60</td></tr></tbody></table>

Correlations among cardiac and metabolic parameters. FFA, free fatty acid; IPGTT AUC: IPGTT's area under curve; Pearson correlation test was realized on all parameters.

between groups was observed for pH during the entire protocol (data not shown).

## 3.7. Correlation analysis

Table 4 presents selected correlations among all parameters. Hepatic total lipids content was positively correlated with AT weight to body weight ratio. LVEDV showed a positively significant correlation with the heart weight to tibia length ratio, comforting ex vivo and in vivo data observed. Basal RPP was negatively correlated with left ventricular mass assessed in vivo. Interestingly, basal and reperfusion RPP were negatively correlated with LVEDV, IPGTT AUC, plasma FFA and hepatic total lipids content. Myocardial perfusion was positively correlated with non-fasting glycemia, plasma FFA and AT weight to body weight ratio.

## 4. Discussion

Recent evidence suggests that in a prediabetic context, AT and MASLD should be considered in the CV risk and complications stratification [8–10]. In addition, it has been recently shown that lifestyle interventions, first line treatments for MASLD and prediabetes, lacked effects on major cardiovascular events over 20 years despite long term diabetes prevention [26]. The aim of the present study was then to evaluate potential cardioprotective effects of RSV supplementation, in comparison with a return to a standard diet, and its relationship with hepatic and AT impairments, in a female diet-induced prediabetic rat model.

### 4.1. HFS induced cardiac alterations and therapeutic approaches cardioprotection

As previously described [32], 5 months of HFS diet induced deleterious cardiac remodeling, characterized by increased left ventricular volume, wall thickness and mass in vivo. These observations were confirmed, as LVEDV was correlated with heart weight to tibia length ratio ex vivo, a relevant cardiac hypertrophy indicator [49]. Deleterious cardiac remodeling characterized by myocardial hypertrophy, increased left ventricular volume, mass and wall thickness has been shown in other prediabetic rodent models [50–52], but also recently in prediabetic patients [53–55]. In our study, HFS diet also induced a compensatory increase in myocardial perfusion, which was previously shown in this model [32,34], and in

other studies [56]. Reduced cardiac efficiency associated with increased mitochondrial uncoupling compared to controls was previously reported in high-fat-diet fed rats [57], but also in prediabetic patients [58]. FFA oxidation generates less ATP per oxygen molecule than glycolysis [59], and myocardial perfusion was correlated with non-fasting hyperglycemia, plasma FFA and AT weight to body weight ratio here. These results corroborate the hypothesis that the rise in perfusion could be a compensatory response to reduced cardiac efficiency due to higher FFA oxidation and reduced glycolysis. However, 5 months of HFS diet caused no significant impact on left ventricular ejection fraction (LVEF), cardiac output or cardiac index in vivo, thus showing no significant systolic dysfunction.

Ex vivo, 5 months of HFS diet reduced the tolerance to ischemia-reperfusion characterized by impaired myocardial function and energy metabolism with reduced basal and reperfusion ATP, PCr and increased PME levels. These characteristics were already found in an earlier study and suggest an impaired energy metabolism [33,34]. Alteration of ischemia-reperfusion tolerance, characterized by reduced glycolytic flux adaptations, has previously been shown in prediabetic rats [60]. Among the various known causal factors of diabetic cardiomyopathy, one of great significance is impaired ATP production [61]. This reduced ischemia-reperfusion tolerance in correlation with increased plasma FFA concentration and glucose intolerance may thus be partly explained by a higher myocardial uptake of FFA. Higher myocardial uptake of FFA is associated with the generation of lipid intermediates such as ceramides and diacylglycerols (DAG), which further impairs insulin sensitivity. The process therefore results in reduced glucose uptake and glycolysis [62], increased oxidative stress and mitochondrial dysfunction in cardiomyocytes [63,64]. Interestingly, basal and post-ischemic cardiac function was negatively correlated with LVEDV, suggesting that the early deleterious cardiac remodeling induced by HFS is already associated with functional impairments. HFS diet also led to increased end-diastolic pressure, as found in previous ex vivo studies in diabetic rat models [65,66]. Acute myocardial infarction following ischemia-induced myocardial edema can lead to stiffening of the myocardial wall and thus to a lower left-ventricular global compliance, resulting in increased left-ventricular end-diastolic pressure, exhibiting furthermore reduced ischemia-reperfusion tolerance [67].

The two therapeutic approaches exhibited different cardioprotective effects. Interestingly, despite the maintained HFS, RSV supplementation protected against deleterious cardiac remodeling in
duced by HFS diet in vivo and ex vivo, unlike return to stan-
dard diet after 3 months of HFS. In type 2 diabetic rats, Fang
et al. [68] previously showed that 16 weeks 50 mg/d/kg resveratrol
supplementation significantly attenuated diabetes induced cardiac
dysfunction and hypertrophy through regulation of mitochondrial
function mediated partly by SIRT1 activation. On the other hand,
2 months of standard diet exhibited no effect, suggesting that this
duration was not enough to reduce cardiac remodeling induced by
3 months of HFS diet. Studies evaluating the cardioprotective ef-
fect of a return to standard diet are scarce. Conversely, in Wistar
rats fed a highly palatable diet for 2 months, Naderali et al. [69]
showed increased heart weight with a return to control values af-
ter 2 months of chow diet. Neither therapeutic approach had an
effect on the compensatory increase in myocardial perfusion ob-
served, suggesting that energy metabolism was not fully recovered.
However, they both exhibited cardioprotective effects on ischemia-
reperfusion tolerance, with improved functional recovery and en-
ergy metabolism characterized by increased ATP and PCr and re-
duced Pi and PME levels. Despite restoration of metabolic param-
eters, RSD did not restore ischemia-reperfusion tolerance to con-
trol levels. This could be explained in part by the absence of ef-
fect on cardiac remodeling, as suggested by the negative corre-
lation between in vivo LVEDV and cardiac function ex vivo. RSV
supplementation restored basal and reperfusion left ventricular
end-diastolic pressure to control levels, unlike return to standard
diet which exhibited no significant effect. Improved myocardial
ischemia-reperfusion tolerance observed here could in part be ex-
plained by increased high energy compounds. It has been previ-
ously reported that resveratrol exerts direct cardioprotective ef-
fects against diabetic cardiomyopathy and ischemia-reperfusion in-
jury through multiple pathways and mechanisms, mainly medi-
ated by SIRT1 activation [70]. Resveratrol supplementation has pre-
viously exhibited cardioprotection through its pleiotropic effects
on metabolism and mitochondrial function [27], inflammation [71]
and apoptosis [72]. Taken all together, these results suggest that
RSV supplementation, despite maintained HFS diet, exhibits a more
important cardioprotective effect than 2 months of return to stan-
dard diet, and further studies are warranted to uncover the mecha-
nisms involved, especially on mitochondrial function and oxidative
stress.

4.2. *Cardioprotection and prediabetic status*

Furthermore, we studied the association of these cardiac al-
terations with prediabetic status. As previously described [32,34],
after 5 months of HFS diet, female rats exhibited a prediabetic
condition with mild hyperglycemia and glucose intolerance com-
pared to control animals. An insignificant tendency to higher body
weight was observed for HFS fed groups, which could be explained
by high heterogeneity of body weights due to HFS diet feeding
and wild type animals [73]. We didn't assess insulin resistance
but further studies in this context should evaluate global and car-
diac insulin resistance as it has been recently suggested that car-
diac insulin resistance plays a predominant role in left ventric-
ular mechano-energetic performance, explaining in part its asso-
ciation with CV morbidity [74]. In addition, as previously men-
tioned, higher myocardial uptake of FFA is associated with de-
creased glucose uptake, glycolysis and mitochondrial dysfunction
[62–64]. These impairments could explain the observation of com-
pensatory increase in myocardial perfusion induced by higher oxy-
gen demand and be involved in the alteration of myocardial func-
tion and high energy compounds induced by HFS feeding. Thus,
restored glucose tolerance may partly explain the cardioprotective
effect of the return to a standard diet, with cardiac function ex vivo
negatively correlated to glucose intolerance and plasma FFA. Low

dose RSV supplementation improved fasting glycemia and glucose
tolerance while 2 months of standard diet restored glycemia and
glucose tolerance to control levels. In fructose-fed Sprague Dawley
rats, *Bagul* et al. [75] reported a complete restoration of the predi-
abetic status (insulin resistance, impaired fasting glucose and glu-
cose tolerance) by 8 weeks RSV supplementation at a higher dose
of 10 mg/d/kg. However, although the effect on glucose metabolism
was less than when returning to the standard diet, RSV supple-
mentation exerted better cardioprotection, suggesting that the car-
dioprotective effects cannot be explained by glucose homeostasis
alone.

4.3. *Cardioprotection and hepatic steatosis*

To further investigate the mechanisms involved in the car-
dioprotection exerted by the therapeutic approaches, we aimed
to assess their effects in a multi-organ approach, including liver
and AT. NAFLD, whose prevalence is in constant rising, is closely
linked to prediabetes, but also with CVD as the leading cause
of death [76,77]. Interestingly, the prevalence of deleterious car-
diac remodeling in NAFLD patients was found to be proportional
to the steatosis grade [78]. Indeed, NAFLD is a known indepen-
dent CV risk factor [10] and was previously found to be associ-
ated with cardiac alterations in this model [32]. Here, 5 months
of HFS diet induced elevated lipid levels and steatosis in the
liver, associated with increased expression levels of mitochondrial,
and peroxisomal β-oxidation genes, as previously found [32]. Not
surprisingly, hepatic total lipids content was correlated with AT
weight to body weight ratio. Indeed, lipids in hepatic steatosis are
known to come mainly from AT [48]. Interestingly, hepatic total
lipids were correlated with basal and post-ischemic cardiac func-
tion, hinting the role of hepatic steatosis on basal cardiac func-
tion and ischemia-reperfusion tolerance. After 5 months of HFS
diet, no significant difference was observed in de novo lipogene-
sis nor inflammatory cytokine gene expression in hepatocytes, sug-
gesting a simple steatosis. Recently, *Fiorentino* et al. [79] reported
a decreased myocardial energy efficiency in non-diabetic patients
with non-alcoholic fatty liver disease which was entirely medi-
ated by the degree of insulin resistance. NAFLD could participate
in the lipotoxic effect mentioned above, caused by the accumu-
lation of toxic lipid intermediates [80-83]. Furthermore, hepatic
triglyceride accumulation was reported to precede cardiac alter-
ations in a HFS mouse model [84], comforting this hypothesis. RSV
supplementation reduced lipid mitochondrial and peroxisomal β-
oxidation gene expression, while 2 months of standard diet re-
stored them to control levels. In STZ induced diabetic rats, 2 weeks
of 10 mg/kg/d resveratrol supplementation exhibited hepatic pro-
tective effects with reduced oxidative stress, decreased liver weight
and reduced inflammation [85]. In a HF diet carp model, RSV al-
leviated liver damage and oxidative stress and improved hepatic
lipid metabolism through its antioxidant properties and activa-
tion of antioxidant enzyme system via Nrf2/NF-κB/PPAR-γ signal-
ing pathway [86]. Recent evidence suggests that sirtuins are an
interesting therapeutic approach against hepatic steatosis with re-
duction of inflammation and oxidative stress [87], which is in line
with our results. Those results suggest that RSV cardioprotection is
associated with hepatic steatosis amelioration despite continuous
HFS feeding in this model.

4.4. *Therapeutic approaches and AT*

On the other hand, AT is known to be an important endocrine
organ involved in CV risk and alterations in prediabetes or type 2
diabetes [8]. Therefore, the effect of both therapeutic approaches
on lipid metabolism and inflammation genes were explored. Five
months of HFS diet increased AT weight and peroxisomal β-oxidation gene expression (Aco). Aco can contribute to lipid oxidation in case of lipid overload, associated with increased ROS production and impaired mitochondrial function [88]. Return to standard diet significantly reduced Aco relative expression, while RSV supplementation had no effect but lowered Ppary expression levels. Previous clinical and preclinical studies demonstrated that resveratrol has an anti-obesity potential by reducing lipogenesis, increasing apoptosis and promoting fatty acid β-oxidation, through Sirt1, AMPK and PGC-1α [89]. Thus, the unchanged Aco expression levels observed in this study may be explained by an insufficient dose to compensate for the continuing HFS diet. In our study, RSV supplementation lowered Ppary expression levels in AT, suggesting reduced adipogenesis and enhanced lipolysis. RSV effects on Ppary are, however, conflicting [90,91]. Sirt1 activation has previously been reported to promote lipolysis by inhibiting PPARγ [92] in adipocytes by deacetylating FOXO1, thus decreasing adipogenesis [93]. Here, RSV supplementation had no effect on AT weight in our study, but further studies are warranted to uncover the mechanisms involved on AT metabolism impairments. We then aimed to evaluate the association of the deleterious cardiac remodeling observed here and AT ATX/Enpp2 expression, which is an AT secreted protein potentially involved in diet induced cardiac remodeling [12,13]. Importantly, it has been previously shown that AT derived ATX induced deleterious cardiac remodeling, fibrosis and mitochondrial dysfunction, whereas blockage of AT derived ATX improved these cardiac injuries [13]. Furthermore, circulating levels of ATX were an independent predictor for non-alcoholic fatty liver disease in obese women, with or without diabetes, exhibiting once more the relevance of the inter-organ crosstalk in this study [94]. Five months of HFS resulted in larger AT ATX expression and higher plasma ATX levels, compared to controls. Interestingly, restoration of AT ATX expression and ATX plasma levels were associated with the RSV cardioprotective effects observed in vivo and ex vivo. To our knowledge, this is the first time that RSV cardioprotection was associated with reduced A derived ATX expression and plasma levels in a prediabetic context. Since ATX expression can be upregulated through inflammation pathways (PI3K/AKT, NFκB) [95], the effect of resveratrol on ATX expression may be explained by NFκB pathway inhibition in adipocytes [96]. However, ATX regulation under RSV supplementation should be further studied to confirm these hypotheses. Two months of return to a standard diet had no effect on AT ATX expression, despite restoring ATX plasma levels. Although, at this time, we cannot fully explain this finding, we can speculate that the 2-month observation was too short to allow full restoration of ATX expression. In addition, restored ATX plasma levels could be in part explained by the reduced AT mass observed as well as other plasma ATX regulation pathways [95].

### 4.5. Limitations of the study

One limitation of our study is that all animals were female and that we could not study sex-specific differences in the potential organ crosstalk, which could be of particular interest for liver and AT due to their role in cardiac alterations. However, our aim was to evaluate cardioprotective effects of RSV supplementation in prediabetic female rats because prediabetic women are still underrepresented among studies in this context and they represent a subject of particular concern due to their higher cardiovascular risk during the prediabetic state. We did not evaluate plasma insulin and insulin resistance in this study, which could help stratify the relationship and role of insulin resistance inter-organ crosstalk on the cardioprotective effects of RSV observed here. Moreover, we did not explore the effects of the therapeutic approaches on total, LDL and HDL cholesterol plasmatic levels as well as hepatic cholesterol

in this study, which are well known to be involved in CV risk [97]. We did not assess plasma Lysophosphatidic Acid (LPA) as the effector of ATX activity [12]. Beyond LPA, future studies should also address typical RSV targets (SIRT1, PGC-1α..) to further uncover the mechanisms involved in RSV cardioprotection. We did not explore the role of estrogens in the cardioprotective effect of resveratrol, which are known to regulate hepatic lipogenesis and glucose metabolism/homeostasis genes [98,99]. RSV has indeed been reported to exhibit an estrogen like effect [100], as well as beneficial effects on physiological parameters in ovariectomized female rats [101]. Further explorations on ovariectomized female Wistar rats could help uncover the involvement of hormones in the cardioprotective RSV effects reported here.

### 5. Conclusion

This study reports a cardioprotective effect of RSV supplementation against HFS diet-induced cardiac remodeling and reduced ischemia-reperfusion tolerance in a prediabetic female rat model. More importantly, RSV supplementation provided stronger protection compared with standard diet restoration. Cardiac remodeling protection and improved ischemia-reperfusion tolerance were associated with better glucose homeostasis, hepatic metabolism and steatosis, as well as reduced AT derived ATX, suggesting an unknown resveratrol action mechanism that needs to be confirmed in further studies. These results further highlight the relevance of inter-organ crosstalk for cardiac alterations in a prediabetic context. Further studies fully uncovering these interactions are, however, warranted, especially in prediabetic women who are exposed to a higher CV risk.

### Funding

This work was supported by Aix-Marseille Université, CNRS (UMR 7339), France Life Imaging (grant ANR-11-INBS-0006) and Institut Carnot Star. Funding was not involved in study design, in collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication.

### Data availability

Data is available on request.

### Declaration of competing interest

None.

### CRediT authorship contribution statement

**Alexis Jouenne:** Writing – original draft, Visualization, Investigation, Formal analysis, Conceptualization. **Joevin Sourdon:** Writing – review & editing, Investigation. **Isabelle Varlet:** Writing – review & editing, Investigation. **Christophe Vilmen:** Methodology. **Frank Kober:** Writing – review & editing, Software, Methodology. **Jean-François Landrier:** Writing – review & editing, Resources. **Monique Bernard:** Writing – review & editing, Resources. **Martine Desrois:** Writing – review & editing, Supervision, Project administration, Investigation, Conceptualization.

### Acknowledgments

We thank Carole Lan (CRMBM) and Kenza Hamici (CRMBM) for experimental supports, and Yann Le Fur (CRMBM) for the spectroscopy analysis software. We thank Julien Astier (C2VN) for biochemical technical support. We also thank Eric Sérée (C2VN) and Laurent Pechere (Ivery laboratory) who supplied the resveratrol.
Supplementary materials

Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.jnutbio.2025.109992.

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