nutrients
Article
Programming Mechanism of Adipose Tissue Expansion in
the Rat Offspring of Obese Mothers Occurs in a
Sex-Specific Manner
Carlos A. Ibáñez 1 , Gabriela Lira-León 1 , Luis A. Reyes-Castro 1 , Guadalupe L. Rodríguez-González 1 ,
Consuelo Lomas-Soria 1,2 , Alejandra Hernández-Rojas 1 , Eyerahí Bravo-Flores 3 , Juan Mario Solis-Paredes 4 ,
Guadalupe Estrada-Gutierrez 5 and Elena Zambrano 1, *
Citation: Ibáñez, C.A.; Lira-León, G.;
Reyes-Castro, L.A.;
Rodríguez-González, G.L.;
Lomas-Soria, C.; Hernández-Rojas, A.;
Bravo-Flores, E.; Solis-Paredes, J.M.;
Estrada-Gutierrez, G.; Zambrano, E.
Programming Mechanism of Adipose
Tissue Expansion in the Rat Offspring
of Obese Mothers Occurs in a
Sex-Specific Manner. Nutrients 2023,
15, 2245. https://doi.org/10.3390/
nu15102245
Academic Editors: Mina Desai and
Michael Ross
Received: 11 March 2023
Revised: 19 April 2023
Accepted: 2 May 2023
Published: 9 May 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Nutrients 2023, 15, 2245. https://doi.org/10.3390/nu15102245
1 Departamento de Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador
Zubirán, Mexico City 14080, Mexico; carlos_albertoibc@comunidad.unam.mx (C.A.I.);
gabsslln@gmail.com (G.L.-L.); luis.reyesc@incmnsz.mx (L.A.R.-C.);
guadalupe.rodriguezg@incmnsz.mx (G.L.R.-G.); mconsuelo@conacyt.mx (C.L.-S.);
alehero03@gmail.com (A.H.-R.)
2 CONACyT-Cátedras, Investigador por México, Departamento de Biología de la Reproducción, Instituto
Nacional de Ciencias Médicas y Nutrición SZ, Mexico City 14080, Mexico
3 Departamento de Inmunobioquímica, Instituto Nacional de Perinatología Isidro Espinosa de los Reyes,
Mexico City 11000, Mexico; eyerahiqfb@yahoo.com.mx
4 Departamento de Investigación en Salud Reproductiva y Perinatal, Instituto Nacional de Perinatología Isidro
Espinosa de los Reyes, Mexico City 11000, Mexico; juan.solis@inper.gob.mx
5 Dirección de Investigación, Instituto Nacional de Perinatología Isidro Espinosa de los Reyes,
Mexico City 11000, Mexico; guadalupe.estrada@inper.gob.mx
* Correspondence: elena.zambranog@incmnsz.mx
Abstract: We investigated whether excessive retroperitoneal adipose tissue (AT) expansion pro-
grammed by maternal obesity (MO) affects adipocyte size distribution and gene expression in
relation to adipocyte proliferation and differentiation in male and female offspring (F1) from control
(F1C) and obese (F1MO) mothers. Female Wistar rats (F0) ate a control or high-fat diet from weaning
through pregnancy and lactation. F1 were weaned onto a control diet and euthanized at 110 postnatal
days. Fat depots were weighed to estimate the total AT. Serum glucose, triglyceride, leptin, insulin,
and the insulin resistance index (HOMA-IR) were determined. Adipocyte size and adipogenic gene
expression were examined in retroperitoneal fat. Body weight, retroperitoneal AT and adipogenesis
differed between male and female F1Cs. Retroperitoneal AT, glucose, triglyceride, insulin, HOMA-IR
and leptin were higher in male and female F1MO vs. F1C. Small adipocytes were reduced in F1MO
females and absent in F1MO males; large adipocytes were increased in F1MO males and females
vs. F1C. Wnt, PI3K-Akt, and insulin signaling pathways in F1MO males and Egr2 in F1MO females
were downregulated vs. F1C. MO induced metabolic dysfunction in F1 through different sex dimor-
phism mechanisms, including the decreased expression of pro-adipogenic genes and reduced insulin
signaling in males and lipid mobilization-related genes in females.
Keywords: maternal obesity; adipocyte size distribution; sexual dimorphism; adipose tissue;
adipogenic capacity; developmental programming
1. Introduction
Epidemiological and experimental studies have demonstrated that maternal under-
and over-nutrition during development predispose offspring to long-term adverse out-
comes in a sex-dependent manner [1]. Specifically, maternal obesity leads to excessive
adipose tissue accumulation in the offspring. We [2–4] and others [5–7] demonstrated that
maternal obesity programs their offspring to gender-specific metabolic alterations. The
expansion pattern of white adipose tissue (WAT) is strongly linked to a greater possibility
https://www.mdpi.com/journal/nutrients
Nutrients 2023, 15, 2245 2 of 19

of developing metabolic alterations. The sex-specific metabolic alterations in programmed

obesity is driven by an altered adipose tissue expansion mechanism. Therefore, elucidating
the molecular mechanisms by which maternal obesity programs offspring’s adipose tissue
expansion is an important issue of physiological and clinical relevance in the context of
the global obesity epidemic [8]. However, the molecular mechanisms underlying maternal
obesity-related increases in adipose mass, including several aspects of the physiological
development of adipose tissue in vivo, have yet to be elucidated [9,10].
Adipose tissue expansion, namely adipogenesis, occurs via the recruitment of adipocyte
precursors, which become newly differentiated adipocytes via increasing the size of existing
adipocytes [11]. The recruitment of plastic adipocyte precursors is an active process in the
fetal rat within the last week of gestation and is accelerated during lactation [12], with both
periods considered windows of vulnerability for developmental programming. However,
the WAT retains a certain expansion capacity during postnatal and adult life, dependent
upon sex, energy balance and perinatal conditions [13]
In rats, the expansion rate of fat depots differs according to their location. Inguinal fat,
for instance, exhibits a high degree of hyperplasia, whereas mesenteric and epididymal
fat depots exhibit a high degree of hypertrophy. In the present study, retroperitoneal fat
tissue was chosen for examination because it is expanded by a balance of hyperplasia
and hypertrophy, and its growth is associated with metabolic alterations [13–16]; also,
it is one of the most sensitive fat depots to sex-specific developmental programming by
maternal obesity [6]. Retroperitoneal adipose tissue expansion is regulated by several
transcription factors, resulting in an increase in adipocyte size (AS) and/or number, and
is linked with insulin resistance and dyslipidemia [17]. Evaluating changes in AS and/or
adipocyte number and the expression profile of adipogenesis-related genes is a widely
accepted method to assess changes in WAT expansion [11].
Considering that the abnormal distribution of AS and its deleterious effects on
metabolic function are different between the male and female offspring of obese mothers,
we hypothesized that gene transcription profiles involved with retroperitoneal adipocyte
proliferation and differentiation could be sex-specifically regulated.

2. Methods
2.1. Animals
All procedures were approved by the Animal Experimentation Ethics Committee of
the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ),
Mexico City, Mexico (ethical approval code, BRE-1868) and were in accordance with the
ARRIVE criteria for reporting animal studies [18,19]. Female albino Wistar rats were
born and maintained in the animal facility of the INCMNSZ, which is accredited by the
Association for Assessment and Accreditation of Laboratory Animal Care International
(AAALAC) and adheres to its requirements. Rats were maintained under a 12 h light–dark
cycle (lights on from 07:00 to 19:00 h) at a constant temperature (22–23 ◦ C) with food and
water given ad libitum.

2.2. Experimental Design

To produce Founder Generation (F0) mothers, 120-day-old female Wistar rats were
randomly mated with proven fertile non-littermates. At birth (day 0), F0 litters were
adjusted to ten pups, including at least four females [2]. At weaning (21-day-old), F0
females were randomly assigned to one of the two experimental groups: the control (F0C)
or maternal obesity (F0MO) group to be fed either a standard laboratory chow diet or a
high-fat diet (HFD) (Figure 1). The C diet consisted of standard laboratory chow (Zeigler
Rodent RQ22-5, Gardners, PA, USA) containing 22.0% protein, 5.0% soy oil fat, 31.0%
polysaccharide, 31.0% simple sugars, 4.0% fiber, 6.0% minerals and 1.0% vitamins (w/w)
(physiological fuel 3.4 kcal/g). The HFD was produced in the specialized dietary facility
of the INCMNSZ with 23.5% protein, 20.0% lard, 5.0% soy oil fat, 20.2% polysaccharide,
Nutrients 2023, 15, x FOR PEER REVIEW 3 of 19

Nutrients 2023, 15, 2245 3 of 19

INCMNSZ with 23.5% protein, 20.0% lard, 5.0% soy oil fat, 20.2% polysaccharide, 20.2%
simple sugars,sugars,
20.2% simple 5.0% fiber,
5.0% 5.0%
fiber, mineral mix, 1.0%
5.0% mineral vitamin
mix, 1.0% mix (w/w),
vitamin physiological
mix (w/w), fuel
physiological
4.8 kcal/g.
fuel 4.8 kcal/g.

Figure
Figure 1.
1. Experimental
Experimentaldesign. Timeline
design. for the
Timeline for study of maternal
the study (F0) and
of maternal offspring
(F0) (F1) experi-
and offspring (F1)
mental groups.
experimental groups.

Based onon the

thefertility
fertilityrate
ratefrom
fromprevious
previous studies
studies in in this
this model
model [2] [2] at 120
at 120 days,
days, 10 fe-
10 female
male rats from
rats from the F0C
the F0C group group
andand 20 from
20 from thethe F0MO
F0MO groupwere
group weremated
matedovernight
overnight (up
(up to
5 days)
days)with
withnon-experimental
non-experimentalmales malesto to
generate
generateoffspring
offspring(F1).(F1).
DailyDaily
vaginal smears
vaginal were
smears
obtained, and the
were obtained, andday
theinday
which a sperm
in which plug plug
a sperm was found was designated
was found was designatedas dayas0day
of con-
0 of
conception.
ception. F0CF0C
andandF0MOF0MO continued
continued to to
bebe fedwith
fed withCCororHFD,
HFD,respectively,
respectively, throughout
pregnancy and lactation (Figure 1). The fertility rate for for F0C
F0C was
was 80%
80% and,
and, for
forF0MO,
F0MO,40%.
40%.
achieve F1
To achieve F1homogeneity,
homogeneity,on onpostnatal
postnataldayday (PND)
(PND) 2, all
2, all F1 F1 litters
litters studied
studied werewere
ad-
adjusted
justed to pups,
to 10 10 pups,
withwith
equalequal numbers
numbers of males
of males andand females
females whenever
whenever possible
possible (F1C(F1C
and
and F1MO).
F1MO). Litters
Litters withwith
lessless
thanthan
9 or9 more
or more than
than 1414 pups
pups wereexcluded
were excludedfromfromthethestudy.
study. F1
F1
litters were weaned at PND 21, housed 2–3 pups per cage, and fed a control diet until the
end of the experiment (PND 110).

2.3. F1:
2.3. F1: Food
Food Intake
Intake
From PND
From PND of95 95
to to
110, offspring
110, food
offspring intake
food was
intake recorded
was forfor
recorded 15 15
consecutive days.
consecutive A
days.
maximum of three rats from the same sex and experimental group (F1C or F1MO)
A maximum of three rats from the same sex and experimental group (F1C or F1MO) were were
housed per cage. The daily amount of food provided was weighed, as was the amount that
housed per cage. The daily amount of food provided was weighed, as was the amount
remained after 24 h. The amount of food consumed daily was averaged to provide a value
that remained after 24 h. The amount of food consumed daily was averaged to provide a
per rat. Food intake was reported as the 15-day average.
value per rat. Food intake was reported as the 15-day average.
2.4. F1: Blood and Tissue Collection
2.4. F1: Blood and Tissue Collection
At PND 110, between 12:00 and 14:00 h, after 6 h of fasting, one male and female
F1 perAt litter
PND (n 110, between
= 8) 12:00 and 14:00
were euthanized h, after 6 h of fasting,
by exsanguination throughone malepuncture
aortic and female F1
under
per litter (n = 8) were euthanized by exsanguination through aortic puncture under
general anesthesia with isoflurane by the same person under identical conditions (Figure 1). gen-
eral
Blood anesthesia with isoflurane
was collected, by the same
and mediastinal, person under
retroperitoneal, identical
omental, conditionsparametrial
mesenteric, (Figure 1).
Blood was collected, and mediastinal, retroperitoneal, omental, mesenteric,
and periovarian or epidydimal fat depots were excised and weighed to determine the parametrial
and
totalperiovarian or epidydimal
visceral adipose fat depots
tissue. One sample were excised
of the and weighed
retroperitoneal fattopad
determine the total
was frozen for
visceral adipose tissue. One sample of the retroperitoneal fat pad was frozen
molecular analysis, whereas another sample from the same pad and region was fixed for for molecular
analysis,
histologicalwhereas another sample from the same pad and region was fixed for histological
analysis.
analysis.
2.5. F1: Biochemical Parameters
Blood samples were centrifuged for 15 min at 4 ◦ C at 2880 RCF, and the serum was
stored at −70 ◦ C until biochemical and hormonal analysis. Glucose (GLUH, # B24985
Nutrients 2023, 15, 2245 4 of 19

Beckman Coulter, Inc., Brea, CA, USA) and triglyceride (TG, #445850 Beckman Coulter,
Inc., Brea, CA, USA) concentrations were determined enzymatically with an automatic
analyzer (Synchron CX Beckman Coulter, Brea, CA, USA). Insulin serum concentrations
were determined by radioimmunoassay (Millipore Radioimmunoassay kit, Burlington, MA,
USA). A homeostatic model assessment for insulin resistance (HOMA-IR) was calculated
using HOMA-IR= [glucose (mmol/L) × insulin (µU/mL)]/22.5 [20,21].

2.6. F1: Adipose Tissue Histology

Retroperitoneal AT samples were fixed using 10% paraformaldehyde in PBS 0.05M,
which were dehydrated and paraffin-embedded. A total of 5 µm thick tissue sections were
mounted on poly-L-lysine-coated slides. The slides were deparaffinated, rehydrated, and
then stained with hematoxylin and eosin [22]. Histology slides were examined under a light
microscope (Olympus BX51, Melville, NY, USA) at 20×magnification. AS was measured
manually by delimiting the adipocyte cross-sectional area in digital images using AxioVisio
LE software real 4.8 version (Zeiss® copyright 2006–2010 Stuttgart, Germany) in at least
180 cells per group corresponding to an average of 25 cells per animal. All histological
measurements were performed by an observer blinded to the nature of the tissue source [6].

2.7. F1: Adipocyte Size Distribution

The cross-sectional area of the adipocytes was measured in µm2 . Histograms of the
relative frequency of 500 µm2 -area intervals were overlaid with their corresponding repre-
sentative gamma distribution functions (GDFs). The AS distribution analysis, based on GDF
modeling, was performed as previously reported [13]. Briefly, GDFs were modeled using pa-
rameter estimators of shape (α) and scale (β), which were calculated as: α = (Mean/Standard
Deviation)2 and β = (Standard Deviation2 /Mean); and plotted using the GAMMA.DIST
function (Microsoft® Excel® for Microsoft 365 MSO Version 2304 Build 16.0.16327.20200 64-bit).
The 10th and 90th percentiles of the GDFs of C groups were used to determine the cut-off
points for small and large adipocytes, respectively. For each rat, the proportions of small and
large AS were calculated from their GDFs as the % of cumulative probability below and above
small and large AS cut-off points.

2.8. F1: Retroperitoneal Adipose Tissue RNA Extraction and Complementary cDNA Synthesis
A total of 0.1 g frozen retroperitoneal AT samples were homogenized with 0.8 mL of a
Tri-Reagent (Zymo, TRI Reagent® #R2050-1-200, Irvine, CA, USA) using a rotor/stator homog-
enizer (Dremel, Tissue Tearor #985-370, Bartlesville, OK, USA) at 2/3 of the maximum speed
setting for 5–10 s in an ice bath. Homogenates were centrifuged at 12,000× g RCF for 10 min
at 4 ◦ C; the surface’s fatty layer was removed, and the supernatant was transferred into an
RNAse-free tube and purified with an RNA Column Purification Kit (Zymo, Direct-zol RNA
Mini-Prep #R2053, Irvine, CA, USA) in accordance with the manufacturer’s specifications.
The purity and concentration of the total RNA were determined using a microvolume
UV/Visible Spectrophotometer (Thermo Scientific, NanoDrop 2000 #ND-2000, Waltham,
MA, USA). Electrophoresis chips (Agilent Technologies, RNA 6000 Nano Kit #5067-1511,
Santa Clara, CA, USA) were used to determine the RNA integrity number (RIN) on a
Bioanalyzer System (Agilent Technologies, Agilent 2100 #G2940AA, Santa Clara, CA, USA).
Samples with: A260/A280 and A260/A230 ratios ≥ 1.8 [23] and no evidence of ribosomal
peak degradation with a RIN ≥ 6.0 [24–26] were used for expression assays.
The total-RNA samples of each rat were processed using a cDNA Conversion Kit
(Qiagen RT2 First Strand Kit #330401, Germantown, MD, USA) in accordance with the
manufacturer’s specifications. Briefly, for each sample the genomic DNA elimination mix
containing 500 ng of the total RNA was incubated at 42 ◦ C for 5 min, followed by 15 min of
reverse transcription at 42 ◦ C, and was stopped for 5 min at 95 ◦ C.
Nutrients 2023, 15, 2245 5 of 19

2.9. F1: Adipose Tissue Expression Profile

Eighty-four adipogenesis-related mRNA transcripts (Table S1) were analyzed using
the SYBR Green qPCR Mastermix (Qiagen, RT2 SYBR Green ROX #330522, Germantown,
MD, USA) and a qPCR array (Qiagen, RT2 Adipogenesis Profiler PARN-049Z #330231, Ger-
mantown, MD, USA) in a multi well-plate RT-PCR system (Applied Biosystems, Step One
Plus #4376600, Waltham, MA, USA) in accordance with the manufacturer’s specifications.
In brief, plated mRNA transcripts were incubated at 95 ◦ C for 10 min to activate DNA Taq
Polymerase, followed by 40 cycles of 15 s at 95 ◦ C and 1 min at 60 ◦ C for the collection of
fluorescence data.
Gene expression was analyzed by the 2−∆∆Ct method and Student’s t-test in the Qiagen
2
RT Profiler PCR Analysis Web Portal https://dataanalysis2.qiagen.com/pcr, accessed on
9 August 2020. RT-qPCR arrays included 5 candidate housekeeping genes (HKG), and
Hprt1 was selected among Actb, B2m, Ldha, and Rplp1 due to its expression stability. Gene
expression with a fold-change of (FC) ≥ 1.4 and a statistical significance of p < 0.05 were
considered as differentially expressed genes (DEGs) [4,27,28]. Common and exclusive DEG
in different groups were identified using the Venny online software https://bioinfogp.cnb.
csic.es/tools/venny/index.html, accessed on 22 December 2020 [29].

F1: Differentially Expressed Genes Interaction

Potential functional interactions of DEGs at the protein level were predicted in the F1C
female and F1MO male. DEGs Refseq accession numbers were uploaded to the Search Tool
for the Retrieval of Interacting Genes/Proteins (STRING) database at https://string-db.org,
accessed on 12 January 2021 [30]. DEG interaction networks were derived from validated
interaction sources, including text mining, experiments, databases, co-occurrence, and
co-expression, applying an interaction score > 0.4. The KEGG identification pathway was
set in the criteria to identify the Predicted Protein Interaction (PPI) in relation to adipose
tissue development and function.

2.10. Statistical Analysis

Physiological and biochemical parameters were expressed as the mean ± standard
error of the mean (SEM) and were assessed by a one-tailed t-test, alongside a median AS
and small and large adipocyte proportions by the Mann–Whitney U-test. These analyses
were performed using Sigma Plot 11.0 software. Cumulative AS were compared through a
Kolmogorov–Smirnov test using GraphPad Prism 7.04 software. Gene expressions were
compared by a t-test using the Qiagen RT2 Profiler PCR Analysis Web Portal. Statistically
significant differences were defined as p < 0.05. F1C male and female body weight and
adipose tissue mass, which are basic and well-characterized biologically meaningful end-
points, were analyzed by a one-sided t-test and were different (p < 0.05); therefore, the sexes
were analyzed separately [31].

3. Results
3.1. F1: Food Intake and Adiposity
F1C males had increased body mass, caloric intake, and total fat compared to F1C
females (Figure 2D–F). Body weight (Figure 2A,D) and food intake (Figure 2B,E) were
similar in both F1C and F1MO males and females. However, adipose tissue mass was
higher in both male and female F1MOs when compared to F1C (Figure 2C,F).

F1: Male and Female Fat Depots

F1C males had increased retroperitoneal, mesenteric, omental and mediastinal fat
compared to F1C females. Omental and mediastinal (p = 0.056; F1C vs. F1MO males) fat
were similar in both F1C and F1MO males and females; epididymal fat was similar between
male F1C and F1MO; retroperitoneal, parametrial and mesenteric fat depots were similar
in female F1C and F1MO. F1MO males had increased retroperitoneal and mesenteric fat
Nutrients 2023, 15, 2245 6 of 19

Nutrients 2023, 15, x FOR PEER REVIEW 6 of 19

depots compared to F1C males, and F1MO females had increased periovarian fat compared
to F1C females (Table 1).

Figure
Figure 2.
2. Adult
Adult offspring
offspring morphometric
morphometric parameters.
parameters. Male
Male and
and female:
female: body
body weight
weight (A,D),
(A,D), food
food
intake (B,E), and adipose tissue mass (C,F). Data are mean ± SEM, n = 8 rats per group, # p < 0.05 vs.
intake (B,E), and adipose tissue mass (C,F). Data are mean ± SEM, n = 8 rats per group, # p < 0.05 vs.
F1C male, * p < 0.05 vs. F1C.
F1C male, * p < 0.05 vs. F1C.

F1: Male and Female Fat Depots

Table 1. Fat depots in male and female F1C and F1MO at 110 PND.
F1C males had increased retroperitoneal, mesenteric, omental and mediastinal fat
comparedFattoDepot (g)
F1C females. F1C
Omental and mediastinal (p = 0.056; F1C vs. F1MO
F1MO males) fat
were similar in both F1C and F1MO males and females; Maleepididymal fat was similar be-
tween male F1C and F1MO; retroperitoneal,
Mediastinal 0.2 ±parametrial
0.02 and mesenteric
0.7 fat depots were
± 0.3
similar in female F1C and F1MO. F1MO males had increased retroperitoneal and mesen-
Retroperitoneal 5.1 ± 0.5 8.2 ± 1.4 *
teric fat depots compared to F1C males, and F1MO females had increased periovarian fat
compared to Omental
F1C females (Table 1). 0.6 ± 0.1 0.7 ± 0.1
Mesenteric 2.5 ± 0.3 3.4 ± 0.4 *
Table 1. FatEpidydimal
depots in male and female F1C and
5.4F1MO
± 0.3 at 110 PND. 6 ± 0.6
Fat Depot (g) F1C
Female F1MO
Mediastinal 0.1 ± 0.02 # Male 0.3 ± 0.2
Mediastinal
Retroperitoneal 2.7 ± 0.2 # 0.2 ± 0.02 0.70.4± 0.3
3.1 ±
Retroperitoneal 5.1 ± 0.5 8.2 ± 1.4 *
Omental 0.4 ± 0.1 # 0.6 ± 0.1
Omental 0.6 ± 0.1 0.7 ± 0.1
Mesenteric 1.6 ± 0.2 # 2.1 ± 0.3
Mesenteric 2.5 ± 0.3 3.4 ± 0.4 *
Parametrial 3.4 ± 0.5 4.3 ± 0.6
Epidydimal 5.4 ± 0.3 6 ± 0.6
Periovarian 1.5 ± 0.2 Female2.4 ± 0.4 *
Data are mean ± SEM, n = 8 rats per group, # p < 0.05 vs. F1C male, * p < 0.05 vs. F1C.
Mediastinal 0.1 ± 0.02 # 0.3 ± 0.2
Retroperitoneal 2.7 ± 0.2 # 3.1 ± 0.4
Omental 0.4 ± 0.1 # 0.6 ± 0.1
Mesenteric 1.6 ± 0.2 # 2.1 ± 0.3
Parametrial 3.4 ± 0.5 4.3 ± 0.6
Periovarian 1.5 ± 0.2 2.4 ± 0.4 *
Data are mean ± SEM, n = 8 rats per group, # p < 0.05 vs. F1C male, * p < 0.05 vs. F1C.
Nutrients 2023, 15, x FOR PEER REVIEW 7 of 19
Nutrients 2023, 15, 2245 7 of 19

3.2.
3.2.F1:
F1:Biochemical
BiochemicalParameters
Parameters
F1C
F1C females hadhigher
females had higherserum
serumglucose
glucoseconcentrations
concentrationsthan
thanF1C
F1Cmales
males(Figure
(Figure3A,F).
3A,F).
Serum glucose concentrations were higher only in F1MO males vs. F1C
Serum glucose concentrations were higher only in F1MO males vs. F1C males (Figure males (Figure
3A,F).
3A,F). However,
However, insulin insulin
(Figure(Figure
3B,G),3B,G), HOMA-IR
HOMA-IR (Figure
(Figure 3C,H),
3C,H), triglyceride
triglyceride (Figure
(Figure 3D,I)3D,I)
and
and leptin
leptin (Figure
(Figure 3E,J) 3E,J)
serumserum concentrations
concentrations were increased
were increased in both in both
male male
and andF1MO
female female
in
F1MO in comparison
comparison to the F1Ctomale
the F1C
andmale andrespectively.
female, female, respectively.

Figure
Figure 3.
3. Offspring
Offspring serum
serum metabolic
metabolic parameters.
parameters. Male
Maleand
andfemale:
female:glucose
glucose(A,F),
(A,F),insulin
insulin(B,G),
(B,G),
HOMA-IR
HOMA-IR (C,H), triglyceride (D,I) and leptin (E,J). Mean ± SEM, n = 8 rats per group,##pp<<0.05
(C,H), triglyceride (D,I) and leptin (E,J). Mean ± SEM, n = 8 rats per group, 0.05vs.
vs.
F1C male, * p < 0.05, ** p < 0.01 vs. F1C.
F1C male, * p < 0.05, ** p < 0.01 vs. F1C.

3.3.
3.3.F1:
F1:Adipocyte
AdipocyteSize
SizeDistribution
Distribution
Cumulative
CumulativeAS ASshowed
showedaashiftshifttotosmaller
smallersize
sizeF1C
F1Cfemales
femalescompared
comparedto toF1C
F1Cmales.
males.
In
In contrast,
contrast, there
there was
was aa shift
shift to
to aa greater
greater size
size in
in F1MO
F1MO females
females and
and males
males compared
compared to to
respective
respectiveF1C.
F1C. For
For both
both males
males andand females,
females, the
the median
median ASAS was
was significantly
significantly greater
greater in
in
F1MO
F1MOthanthanininF1C
F1C(Figure
(Figure 4A,B).
4A,B). In In all
allgroups,
groups, the
the observed
observedASASdistribution
distributionfit
fitto
togamma
gamma
distribution
distributionexhibited
exhibitedaapeak
peaklocated
locatedat atthe
theleft
leftof
ofthe
thecenter
centerand
andaamore
more gradual
gradual tapering
tapering
tothe
to theright
rightwith
withmore
moredispersion
dispersionin inF1MO
F1MOthanthanin inF1C
F1Cmales
malesandandfemales
females(Figure
(Figure4C,D).
4C,D).
Adipocyte size
Adipocyte cut-off points were established at 1022 µm22 for
size cut-off for small
small and 5830 µm22for
and 5830 forlarge
large
adipocytesin
adipocytes in males,
males, and
and 813 µm22 for
813 µm forsmall
smalland
and4736 µm22for
4736 µm forlarge
largeadipocytes
adipocytesin infemales
females
(Figure 4C).
(Figure 4C).

3.4. F1: Adipogenic Expression Profile

The adipogenic expression profile (Table S2) showed a total of 65 DEGs in F1C females
vs. F1C males (Figure 5A), 58 in F1MO males vs. F1C males (Figure 5B) and 1 in F1MO
females vs. F1C females (Figure 5C). All DEGs were downregulated in the F1C female vs.
F1C male and F1MO male vs. F1 male, while the 10 most significant were: Retn, Slc2a4,
Fasn, Insr, Tsc22d3, Nr1h3, Lipe, Cebpa, Ncor1, and Mapk14. Only Egr2 expression was
downregulated in the F1MO female vs. F1C females (Table 2).
Nutrients 2023,15,
Nutrients2023, 15,2245
x FOR PEER REVIEW 88 of
of 19
19

Figure 4.
Figure 4. Offspring
Offspring adipocyte
adipocyte size
size distribution.
distribution. Male
Male and
and female
female median
median adipocyte
adipocyte size,
size, with
with their
their
interquartile range, 10th and 90th percentile (A). Adipocyte size cumulative distributions (B),
interquartile range, 10th and 90th percentile (A). Adipocyte size cumulative distributions (B), relative rela-
tive frequency histograms with their representative gamma distribution functions with small and
frequency histograms with their representative gamma distribution functions with small and large
large adipocyte proportions shown as shaded regions (C). Representative H&E-stained photomi-
adipocyte proportions shown as shaded regions (C). Representative H&E-stained photomicrographs
crographs of retroperitoneal adipose tissue at 20× magnification (D). The Mann–Whitney test was
of retroperitoneal
used adipose
to compare median adipocyte 20×and
tissue atsize magnification
proportions(D). The and
of small Mann–Whitney test was
large adipocytes, used
and the to
two-
compare median adipocyte size and proportions of small and large adipocytes, and
sample Kolmogorov–Smirnov test was used to compare the cumulative distributions. Small and the two-sample
Kolmogorov–Smirnov test points
large adipocyte size cut-off was usedweretodefined
compare as the cumulative
the 10th and 90thdistributions. Small
percentile of F1C and large
groups. n=8
adipocyte
per group.size
# p <cut-off points
0.05 vs. were defined
F1C male; * p < 0.05asvs. 10th and 90th percentile of F1C groups. n = 8 per
theF1C.
group. # p < 0.05 vs. F1C male; * p < 0.05 vs. F1C.
3.4. F1: Adipogenic Expression Profile
Table A Top 10ofdifferentially
2. total 65 DEGs were expressed genes in
identified (DEG)
F1Crelated
femalesto adipogenesis
vs. F1C malesin F1C females
(Figure vs.58
5A): F1C
in
males and F1MO males vs. F1C males. The 10 most significant (by p-value)
F1MO males vs. F1C males (Figure 5B) and 1 in F1MO females vs. F1C females (Figure and common DEGs
in F1C
5C). females
All DEGsand wereF1MO males are shown.
downregulated in theF1MO females vs.
F1C female vs. F1C
F1C male
females
andonly exhibited
F1MO maleone
vs.
single DEG.
F1 male, while the 10 most significant were: Retn, Slc2a4, Fasn, Insr, Tsc22d3, Nr1h3, Lipe,
Cebpa, Ncor1, and Mapk14. Only Egr2 expressionF1C
was downregulated in the F1MO female
F1MO vs. F1C
vs. F1C females (Table 2).
Fold p-Value Fold p-Value
Gene Description
Regulation (t-Test) Regulation (t-Test)
Table 2. Top 10 differentially expressed genes (DEG) related to adipogenesis in F1C females vs. F1C
Female vs. Male Male
males and F1MO males
Retn
vs. F1C males. The 10
Resistin
most significant
−65.4
(by p-value)
0.000001
and common
−7.2
DEGs in
0.0009
F1C females andSolute
F1MO males are shown.
carrier family 2, F1MO females vs. F1C females only exhibited one single
Slc2a4 −44.9 0.000001 −30.9 0.0001
DEG. member 4
Fasn Fatty acid synthase −29.1 0.00004 −14.2 0.0004
Insr Insulin receptor −27.9 F1C
0.00005 F1MO vs. 0.002
−25.4 F1C
TSC22 domain family,
Tsc22d3 −14.3
0.00005 −11.6 0.004p-
member 3 Fold Regula- p-Value Fold Regula-
Gene
Nr1h3 Description
Nuclear −12.4
receptor 1H3 0.00008 −13.4 Value
0.0008
Lipe −11.2 tion 0.00001
Lipase, hormone sensitive (t-Test) −12.9tion 0.0004
(t-Test)
CCAAT/enhancer binding
Cebpa −10.6 Female 0.00004
vs. Male −7.5 Male 0.002
protein α
Retn Nuclear receptor
Resistin
Ncor1 −5.2 −65.4 0.00009
0.000001 −6.0 −7.2 0.0009
0.002
co-repressor 1
Slc2a4 SoluteMitogen
carrieractivated
familyprotein
2, member
4 −44.9 0.000001 −30.9 0.0001
Mapk14 −5.0 0.00003 −5.5 0.0006
Fasn Fattykinase 14
acid synthase −29.1 0.00004 −14.2 0.0004
Female
Insr
Egr2 Insulin receptor
Early growth response 2 −27.9 0.00005 −7.6−25.4 0.002
0.006
Tsc22d3 TSC22 domain family, member 3 −14.3 0.00005 −11.6 0.004
 Egr2 Early growth response 2 −7.6 0.006

The union and intersection analysis of DEG sets showed that 56 DEGs were regulated
by sex as well as maternal diet, 9 DEGs (Bmp4, Fos, Runx1t1, Klf4, Ddit3, Bmp7, Sfrp1, Pparg,
and Adipoq) were regulated only by sex, 2 DEGs (Gata3 and Irs1) were regulated only by
Nutrients 2023, 15, 2245 9 of 19
maternal diet within males, and 1 DEG (Egr2) was regulated only by the maternal diet
within females (Figure 5D).

Figure 5. Offspring adipogenic expression profile and Venn diagram of differentially expressed
Figure 5. Offspring adipogenic expression profile and Venn diagram of differentially expressed genes
genes (DEGs). ‘Volcano Plots’ showing the gene expression in (A) F1C female, (B) F1MO male and
(DEGs). ‘Volcano Plots’ showing the gene expression in (A) F1C female, (B) F1MO male and (C) F1MO
(C) F1MO female. Downregulated (pink and blue dots) and non-differentially expressed (black dots)
female. Downregulated
genes are (pink and
shown. Fold-change blue
(FC dots)
= 1.5) and
and non-differentially
statistical expressed
significance (black
(p = 0.05) cut-offdots) genes
values are
(gray
shown. Fold-change
lines) are indicated.(FC = 1.5) andprofile
Expression statistical significance
analyzed (p = 0.05)arrays
in RT-qPCR cut-offofvalues (gray related
84 genes lines) are
to
indicated. Expression profile analyzed in RT-qPCR arrays of 84 genes related to adipogenesis (RT2
Profiler PCR array PARN-Z49, Qiagen). (D) Overlapping of DEGs sets in F1C female (blue), F1MO
males (yellow) and F1MO females (green) are shown.

The union and intersection analysis of DEG sets showed that 56 DEGs were regulated
by sex as well as maternal diet, 9 DEGs (Bmp4, Fos, Runx1t1, Klf4, Ddit3, Bmp7, Sfrp1, Pparg,
and Adipoq) were regulated only by sex, 2 DEGs (Gata3 and Irs1) were regulated only by
maternal diet within males, and 1 DEG (Egr2) was regulated only by the maternal diet
within females (Figure 5D).

Differentially Expressed Genes Interaction

According to STRING v11.0 PPI analysis, significant connectivity among DEGs was
found as F1C females showed 62 genes with 345 interactions, and F1MO males had 56 genes
with 268 interactions (PPI enrichment p < 1.0 × 10−16 ). Similar gene sets in F1C females vs.
F1C males; F1MO males vs. F1C males were involved in the Wnt pathway, 11 and 13 DEGs,
respectively, and in insulin signaling pathways, 10 and 13 DEG, respectively (Figure 6).
However, F1C females showed 13 DEGs to be involved in the AMPK signaling pathway
 Differentially Expressed Genes Interaction
According to STRING v11.0 PPI analysis, significant connectivity among DEGs was
found as F1C females showed 62 genes with 345 interactions, and F1MO males had 56
genes with 268 interactions (PPI enrichment p < 1.0 × 10−16). Similar gene sets in F1C fe-
Nutrients 2023, 15, 2245 males vs. F1C males; F1MO males vs. F1C males were involved in the Wnt pathway, 10 of11
19
and 13 DEGs, respectively, and in insulin signaling pathways, 10 and 13 DEG, respectively
(Figure 6). However, F1C females showed 13 DEGs to be involved in the AMPK signaling
pathway
with a falsewith a falserate
discovery discovery
(FDR) rate
≤ 8.04 × 10≤−8.04
(FDR) 6 (Figure
× 10−66A),
(Figure
while6A), while
F1MO F1MO
males males
showed
14showed 14 DEGsin
DEGs involved involved
PI3K-Aktin PI3K-Akt
signaling signaling
with an FDRwith≤an FDR
1.56 − 6 × 10 (Figure
× ≤101.56(Figure
−6
6B). 6B).

Figure
Figure 6.6.Functional
Functional interaction
interaction of
ofdifferentially
differentiallyexpressed
expressedgenes (DEGs).
genes Known
(DEGs). and and
Known predicted pro-
predicted
tein–protein interactions (PPI) of downregulated DEGs in (A). F1C females and (B). F1MO males.
protein–protein interactions (PPI) of downregulated DEGs in (A). F1C females and (B). F1MO males.
Interactions (lines) between DEGs (nodes) and the association strength (line thickness) are shown.
Interactions (lines) between DEGs (nodes) and the association strength (line thickness) are shown.
KEGG pathways related to adipose tissue development and function are indicated (highlighted
KEGG pathways
nodes). related
PPI network to adipose
analysis tissue
performed indevelopment
STRING v11.0.and function are indicated (highlighted
nodes). PPI network analysis performed in STRING v11.0.
4. Discussion
4. Discussion
There is now compelling evidence that environmental factors, such as inadequate
There is now compelling evidence that environmental factors, such as inadequate
maternal nutrition during pregnancy and/or in early neonatal life, may increase off-
maternal nutrition during pregnancy and/or in early neonatal life, may increase offspring’s
spring’s susceptibility to developing type 2 diabetes, hypertension, and obesity [32–35].
susceptibility to developing type 2 diabetes, hypertension, and obesity [32–35]. We previ-
We previously reported that obese mothers show metabolic dysfunction at 19 days of ges-
ously reported that obese mothers show metabolic dysfunction at 19 days of gestation [36],
tation [36], at the end of lactation [2] and that milk production and the % of water and
at the end of lactation [2] and that milk production and the % of water and carbohydrate
carbohydrate content were lower in obese mothers, whereas milk leptin, total fat, arachi-
content were lower in obese mothers, whereas milk leptin, total fat, arachidonic acid and
donic acid and monosaturated fatty acids were higher, with no changes in protein content
monosaturated fatty acids were higher, with no changes in protein content [37] explaining
[37] explaining the adverse metabolic outcomes of offspring. However, in many studies,
the adverse metabolic outcomes of offspring. However, in many studies, sex differences
sex differences in offspring are often overlooked as a significant variable in disease devel-
in offspring are often overlooked as a significant variable in disease development. In the
opment. In the present study, we observed sexual dimorphism in both F1C males and
present study, we observed sexual dimorphism in both F1C males and females and between
females and between F1MO and F1C. These findings are consistent with our previous
F1MO and F1C. These findings are consistent with our previous studies, in which the male
studies, in which the male offspring of obese mothers but not females had greater fat mass,
offspring of obese mothers but not females had greater fat mass, leptin, and triglyceride
leptin, and triglyceride concentrations at PND 36 [2]. In addition, we reported that MO
concentrations at PND 36 [2]. In addition, we reported that MO programs insulin, glucose,
and lipid signaling in the liver of young adult offspring in a sex-dependent manner, leading
to liver dysfunction and insulin resistance [3,4] and that the male and female offspring of
obese mothers have different aging metabolic trajectories [3].
Body fat distribution differs by sex and is a significant indicator of health; the ac-
cumulation of retroperitoneal adipose tissue, for example, raises the risk of developing
cardiometabolic diseases. Sex variations are due to the interaction of sex chromosomes,
genetic diversity, sex hormones, and the environment. Therefore, a better understanding
of the molecular mechanisms driving sexual dimorphism could contribute to the devel-
opment and implementation of interventions to prevent metabolic and cardiovascular
disease in offspring born to obese mothers [38]. Several studies have proposed that male
Nutrients 2023, 15, 2245 11 of 19

and female offspring have different susceptibilities to metabolic alterations in relation to

insulin resistance in response to maternal HFD consumption [3,5], although the reasons for
this difference are unclear. After sexual maturity, the expansion of retroperitoneal adipose
tissue plays a crucial role in the development of insulin resistance [16,39]. Therefore, the
observed metabolic differences between male and female F1MO young adults suggest that
the expansion of retroperitoneal adipose tissue is one of the mechanisms by which MO
programs the offspring’s metabolic phenotype in a sex-dependent manner.
Increased insulin concentrations, HOMA-IR, and adiposity were observed in male
and female F1MO, with augmented serum glucose in males but not in females. Previous
reports indicated that hyperinsulinemia and insulin resistance developed before the onset
of obesity in F1MO [40]. However, the failure to observe elevated glucose concentrations in
F1MO females despite having increased visceral adiposity may be related to the increased
expandability capacity of the female retroperitoneal adipose tissue due to an increase in
the number of adipocytes [41,42]. In this regard, adipose tissue hyperplasia has been
significantly associated with better glucose and insulin concentrations than adipose hyper-
trophy [43]. Therefore, the increased proportion of small adipocytes in females vs. males
might contribute to the reduced effect of insulin resistance in young adult females [44].
Several studies have shown that, in contrast to males, females who gain more relative
body weight or adiposity in response to HFD do not develop hyperglycemia and are more
glucose tolerant [45–47]. The association of higher adipose tissue expandability capacity,
due to increased adipocyte precursor proliferation, with improved glucose homeostasis is
not well understood, though it likely involves several mechanisms, such as protection from
adipose tissue hypertrophy and reduced ectopic lipid accumulation [48].
In accordance with a previous systematic review and meta-analysis [49], triglyceride
serum concentrations increased similarly in F1MO males (32%) and females (36%) when
compared to the respective F1C groups. As triglyceride serum concentration reflects the
lipid rate of entry and removal into the blood, hypertriglyceridemia may be considered the
central pathophysiologic feature of the abnormal lipid profile. Although increased serum
triglyceride is frequently associated with dietary intake [50], in the present study, all F1MO
were weaned to the same C diet; therefore, the main factors leading to increased serum
triglyceride in F1MO may be due to perturbation in lipolysis and lipogenesis balance in the
liver and adipose tissue, which determine the lipid uptake and its release to the blood [4].
Maternal diet is important to offspring health, but the postnatal environment also
influences the phenotype. The mice offspring of obese mothers (first-hit) were fed a high-fat
diet (second-hit) after weaning and exhibited triglyceride profile changes between adipose
depots and sexes [51]. We have published that the postnatal consumption of a high-fat diet
by the offspring of obese mothers exacerbates body fat accumulation as well as a decrease
in small and an increase in large adipocytes in retroperitoneal adipose tissue, resulting in
larger metabolic abnormalities [14].
Previous studies using the same experimental model (young adult offspring) showed
marked characteristics of fatty liver in males but not in females F1MO [4]. The increased
liver weight, total fat, and the triglyceride content seen in F1MO males was attributed
to a decrease in hepatic lipid metabolism capacity. In contrast, the slight rise in liver
triglyceride concentration found in F1MO females was attributed to enhanced hepatic de
novo lipogenesis, as shown by liver transcriptome profiles [4]. Our previous results [4]
suggest that a reduced lipolysis rate in the liver could significantly contribute to increased
triglyceride concentrations in male and female F1MO, which cannot be counterbalanced by
liver lipid uptake and lipolysis and might be worsened by increased hepatic lipogenesis and
lipid release in F1MO females. Impaired lipid mobilization from adipocytes is associated
with higher lipogenesis in the liver as a result of insulin’s failure to regulate lipolysis in the
adipose tissue [52].
Histologically, we found retroperitoneal adipose tissue hypertrophy in both male and
female F1MO, which was consistent with prior studies [13,53–55]. It has been shown that
adipose tissue hypertrophy and insulin resistance indicates the presence of lipid-overloaded
Nutrients 2023, 15, 2245 12 of 19

adipocytes in the offspring of obese mothers [56]. Although F1MO males and females
exhibited a greater median AS, these changes themselves were not capable of explaining
the different metabolic effects observed. Due to the heterogeneity of AS distribution among
the groups, cumulative frequencies differed more than the median AS; consequently, a
better approach to understanding the AS would be to analyze the relative frequencies
that are characterized by heavy and light tails. Gamma distribution modeling provides a
statistical method to characterize AS distribution. In this work, the retroperitoneal adipose
tissue of MO males exhibited a greater proportion of large adipocytes than MO females,
which could be related to a sex-specific offspring response to the maternal nutritional state.
The AS fit to a gamma distribution model in all groups, agreeing with previous
works [13,57], in which the analysis of AS distributions revealed that they were more
asymmetric in F1C groups and less asymmetric and with more spread in F1MO males and
females compared to F1C. The AS distribution asymmetry was characterized by a short
and heavy tail on the left, and a long and thin tail on the right, which could be described as
having a short-range size for small adipocytes and a long-range size for large adipocytes in
all groups. The small size was associated with newly generated adipocytes, and large sizes
were related to adipocyte lipid storage capacity. Small and large adipocyte proportions are
linked to adipocyte precursor proliferation and adipocyte late differentiation, respectively.
Therefore, adipocyte extreme sizes located at AS distribution tails are important parameters
for evaluating the mechanisms of expansion in the adipose tissue at the cellular level [13].
Currently, it is recognized that the proportions of different adipocyte size range, namely
small, large, and very large, are indications of the metabolic state [58]. Large adipocytes
are linked to energy storage, whereas small adipocytes are connected to lower insulin
resistance [59,60].
In the present study, the histological analysis of retroperitoneal adipose tissue samples
from F1 rats revealed an AS cumulative distribution shift toward smaller sizes, leading
to a lower median AS in F1C females compared to males. Consequently, the adipocyte
cut-off for a small size in males increased by 26% compared to females, and the cut-off
value for large adipocytes was 23% greater in males than in females, indicating that females
under physiological conditions had a higher proportion of small adipocytes in a relative
short size range and a lower proportion of large adipocytes in retroperitoneal adipose
tissue than males, suggesting a more proliferative adipose tissue capacity in females than
males. It is well-accepted that estradiol stimulates the proliferation of preadipocytes in
both males and females; however, female preadipocytes are more responsive to estradiol
and proliferate more rapidly than male preadipocytes [61]. According to experimental
models using 3T3-L1 cells, it has been shown that estradiol facilitates adipocyte prolif-
eration and differentiation [62]. Therefore, the increased abundance of small adipocytes
in F1C females to F1C males may be due to female estradiol levels. This is in agreement
with human studies, in which the preadipocyte proliferation rate is stimulated more by
estradiol in retroperitoneal adipose tissue in women than in men [63]. Supporting these
findings, ovarian hormone withdrawal models with ovariectomized (OVX) rats exhibited
a higher body weight and visceral adiposity, while OVX rats with estradiol replacement
exhibited a substantial body weight decrease with a modest tendency to improve insulin
sensitivity [41,64]. In another study, OVX rats with and without estradiol replacement
showed similar AS in retroperitoneal fat depots [64], suggesting that the observed differ-
ences between F1C males and females in AS distributions in the present study could be
attributed to other sex-related factors than estradiol levels. In this regard, the observed
decrease in the AS cut-off for small adipocytes in females respective to males could be
related to the increased differentiation of preadipocytes, confirming the female’s higher
adipogenic capacity compared to males.
In the present study, the adipogenic expression profile of F1C females vs. F1C males
reflected a greater expansion capacity in female than male retroperitoneal adipose tissue,
and an increased amount of adipocyte precursors and newly differentiated adipocytes
contributed, at least partially, to a lower adipocyte lipid accumulation and led to a shift
Nutrients 2023, 15, 2245 13 of 19

in AS distribution to a smaller size [42]. Wnt signaling plays a central role in adipose
tissue development, which is required for lipogenic gene expression during terminal
adipocyte differentiation via β-catenin-signaling and for regulating de novo lipogenesis
by Fasn expression [65–67]. The downregulation of genes associated with Wnt signaling
observed in F1C females vs. F1C males was in line with prior rodent studies that identified
the sexually dimorphic expression of genes related to adipose tissue morphogenesis [68]:
therefore, the lower Fasn expression observed in C females vs. C males, was related to Wnt
signaling disruption.
AMPK signaling is a fuel-sensing enzyme and is a major regulator of cell cycle and
energy homeostasis [69]. AMPK activation in adipose tissue was linked to the inhibition of
white preadipocyte differentiation [70]. In the current study, AMPK signaling genes were
downregulated in F1C females vs. F1C males; this downregulation in females suggested a
more active preadipocyte differentiation than in males. Previous studies showed that Retn
expression was higher in males than females, induced during adipogenesis and elevated
in obesity [71], which is in line with our findings in which Retn gene expression was
lower in F1C females compared to F1C males, suggesting a higher rate of preadipocyte
differentiation in females. The solute carrier family 2 (facilitated glucose transporter),
member 4 (Slc2a4), previously known as Glut4, which is encoded by the Slc2a4 gene, is
predominantly expressed in adipose tissue and muscle. Nevertheless, Slc2a4 expression
during adipocyte development is extremely low, particularly in undifferentiated precursor
cells [72]. The observed reduced expression of Slc2a4 in F1C females compared to F1C
males could be associated with an increased content of adipocyte precursors in adipose
tissue samples rather than an impairment of insulin signaling, given that there was no
evidence of insulin resistance in terms of HOMA-IR.
Clearly, under physiological conditions, there are sexual differences in the mechanisms
that control the expansion of adipose tissue. Therefore, suboptimal conditions during
pregnancy and lactation, such as maternal obesity, lead to sex-specific changes in the
expression of adipogenesis-related genes in offspring [6]. Lecoutre et al. showed that
maternal obesity in the rat programs increased adiposity in a sex-specific manner and that
perirenal adipose tissue was more sensitive than gonadal adipose tissue to maternal obesity
programming, as evidenced by adipocyte hypertrophy and a decrease in adipogenic genes
(Pparγ) in male offspring [6]. However, the molecular mechanisms that explain the insulin
resistance of male progeny of obese mothers have not yet been fully understood. In the
present study, the adipogenic profile identified genes associated with insulin resistance that
were substantially related to one another (Figure 6). Our research supports the fact that
sex-specific insulin resistance in response to maternal obesity originates predominantly in
adipose tissue [12]; however, additional evidence is required to confirm this hypothesis.
Although adipogenesis regulation is strongly linked to Pparγ expression, this process
is mediated by the recruitment of its co-activators and co-repressors, which are considered
the transcriptional switches for adipocyte differentiation and lipogenesis programmed in
the offspring of obese mothers [73]. MO leads to epigenetic modifications in the main adi-
pogenesis regulator genes in offspring. The adipose tissue of male rat offspring from obese
mothers exhibited hypermethylation and histone modification of the Pparγ2 promoter,
which led to Pparγ2 downregulation, supporting the cellular memory concept in which
adipocyte precursors retained epigenetic marks, predisposing offspring to obesity [74–76].
Indeed, the DNA methylation of the Zfp42 promoter has been reported to decrease in the
adipose tissue of male offspring of obese mothers [77]. Microarray expression analysis
demonstrated that the increased expression of insulin signaling cascade genes in female
adipose tissue contributed to reduced insulin resistance and diabetes risk compared to
males [78].
Our results showed that in MO males compared to C males, the retroperitoneal AT
downregulation of Wnt signaling was accompanied by pronounced hypertrophy and indica-
tors of insulin resistance. Several studies employing rodent obesity models have supported
the notion that Wnt signaling is suppressed during the hypertrophic stages [12,70–72]. A
Nutrients 2023, 15, 2245 14 of 19

mice study reported that after several weeks of consuming a high-fat diet, the adipocyte
precursors remained quiescent, and the suppression of adipocyte precursor proliferation
was associated with Wnt signaling inhibition and adipose tissue hypertrophy [79]. Other
studies have suggested that the activation of the PDGFRα pathway in adipose tissue
suppresses Wnt/β-catenin signaling during the final stages of adipogenesis, which has
been linked to an early onset of insulin resistance [80,81]. Indeed, a reduction in the Wnt
signaling-related expression was observed in young healthy individuals with certain low
insulin sensitivity [82]. Alterations in Wnt signaling have also been reported to cause
insulin resistance in cultured preadipocytes, indicating a crosstalk between Wnt and insulin
signaling [83].
The PI3K/AKT pathway is crucial for insulin action in adipose tissue, and its proper
signaling is required for insulin sensitivity [84]. In fact, several anti-cancer PI3K inhibitors
induce hyperglycemia and insulin resistance [85]. In accordance with the adipogenic
expression profile, several PI3K/AKT signaling-related genes were downregulated in
F1MO males compared to F1C males. The observed insulin resistance in F1MO males may
be mediated by an impairment of PI3K/AKT signaling.
The Egr2, formerly known as Krox20, encodes the Early Growth Response 2: a zinc-
finger transcription factor that is necessary for adipogenesis [86]. The present work showed
Egr2 downregulation with a reduction in the small adipocyte proportion in F1MO females
vs. F1C females, supporting the association between Egr2 suppression and lower adipocyte
precursor differentiation. However, the reduction in early adipogenesis was compensated
by an increased lipid accumulation in differentiated adipocytes, resulting in a greater
proportion of large adipocytes in F1MO females compared to F1C females.
In young adults, F1MO females exhibited fewer adverse metabolic outcomes than
F1MO males, as explained by the increased adipogenesis. However, metabolic alterations
worsened with age in the male and female F1 of obese mothers [3] due to a subsequent
depletion in the adipocyte progenitor pool [11]. Other authors have reported that adipose
tissue gene expression exhibited sexual dimorphism regardless of diet, fat depot, and
function [78]. Growing data suggest that sex-specific differences in the functionality of
each kind of adipose tissue were reliant not only on the amount of a particular fat depot
but also on their differential gene expression patterns [87,88]. Differences in sex hormone
concentrations, such as estradiol, and the amount and sensitivity of its receptors expressed
in adipose tissue led to the sex differences of master adipogenesis regulator gene expres-
sion, such as Pparγ [61]. However, the sex chromosome complement has been proposed
as another determinant of sex morphologic and physiologic differences in WAT. The pro-
posed mechanism driving these differences involves the inactivation and imprinting of X
chromosomal genes [89] and miRNA regulation of adipogenesis [90], but further studies
providing additional insights on AT sexual dimorphism are needed.

5. Conclusions
Maternal obesity leads to sex-specific changes in AS distribution and adipogenic profile
in offspring, and these differences can help explain the intergenerational transmission of
metabolic alterations. Reducing the transmission of obesity and its metabolic comorbidities,
such as insulin resistance and dyslipidemia, is a significant challenge for the scientific
community. Consequently, a greater understanding of the mechanisms involved in the
programming and regulation of adipocyte gene expression is needed. In the present study,
adipogenic profile analysis allowed us not only to identify molecular differences in the
programming mechanism of adipose tissue expansion, such as the decreased expression
of pro-adipogenic genes in F1MO males and lipid mobilization-related genes in F1MO
females (Figure 7) but also that the mechanisms implicated in the metabolic alterations
of F1MO were sex-specific. In F1MO males, this was mainly due to decreased insulin
signaling in retroperitoneal adipose tissue. Therefore, interventions to counteract the
adverse metabolic effects observed in F1MO should be designed in accordance with the
 in the programming and regulation of adipocyte gene expression is needed. In the present
study, adipogenic profile analysis allowed us not only to identify molecular differences in
the programming mechanism of adipose tissue expansion, such as the decreased expres-
sion of pro-adipogenic genes in F1MO males and lipid mobilization-related genes in
F1MO females (Figure 7) but also that the mechanisms implicated in the metabolic altera-
Nutrients 2023, 15, 2245 15 of 19
tions of F1MO were sex-specific. In F1MO males, this was mainly due to decreased insulin
signaling in retroperitoneal adipose tissue. Therefore, interventions to counteract the ad-
verse metabolic effects observed in F1MO should be designed in accordance with the pro-
programmed metabolic
grammed metabolic risk risk underpinning
underpinning the programmed
the programmed adipose
adipose tissuetissue mechanism
mechanism of ex-of
expansion in response to maternal obesity.
pansion in response to maternal obesity.

Figure 7. Adipogenic
Adipogenicsexual
sexualdimorphism
dimorphismininF1CF1Cand
andF1MO.
F1MO.Maternal
Maternalobesity
obesityprograms
programsin in
a sex-
a sex-
specific manner adipocyte size and gene expression, suggesting a down-regulation of adipogenesis
specific manner adipocyte size and gene expression, suggesting a down-regulation of adipogenesis
in males
in males and
and down-regulation
down-regulation of
of lipid
lipidmobilization
mobilizationininfemales.
females.

Supplementary Materials: The following supporting information can be downloaded at:

Supplementary
www.mdpi.com/xxx/s1,Materials: The
Table S1:following supporting
Target genes included information can be downloaded
in the Adipogenesis qPCR arrayat: https://
(Qiagen,
www.mdpi.com/article/10.3390/nu15102245/s1,
RT2 Adipogenesis Profiler PARN-049Z #330231); Table TableS2:
S1:Gene
Targetexpression
genes included in to
related theadipogenesis
Adipogenesis
qPCR 2 Adipogenesis Profiler PARN-049Z #330231); Table S2: Gene expression
in F1Carray
female (Qiagen,
vs. F1CRTmale, F1MO vs. F1C male, and F1MO vs. F1C female. Fold regulation and
related to adipogenesis
their statistical in F1C
significance female by
are shown vs. P-value.
F1C male, F1MO vs. F1C male, and F1MO vs. F1C female.
Fold regulation and their statistical significance are shown by p-value.
Author Contributions: C.A.I., G.L.-L., L.A.R.-C., G.L.R.-G., C.L.-S., A.H.-R., E.B.-F. and J.M.S.-P. re-
searchedContributions:
Author data, C.A.I., E.B.-F. and
C.A.I., J.M.S.-P.
G.L.-L., data analysis,
L.A.R.-C., E.Z. C.L.-S.,
G.L.R.-G., study design, C.A.I.,
A.H.-R., G.L.R.-G.
E.B.-F. and
and J.M.S.-P.
E.Z. preparation
researched data, of manuscript,
C.A.I., C.A.I.,
E.B.-F. and G.L.R.-G.,
J.M.S.-P. data J.M.S.-P.,
analysis, G.E.-G. anddesign,
E.Z. study E.Z. review of G.L.R.-G.
C.A.I., manuscript,
and
E.Z. final approval
preparation ofofmanuscript,
the manuscript. AllG.L.R.-G.,
C.A.I., authors have read and
J.M.S.-P., agreed
G.E.-G. andtoE.Z.
the published version of
review of manuscript,
the manuscript.
E.Z. final approval of the manuscript. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was supported by the Newton Fund RCUK-CONACyT (FONCICYT/49/2016).
Funding: This work was supported by the Newton Fund RCUK-CONACyT (FONCICYT/49/2016).
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki and approved by the Animal Experimentation Ethics Committee of the
Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), Mexico City,
Mexico (BRE-1868).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data supporting the research for this study are available within
the manuscript.
Conflicts of Interest: The authors have declared that no competing interest exist.
Nutrients 2023, 15, 2245 16 of 19

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