Associating Inulin with a Pea Protein Improves Fast-Twitch Skeletal Muscle Mass and Muscle Mitochondrial Activities in Old Rats

Abstract

Aging is associated with a decline in muscle mass and function, leading to increased risk for mobility limitations and frailty. Dietary interventions incorporating specific nutrients, such as pea proteins or inulin, have shown promise in attenuating age-related muscle loss. This study aimed to investigate the effect of pea proteins given with inulin on skeletal muscle in old rats. Old male rats (20 months old) were randomly assigned to one of two diet groups for 16 weeks: a ‘PEA’ group receiving a pea-protein-based diet, or a ‘PEA + INU’ group receiving the same pea protein-based diet supplemented with inulin. Both groups showed significant postprandial stimulation of muscle p70 S6 kinase phosphorylation rate after consumption of pea proteins. However, the PEA + INU rats showed significant preservation of muscle mass with time together with decreased MuRF1 transcript levels. In addition, inulin specifically increased PGC1-α expression and key mitochondrial enzyme activities in the plantaris muscle of the old rats. These findings suggest that dietary supplementation with pea proteins in combination with inulin has the potential to attenuate age-related muscle loss. Further research is warranted to explore the underlying mechanisms and determine the optimal dosage and duration of intervention for potential translation to human studies.

Full text

Citation: Salles, J.; Gueugneau, M.;
Patrac, V.; Malnero-Fernandez, C.;
Guillet, C.; Le Bacquer, O.; Giraudet,
C.; Sanchez, P.; Collin, M.-L.; Hermet,
J.; et al. Associating Inulin with a Pea
Protein Improves Fast-Twitch
Skeletal Muscle Mass and Muscle
Mitochondrial Activities in Old Rats.
Nutrients 2023,15, 3766. https://
doi.org/10.3390/nu15173766
Academic Editors: Manfred
Lamprecht, Claire Williams and
Luis Cisneros-Zevallos
Received: 28 June 2023
Revised: 21 July 2023
Accepted: 27 July 2023
Published: 28 August 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
Article
Associating Inulin with a Pea Protein Improves Fast-Twitch
Skeletal Muscle Mass and Muscle Mitochondrial Activities in
Old Rats
Jérôme Salles 1, * , Marine Gueugneau 1, Véronique Patrac 1, Carmen Malnero-Fernandez 2, Christelle Guillet 1,
Olivier Le Bacquer 1, Christophe Giraudet 1, Phelipe Sanchez 1, Marie-Laure Collin 1, Julien Hermet 1,
Corinne Pouyet 1,3, Yves Boirie 1,4 , Heidi Jacobs 2and Stéphane Walrand 1,4
1Unitéde Nutrition Humaine (UNH), UniversitéClermont Auvergne, INRAE, CRNH Auvergne,
63000 Clermont-Ferrand, France; marine.gueugneau@inrae.fr (M.G.); veronique.patrac@inrae.fr (V.P.);
christelle.guillet@uca.fr (C.G.); olivier.le-bacquer@inrae.fr (O.L.B.); christophe.giraudet@inrae.fr (C.G.);
phelipe.sanchez@uca.fr (P.S.); m-laure.collin@uca.fr (M.-L.C.); julien.hermet@inrae.fr (J.H.);
corinne.pouyet@inrae.fr (C.P.); yves.boirie@inrae.fr (Y.B.); stephane.walrand@inrae.fr (S.W.)
2Cosucra-Groupe Warcoing S.A., 7740 Warcoing, Belgium; cmalnero-fernandez@cosucra.com (C.M.-F.);
hjacobs@cosucra.com (H.J.)
3Unitéde Nutrition Humaine (UNH), UniversitéClermont Auvergne, INRAE, PlateForme d’Exploration du
Métabolisme, MetaboHUB-Clermont, 63000 Clermont-Ferrand, France
4CHU Clermont-Ferrand, Service Nutrition Clinique, 63000 Clermont-Ferrand, France
*Correspondence: jerome.salles@inrae.fr; Tel.: +33-4-73-178274; Fax: +33-4-73-17-8222
Abstract:
Aging is associated with a decline in muscle mass and function, leading to increased risk
for mobility limitations and frailty. Dietary interventions incorporating specific nutrients, such as
pea proteins or inulin, have shown promise in attenuating age-related muscle loss. This study aimed
to investigate the effect of pea proteins given with inulin on skeletal muscle in old rats. Old male
rats (20 months old) were randomly assigned to one of two diet groups for 16 weeks: a ‘PEA’ group
receiving a pea-protein-based diet, or a ‘PEA + INU’ group receiving the same pea protein-based
diet supplemented with inulin. Both groups showed significant postprandial stimulation of muscle
p70 S6 kinase phosphorylation rate after consumption of pea proteins. However, the PEA + INU rats
showed significant preservation of muscle mass with time together with decreased MuRF1 transcript
levels. In addition, inulin specifically increased PGC1-
α
expression and key mitochondrial enzyme
activities in the plantaris muscle of the old rats. These findings suggest that dietary supplementation
with pea proteins in combination with inulin has the potential to attenuate age-related muscle loss.
Further research is warranted to explore the underlying mechanisms and determine the optimal
dosage and duration of intervention for potential translation to human studies.
Keywords:
inulin; pea protein; sarcopenia; skeletal muscle; protein synthesis; mitochondrial activity
1. Introduction
As the global population continues to grow, there is increasing pressure on environ-
ment and food resources. The production of animal-based protein, such as beef and lamb, is
associated with significant environmental challenges [
1
]. Plant-based agriculture uses less
natural resources such as water and land, generates fewer greenhouse gas emissions, has a
lower carbon footprint, and helps preserve biodiversity [
2
]. By incorporating more plant
protein into the food system, we can mitigate environmental degradation and work towards
a more sustainable future. Plant protein also diversifies the food supply and contributes
to global food security. Growing plant-based proteins is less resource-intensive and more
adaptable to different regions, making it accessible to a broader range of communities [3].
Plant-based protein sources are rich in certain essential amino acids, vitamins, minerals,
and dietary fiber, making them a valuable source of nutrition. They contribute to a well-
balanced diet and support various bodily functions, including muscle growth and repair,
Nutrients 2023,15, 3766. https://doi.org/10.3390/nu15173766 https://www.mdpi.com/journal/nutrients

Nutrients 2023,15, 3766 2 of 16
and overall health [
4
]. Higher consumption of plant-based protein sources is associated
with several health benefits, including reduced risk for chronic diseases such as heart
disease, type 2 diabetes, and certain types of cancer [
5
]. Plant-based diets have been
linked to lower cholesterol levels, improved weight management, and better overall health
outcomes [5].
Sarcopenia refers to the age-related loss of skeletal muscle mass, strength, and function.
The condition is characterized by a progressive decline in skeletal muscle mass and function,
which can lead to decreased mobility, increased risk of falls and fractures, and a decline
in overall physical performance. Loss of muscle tissue is a common condition that affects
many older adults, typically starting around the age of 40 and becoming more pronounced
after the age of 65. Although sarcopenia is a natural part of the aging process, it can
be accelerated by certain factors, such as physical inactivity, poor nutrition, hormonal
changes, and chronic diseases [
6
,
7
]. Symptoms of sarcopenia include muscle weakness,
reduced stamina and resistance, difficulty performing everyday tasks, and changes in body
composition (increased body fat and decreased muscle mass). However, these symptoms
vary in severity among individuals [
8
]. The causes of sarcopenia cover in particular a
deregulation of muscle protein metabolism and an alteration of muscle mitochondrial
function.
Preventing and managing aging-related loss of skeletal muscle demands a multi-
faceted approach that includes regular exercise, proper nutrition, and lifestyle modifications.
Among these, adequate protein intake is essential for muscle maintenance in older subjects,
mainly through the regulation of muscle synthesis rate [
6
]. Plant proteins can play a
valuable role in managing sarcopenia, especially when incorporated into a well-balanced
diet that meets individual nutritional needs. While animal-based protein sources have
traditionally been considered the gold standard for muscle health, plant-source proteins can
provide a variety of essential amino acids necessary for muscle synthesis and maintenance.
For instance, legumes such as peas, beans, lentils and chickpeas are valuable sources of
plant proteins [
4
], especially when provided with grain protein to balance the amino acid
profile. We previously demonstrated that net protein utilization in old rats fed only legume-
enriched pasta diets was similar to those fed a casein diet [
9
]. The legume-enriched pasta
diet was found to induce a similar muscle protein synthesis rate and an equivalent effect
on muscle weight and muscle protein accretion in old rats compared to a casein diet [
9
].
Blending wheat and legumes in a food staple, such as pasta, improved its essential amino
acid profile by making it more adequate for muscle synthesis rate and muscle protein
accretion, especially for older individuals. Sources of plant proteins also offer dietary
fiber, vitamins, minerals, and antioxidants that all support overall health. Studies have
shown that the consumption of some of these nutrients makes it possible to optimize
the effectiveness of dietary proteins on the regulation of muscle protein metabolism. For
example, in old rats, regular antioxidant intake increased the effect of protein intake on
muscle protein synthesis rate [
10
]. This same action has also been demonstrated for other
micronutrients, such as vitamin D [11–13].
Inulin is composed of chains of fructose molecules linked together and belongs to a
group of carbohydrates known as fructans. Inulin is not digestible by human digestive
enzymes, which means it reaches the colon intact, where it can serve as a prebiotic. Ben-
eficial gut bacteria, such as bifidobacteria and lactobacilli, can ferment inulin, producing
short-chain fatty acids (SCFA) as by-products, which have several health benefits for the
human body, such as anti-inflammatory effects or blood glucose regulation. A recent study
has shown that the prebiotic 1-kestose drove recovery from muscle atrophy in older patients
with sarcopenia [
14
]. 1-kestose is the inulin precursor naturally present in various fruits and
vegetables and it can be readily extracted from many plants [
15
]. All patients administered
1-kestose for 12 weeks showed an increase in skeletal muscle mass index and a decrease in
body fat percentage. The study went on to explore the gut microbiota and found that the
Bifidobacterium longum population was significantly increased in the intestine after 1-kestose
administration [
14
]. However, the change in gut microbial composition was subtle and

Nutrients 2023,15, 3766 3 of 16
transient, so further studies are needed in order to firmly conclude that prebiotics have a
beneficial effect on muscle mass and function, and to define the mechanisms underlying
this action.
Here we hypothesized that consumption of a prebiotic such as inulin in addition to a
plant-based diet rich in pea protein would increase the effect of the plant protein on muscle
mass in old rats and that this effect would be associated with profound changes in muscle
protein and energy metabolism. We thus designed this study to establish the links between
consumption of a pea protein diet with or without dietary fiber, i.e., inulin, on muscle
protein turnover and mitochondrial activity in old rats.
2. Materials and Methods
2.1. Animal Experiment
Animal procedures were performed in accordance with the European guideline on
the care and use of laboratory animals (2010-63UE). Experimental protocols were reviewed
and approved by the local institutional animal care and use committee (authorization
number: APAFIS#5329-2016051115541284 v2). Animals were housed in the INRAE’s
Human Nutrition Research animal facility (agreement No. E6334515).
A total of forty 20-month-old male Wistar rats were purchased from the Janvier Labs
breeding facility (Le Genest-St-Isle, France). All animals came from the same batch, and
were bred under the same conditions throughout their lives. The rats were individually
housed in plastic cages and maintained at 22
◦
C under a 12 h dark–12 h light cycle with
free access to water, as previously described [
16
]. All the rats were fed a maintenance
diet (A04, Safe, Augy, France) ad libitum for two weeks. After this adaptation period,
the rats were randomized into two groups according to body weight, fat mass and lean
mass. Animals were assigned (n = 20 per group) to a diet containing 14% pea protein
(PEA rats), i.e., PISANE
™
C9 (Cosucra, Warcoing, Belgium) or a diet containing 14%
pea protein supplemented with 7.5% inulin (FIBRULINE
™
Instant, Cosucra, Warcoing,
Belgium) (
PEA + INU rats
) for 16 weeks. Diets were produced by the “Sciences de l’Animal
et de l’Aliment de Jouy” (SAAJ) experimental unit (INRAE, Jouy en Josas, France) (Table 1).
Body weight and food intake were measured weekly. At the end of the experiment, the
remaining rats (PEA rats; n = 14 and PEA + INU; rats n = 12) were fasted overnight but
with free access to water. Each rat group was then randomly divided into two subgroups
that either were kept in the fasted state or given a nutrient mixture by oral gavage as
a nutrient stimulus (postprandial state). The nutrient mixture contained amino acids,
sucrose and glucose (see Table 2for composition data) and was administered at a volume
corresponding to 1 mL/100 g body weight [
17
–
19
]. The nutrient bolus contained
≈
33%
of the daily amount of leucine ingested by both rat groups (PEA and PEA + INU rats)
during the experimental protocol to stimulate muscle protein synthesis, and carbohydrates
to supply energy and induce acute insulin secretion. Fasted rats and postprandial-state rats
were anesthetized by isoflurane inhalation (60 min after oral gavage for postprandial-state
rats). Blood samples were collected from the abdominal aorta, and plasma was prepared by
centrifugation. Liver, heart, adipose tissues and hindlimb skeletal muscles were removed,
weighed, snap-frozen in liquid nitrogen, and stored at −80 ◦C for later analysis.
Table 1. Composition of the experimental diets.
PEA PEA + INU
Diet composition (g/100 g)
Pea protein 14 14
Fat (soybean oil) 6 6
Carbohydrates 68 68
Cellulose 7.5 0
Inulin 0 7.5
Vitamin and mineral mix 4.5 4.5

Nutrients 2023,15, 3766 4 of 16
Table 2. Composition of the nutrient mixture used for oral gavage.
Component Final Concentration or Volume
Leucine 16 g/L
Sucrose 191 g/L
Glucose 191 g/L
Glutamine 0.3 g/L
Amino acid solution
(M5550–Sigma Aldrich) 2 mL
Distilled water q.s. 50 mL
2.2. Body Composition Measurement
Body composition was measured to assess the effect of the two diets on the change
of lean mass and fat mass of the aged animals. Fat mass and lean body mass (g) were
determined on live non-anesthetized rats using an EchoMRI-100 body composition analyzer
(Echo Medical Systems LLC, Houston, TX, USA).
2.3. Protein Quality Evaluation
Dietary quality of proteins can be defined by the nitrogen balance, i.e., an estimate of
body nitrogen retention. In the last week of the experiment, rats were placed in metabolic
cages for the last 4 days. Urine and feces were collected, and total excreted nitrogen
was determined by at Institut UniLaSalle (Beauvais, France) by the Dumas method [
20
].
Nitrogen balance (NB) was calculated using the equation [21].
NB(g)=NI −(FN +UN)
where NI is nitrogen intake, FN is fecal nitrogen, and UN is urinary nitrogen. Fecal and
urinary endogenous nitrogen excretion were deduced from a group of old rats that received
a nitrogen-free diet during the metabolic cage period [16].
2.4. Plasma Analyses
The overall metabolic effects of the two diets were assessed by determining blood tests.
Plasma levels of glucose and triglycerides were determined using a Konelab 20 analyzer
(Thermo-Electron Corporation, Waltham, MA, USA). ELISA kits (Alpco Diagnostics, Salem,
NH, USA) were used to determine plasma insulin levels in both fasted rats and postprandial-
state rats.
2.5. Protein Synthesis Measurement
One of the dynamic markers of muscle protein turnover is the evaluation of the rate of
protein synthesis using isotopic tracers. Muscle protein synthesis was studied by measuring
the rate of incorporation of a stable isotope, i.e., an AA L-[
13
C
6
]-labeled phenylalanine
(Eurisotop Saint-Aubin, France), into muscle proteins using the flooding dose method.
Fasted rats and postprandial rats (at 10 min after oral gavage) were injected subcutaneously
with a large dose of L-[
13
C
6
] phenylalanine (50% mol excess, 150
µ
mol/100 g) to flood the
precursor pool for protein synthesis. Tracer incorporation time was 50 min in both groups.
A 50-mg piece of plantaris muscle was used to isolate and hydrolyze total mixed proteins as
previously described [
9
,
16
,
22
–
24
]. After derivatization, L-[
13
C
6
] phenylalanine enrichments
in hydrolyzed proteins and in tissue fluid were assessed using gas chromatography–mass
spectrometry (Hewlett-Packard 5971A; Hewlett-Packard Co., Palo Alto, CA, USA). The
absolute synthesis rate (ASR) of proteins was calculated using the equation:
ASR =Ei
Ep ×t×100 ×TPC
where Ei is enrichment as atom percent excess of L-[
13
C
6
] phenylalanine derived from
phenylalanine from proteins at time t (minus basal enrichment), Ep is mean enrichment in

Nutrients 2023,15, 3766 5 of 16
the precursor pool (tissue fluid L-[
13
C
6
] phenylalanine), t is incorporation time in hours,
and TPC is the total protein content in mg. ASR data were expressed as milligrams of
proteins per hour (mg/h).
2.6. Quantitative RT-PCR Analysis
Some muscle metabolic markers altered by dietary protein quality have been deter-
mined by measuring their transcript level. This is the case for markers of muscle proteolysis
and mitochondrial biogenesis. The protocol used here for total RNA extraction and mRNA
analysis has been detailed elsewhere [
16
,
23
,
25
]. Briefly, total RNA was extracted from a
piece of frozen plantaris or perirenal adipose tissue using Tri-Reagent according to the
manufacturer’s instructions (Euromedex, Mundolsheim, France). RNA was quantified
by spectrophotometry at 260 nm. Concentrations of mRNAs corresponding to genes of
interest were measured by reverse transcription followed by PCR amplification. Total RNA
was reverse-transcribed using SuperScript III reverse transcriptase and a combination of
random hexamer and oligo dT primers (Invitrogen, Life Technologies, Saint-Aubin, France).
PCR amplification was performed using 2
×
Rotor-Gene SYBR Green PCR master mix and
a Rotor-Gene Q system (Qiagen, Courtaboeuf, France). Relative mRNA concentrations
were analyzed using Rotor-Gene software (version 2.3.1) and the linear standard curve
method. Table 3lists the primers used for real-time PCR amplification. mRNA contents
were normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT) level. Data
were expressed in arbitrary units.
Table 3. Primer sequences used for quantitative analysis of gene expression.
Gene Name Forward and Reverse Primers
MAFbx
Muscle atrophy F-box
For 50-AGTGAAGACCGGCTACTGTGGAA-30
Rev 50-TTGCAAAGCTGCAGGGTGAC-30
MuRF1
Muscle RING finger-1
For 50-GTGAAGTTGCCCCCTTACAA-30
Rev 50-TGGAGATGCAATTGCTCAGT-30
PGC1α
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha
For 50-AGTTTTTGGTGAAATTGAGGAAT-30
Rev 50-TCATACTTGCTCTTGGTGGAAGC-30
HPRT
Hypoxanthine-guanine phosphoribosyltransferase
For 50-AGTTGAGAGATCATCTCCAC-30
Rev 50-TTGCTGACCTGCTGGATTAC-30
2.7. Western Blot Analysis
To complete the evaluation of muscle protein synthesis, we evaluated the activation
state of its main regulatory pathway, the p70 S6 kinase pathway. Plantaris muscles were
homogenized in an ice-cold lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10 mM
EDTA, 10 mM NaPPi, 25 mM
β
-glycerophosphate, 100 mM NaF, 2 mM Na orthovanadate,
10% glycerol, 1% Triton X-100) containing a protease inhibitor cocktail (1%) as previously
described [
12
,
25
,
26
]. Denatured proteins were separated by SDS-PAGE on a polyacry-
lamide gel and transferred to a polyvinylidene membrane (Millipore, Molsheim, France).
Immunoblots were incubated in a blocking buffer and then probed with anti-phospho
p70 S6 kinase (Thr389) and anti-total p70 S6 kinase primary antibodies (Cell Signaling
Technology, Ozyme, Saint-Quentin-en-Yvelines, France). The immunoblots were then
incubated with horseradish peroxidase-conjugated swine anti-rabbit immunoglobulins
(DAKO, Trappes, France). Luminescent visualization was done using ECL Western Blot-
ting Substrate (Pierce, Thermo Fisher Scientific, Courtaboeuf, France) and a Fusion Fx
imaging system (Vilber Lourmat, Collegien, France). Density of the bands was quantified
using MultiGauge
3.2 software
(Fujifilm Corporation). The phosphorylation state of p70-S6
kinase in the plantaris muscle exhibited a strong difference between the fasting and post-
prandial states. Consequently, samples from fasted and postprandial rats were separated
onto individual gels, while a consistent mixture of all samples was loaded on each gel to
facilitate comparison of the blots. The values represent the ratio of the phosphorylated
protein levels to total protein levels, and are expressed in arbitrary units.

Nutrients 2023,15, 3766 6 of 16
2.8. Mitochondrial Enzymatic Assays
Since dietary protein, especially plant-source protein, can change mitochondrial ac-
tivity [
16
], we assessed mitochondrial function. Fifty mg of frozen rat plantaris muscles
was homogenized in a glass–glass Potter in 9 volumes of homogenization buffer (225 mM
mannitol, 75 mM sucrose, 10 mM Tris-HCl, 10 mM EDTA, pH 7.2) and spun down at
650
×
gfor 20 min at 4
◦
C. The supernatant was kept, and the pellet was suspended in
9 volumes
of homogenization buffer and re-submitted to the same spin-down procedure.
Both supernatants were pooled and used for the assay. After protein quantification, activi-
ties of complex 1, citrate synthase and 3-hydroxyacyl-CoA dehydrogenase (HAD) were
spectrophotometrically assayed as previously described [
23
,
25
,
27
,
28
]. Complex I and HAD
activities were spectrophotometrically assayed in the supernatant fraction by following the
oxidation of nicotinamide adenine dinucleotide, reduced form (NADH). Citrate synthase
activity was measured by following the reduction of 5,5-dithiobis(2-nitrobenzoic acid)
(DTNB) [23,25,27–29]. All activities were expressed as fold change vs. the value found for
the PEA group.
2.9. Statistics
All data were expressed as means
±
SEM. Animals that died or developed tumors
during the experiment were excluded from the analysis. In detail, while we had 20 rats per
group at baseline, the number of rats remaining at the end of the experiment was
14 PEA
rats and 12 PEA + INU rats. The data were analyzed for homogeneity of variance and
normality. Homogeneous data were analyzed using an unpaired t-test. Heterogeneous
data were analyzed using the non-parametric Mann–Whitney U test. To compare the initial
values of body weight, fat mass and lean mass with the final values, data were analyzed
by a paired t-test. Differences were considered significant at p< 0.05. Statistical analysis
was performed using NCSS 2020 software (NCSS LLC., Kaysville, UT, USA) and StatView
software (version 4.02; Abacus Concepts, Berkeley, CA, USA).
3. Results
3.1. Body Weight and Composition Changes and Final Relative Tissue Weights
There was no significant between-group difference in food intake over the course of
the study (21.8
±
0.6 g/day and 21.3
±
0.6 g/day for PEA and PEA + INU rats, respectively).
At the beginning and at the end of the study, there was no between-group difference in
body weight, fat mass and lean mass. PEA and PEA + INU rats tended to gain body weight,
significantly gained fat mass and significantly lost lean mass over the course of the 16-week
experimental period, and these changes were similar between groups (Table 4).
Table 4. Body weight, fat mass and lean mass changes after 16 weeks of the experimental study.
PEA
(n = 14)
PEA + INU
(n = 12) p
Body weight (g)
initial 598 ±19 577 ±18 0.4
final 612 ±27 588 ±21 0.5
change +13 ±16 +12 ±12 0.9
Fat mass (g)
initial 108 ±8 89 ±8 0.1
final 140 ±16 #115 ±12 ## 0.2
change +33 ±12 +26 ±7 0.6
Lean mass (g)
initial 438 ±12 436 ±13 0.9
final 414 ±13 ### 418 ±14 ## 0.8
change −23 ±4−18 ±5 0.4
Body weight change = (final body weight) minus (initial body weight). Fat mass change = (final fat mass)
minus (initial fat mass). Lean mass change = (final lean mass) minus (initial lean mass). Results are expressed
as
means ±SEM.
pindicates the p-value for the statistical analysis comparing PEA rats and PEA + INU rats.
#significantly
different from initial value with p< 0.05.
##
significantly different from initial value with p< 0.01.
### significantly different from initial value with p< 0.001.

Nutrients 2023,15, 3766 7 of 16
Tissue mass-to-body weight ratios were calculated for different lean soft and adipose
tissues and reported in Table 5. The relative weights of fast-twitch skeletal muscles, i.e.,
plantaris, tibialis and gastrocnemius, were significantly higher in PEA + INU rats than
in PEA rats (+16%, +16% and +18%, p< 0.05, respectively). Conversely, PEA + INU diet
tended to decrease the relative weight of perirenal adipose tissue (p= 0.06). Note that
relative weights of soleus (slow-twitch skeletal muscle), liver, heart and subcutaneous
adipose tissue were similar in both groups (Table 5).
Table 5.
Relative tissue weights in PEA and PEA + INU rats after 16 weeks of the experimental study.
PEA
(n = 14)
PEA + INU
(n = 12)
Plantaris (mg/100 g body weight) 46.0 ±1.6 53.5 ±3.4 *
Tibialis (mg/100 g body weight) 70.6 ±2.7 82.0 ±4.1 *
Gastrocnemius (mg/100 g body weight) 202.1 ±9.0 238.9 ±15.4 *
Soleus (mg/100 g body weight) 29.8 ±1.9 33.9 ±2.9
Perirenal adipose tissue (g/100 g body weight) 3.0 ±0.3 2.2 ±0.2
Subcutaneous adipose tissue (g/100 g body weight) 2.3 ±0.2 1.9 ±0.3
Liver (g/100 g body weight) 2.3 ±0.1 2.2 ±0.1
Heart (g/100 g body weight) 0.3 ±0.0 0.3 ±0.0
Results are expressed as means ±SEM. * significantly different from PEA rats at p< 0.05.
3.2. Protein Quality Evaluation
Nitrogen intake and fecal and urinary nitrogen contents were measured during the
metabolic cage period (Table 6). Nitrogen intake and urinary nitrogen content were similar
between the two rat groups whereas fecal excreted nitrogen was significantly higher
for
PEA + INU
rats than PEA rats. Fecal nitrogen content-to-nitrogen intake ratio was
significantly higher in PEA + INU rats than in PEA rats (+25%, p< 0.05) whereas urinary
nitrogen content-to-nitrogen intake tended to be lower in PEA + INU rats than in PEA rats
(
−
16%,
p= 0.05;
Table 6). As a result, the nitrogen balance, which is the difference between
nitrogen intake and nitrogen loss by both fecal and urinary routes, was slightly greater in
rats fed the PEA + INU diet than in rats fed the PEA diet (p= 0.07).
Table 6. Evaluation of protein quality during the 4-day period in metabolic cages.
PEA
(n = 14)
PEA + INU
(n = 12)
Nitrogen intake (g) 1.65 ±0.09 1.86 ±0.11
Fecal nitrogen (g) 0.13 ±0.01 0.18 ±0.01 *
Urinary nitrogen (g) 0.92 ±0.07 0.86 ±0.06
FN/NI 0.08 ±0.01 0.10 ±0.01 *
UN/NI 0.56 ±0.03 0.47 ±0.03
Nitrogen balance (g) 0.60 ±0.07 0.81 ±0.09
NI = Nitrogen Intake. FN = Fecal Nitrogen. UN = Urinary Nitrogen. Results are expressed as means
±
SEM.
* significantly different from PEA rats at p< 0.05.
3.3. Muscle Protein Synthesis in Fasted and Postprandial State and Markers of Muscle Proteolysis
To investigate the molecular mechanisms involved in the increase of relative weight of
fast-twitch skeletal muscles in PEA + INU rats compared to PEA rats, we explored protein
synthesis and gene expression of markers of muscle proteolysis in plantaris muscles in
fasted state and postprandial state. To mimic the postprandial state, we administered a
solution of amino acids and carbohydrates by oral gavage. Oral administration induced
a significant increase in plasma glucose levels and, consequently, a significant increase
in plasma insulin concentration in both rat groups (Table 7). Note that plasma levels
of glucose, insulin and triglycerides were not significantly different in both PEA and
PEA + INU rats in both fasted state and postprandial state (Table 7). Analysis of the
data for groups pooled together found that the oral gavage with the nutrient mixture

Nutrients 2023,15, 3766 8 of 16
enhanced muscle absolute synthesis rate (ASR) in comparison with the fasted state (+42%,
p< 0.05)
. However, analysis of the data for individual groups found that oral gavage did
not significantly increase muscle ASR compared to the fasted state (Figure 1A), despite a
strong activation by phosphorylation of p70 S6 kinase, an intermediate of the translation
initiation step, in the postprandial state compared to the fasted state (Figure 1B,C). There
was no between-diet (PEA vs. PEA + INU) difference in muscle ASR (Figure 1A) or p70
S6kinase phosphorylation level (Figure 1B,C) in the fasted state or in the postprandial state.
We then assessed the involvement of the ubiquitin-proteasome pathway in the regulation
of skeletal muscle mass in PEA and PEA + INU rats by measuring transcript levels of
MuRF1 and MAFbx. MAFbx mRNA expression was unaffected by both experimental diets
and both nutritional states (Figure 1D). MuRF1 transcript levels were similar between diet
groups in the fasted state but significantly lower in plantaris muscles of PEA + INU rats
compared to PEA rats in the postprandial state (Figure 1E). Oral gavage with the nutrient
mixture induced a significant downregulation of muscle MuRF1 mRNA expression in
PEA + INU rat muscles but not in PEA rat muscles (Figure 1E).
Nutrients 2023, 15, 3766 9 of 17
Figure 1. Protein synthesis rate, p70 S6 kinase phosphorylation rate, and expression of ubiquitin-
proteasome pathway markers in plantaris muscles of fasted and postprandial old rats fed either
PEA or PEA + INU diets. All rats were fasted overnight. PEA rats and PEA + INU rats were randomly
divided into groups that either were kept in the fasted state or given a nutrient mixture by oral
gavage (postprandial state). (A) Absolute synthesis rate was measured by tracer enrichment in
plantaris muscles after incubation with L-[13C6] phenylalanine. (B,C) Ratio of phosphorylated p70
S6 kinase to total p70 S6 kinase content in the same plantaris muscles was determined by Western-
bloing. Gene expressions of the two ubiquitin E3 ligases MAFbx (D) and MuRF1 (E) were assessed
by quantitative RT-PCR. Data are expressed as means ± SEM. * significantly different from PEA rats
at identical nutritional status (fasted or postprandial) at p < 0.05. # significantly different from the
same rat group in the fasted state at p < 0.05. ### significantly different from the same rat group rats
in the fasted state at p < 0.001. A.U.: arbitrary units. M: Mix of all samples. PEA rat group at the
fasted state, n = 8; PEA rat group at the postprandial state, n = 6; PEA + INU rat group at the fasted
state, n = 7; PEA + INU rat group at the postprandial state, n = 5.
Table 7. Metabolic parameters in plasma of PEA and PEA + INU rats in fasted vs. postprandial
state.
PEA PEA + INU
Fasted State
(n = 8)
Postprandial
State
(n = 6)
Fasted State
(n = 7)
Postprandial
State
(n = 5)
Glucose (g/L) 0.970 ± 0.108 1.574 ± 0.072 ***
0.795 ± 0.112 1.445 ± 0.083 ***
Insulin (ng/mL)
0.553 ± 0.102 2.531 ± 0.456 ***
0.564 ± 0.125 2.037 ± 1.066 *
Triglycerides
(g/L) 0.604 ± 0.176 0.745 ± 0.133 0.682 ± 0.139 0.486 ± 0.036
Results are expressed as means ± SEM. * Mean values significantly different between fasted rats and
postprandial rats fed with the same diet at p < 0.05. *** Mean values significantly different between
fasted rats and postprandial rats fed with the same diet at p < 0.001.
3.4. Muscle Mitochondrial Activity
We investigated the effect of inulin supplementation on muscle mitochondrial func-
tion in old rats by measuring the maximal activities of complex 1, which is an electron
transport chain complex, citrate synthase, which is a mitochondrial matrix enzyme used
as a marker of mitochondrial density, and 3-hydroxyacyl-CoA dehydrogenase (HAD),
which is a key enzyme of the mitochondrial β-oxidation pathway. Complex 1, citrate syn-
thase and HAD activity showed no nutritional state-related changes, so we pooled the
Figure 1.
Protein synthesis rate, p70 S6 kinase phosphorylation rate, and expression of ubiquitin-
proteasome pathway markers in plantaris muscles of fasted and postprandial old rats fed either PEA
or PEA + INU diets. All rats were fasted overnight. PEA rats and PEA + INU rats were randomly
divided into groups that either were kept in the fasted state or given a nutrient mixture by oral gavage
(postprandial state). (
A
) Absolute synthesis rate was measured by tracer enrichment in plantaris
muscles after incubation with L-[
13
C
6
] phenylalanine. (
B
,
C
) Ratio of phosphorylated p70 S6 kinase to
total p70 S6 kinase content in the same plantaris muscles was determined by Western-blotting. Gene
expressions of the two ubiquitin E3 ligases MAFbx (
D
) and MuRF1 (
E
) were assessed by quantitative
RT-PCR. Data are expressed as means
±
SEM. * significantly different from PEA rats at identical
nutritional status (fasted or postprandial) at p< 0.05. # significantly different from the same rat group
in the fasted state at p< 0.05. ### significantly different from the same rat group rats in the fasted
state at p< 0.001. A.U.: arbitrary units. M: Mix of all samples. PEA rat group at the fasted state,
n = 8;
PEA rat group at the postprandial state, n = 6; PEA + INU rat group at the fasted state, n = 7;
PEA + INU rat group at the postprandial state, n = 5.

Nutrients 2023,15, 3766 9 of 16
Table 7.
Metabolic parameters in plasma of PEA and PEA + INU rats in fasted vs. postprandial state.
PEA PEA + INU
Fasted State
(n = 8)
Postprandial State
(n = 6)
Fasted State
(n = 7)
Postprandial State
(n = 5)
Glucose (g/L) 0.970 ±0.108 1.574 ±0.072 *** 0.795 ±0.112 1.445 ±0.083 ***
Insulin (ng/mL) 0.553 ±0.102 2.531 ±0.456 *** 0.564 ±0.125 2.037 ±1.066 *
Triglycerides (g/L)
0.604 ±0.176 0.745 ±0.133 0.682 ±0.139 0.486 ±0.036
Results are expressed as means
±
SEM. * Mean values significantly different between fasted rats and postprandial
rats fed with the same diet at p< 0.05. *** Mean values significantly different between fasted rats and postprandial
rats fed with the same diet at p< 0.001.
3.4. Muscle Mitochondrial Activity
We investigated the effect of inulin supplementation on muscle mitochondrial func-
tion in old rats by measuring the maximal activities of complex 1, which is an electron
transport chain complex, citrate synthase, which is a mitochondrial matrix enzyme used
as a marker of mitochondrial density, and 3-hydroxyacyl-CoA dehydrogenase (HAD),
which is a key enzyme of the mitochondrial
β
-oxidation pathway. Complex 1, citrate
synthase and HAD activity showed no nutritional state-related changes, so we pooled the
muscle activity measurements done in fasted state with the muscle activity measurements
done in postprandial state for each of the three enzymes. Complex 1 activity tended to be
enhanced in plantaris homogenates of PEA + INU rats compared to PEA rats (p= 0.07)
(Figure 2A).
Inulin supplementation induced an increase in citrate synthase and HAD activ-
ity in plantaris muscles (+18% and +28% vs. PEA rats, respectively, p< 0.01; Figure 2A). To
investigate the molecular mechanisms involved in the modulation of muscle mitochondrial
function in response to dietary inulin supplementation, we analyzed the gene expression
levels of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1
α
) to
estimate mitochondrial biogenesis in plantaris muscles. In line with the increase in muscle
mitochondrial function in inulin-supplemented old rats, plantaris muscles of PEA + INU
rats showed a strong increase of PGC1
α
transcripts compared to PEA rats (+64%, p< 0.05;
Figure 2B).
Nutrients 2023, 15, 3766 10 of 17
muscle activity measurements done in fasted state with the muscle activity measurements
done in postprandial state for each of the three enzymes. Complex 1 activity tended to be
enhanced in plantaris homogenates of PEA + INU rats compared to PEA rats (p = 0.07)
(Figure 2A). Inulin supplementation induced an increase in citrate synthase and HAD ac-
tivity in plantaris muscles (+18% and +28% vs. PEA rats, respectively, p < 0.01; Figure 2A).
To investigate the molecular mechanisms involved in the modulation of muscle mitochon-
drial function in response to dietary inulin supplementation, we analyzed the gene ex-
pression levels of peroxisome proliferator-activated receptor gamma coactivator 1 alpha
(PGC1α) to estimate mitochondrial biogenesis in plantaris muscles. In line with the in-
crease in muscle mitochondrial function in inulin-supplemented old rats, plantaris mus-
cles of PEA + INU rats showed a strong increase of PGC1α transcripts compared to PEA
rats (+64%, p < 0.05; Figure 2B).
Figure 2. Mitochondrial function and biogenesis in plantaris muscles of PEA and PEA + INU old
rats. (A) Mitochondrial function was assessed by measuring complex 1, citrate synthase, and 3-hy-
droxyacyl-CoA dehydrogenase (HAD) activity in plantaris muscles. Enzyme activities were ex-
pressed as fold change vs. the value found for the PEA rat group. (B) Mitochondrial biogenesis was
determined by measuring PGC1α mRNA expression using quantitative RT-PCR and was expressed
as fold change vs. value found for the PEA rat group. Data are expressed as means ± SEM. * signifi-
cantly different from PEA rats at p < 0.05. ** significantly different from PEA rats at p < 0.01. (A.U.):
arbitrary units. PEA rat group, n = 14; PEA + INU rat group, n = 12.
4. Discussion
This study demonstrates that pea protein combined with inulin improves muscle
anabolism and muscle mitochondrial activity, suggesting potential benefits for muscle
health in old rats. The weight of fast-twitch muscles, which are precisely the muscles most
damaged by aging, was higher in old rats following consumption of a diet based on pea
protein plus dietary fiber (inulin) compared with pea protein given without dietary fiber.
Mitochondria are the energy powerhouses of cells, including muscle cells, so as pea pro-
tein with inulin improves mitochondrial function and efficiency, it could potentially also
enhance energy production, which is crucial for optimal muscle function and perfor-
mance. These findings align with the potential benefits of plant proteins and inulin sepa-
rately identified in previous specific studies [9,16,30]. Pea protein is a good source of es-
sential amino acids, which are crucial for muscle protein turnover. Inulin, acting as a
prebiotic, can positively influence gut microbiota, leading to reduced inflammation and
metabolic improvements.
The primary aim of this study was to evaluate the quality and impact of pea protein
enriched with inulin on protein retention in old rats compared to pea protein given with-
out inulin. The nutritional value of proteins is dependent on their amino acid composition
and how readily they can be digested, absorbed, and incorporated into body proteins [4].
As expected, pea protein and inulin lead to a higher fecal nitrogen excretion in old rats,
Figure 2.
Mitochondrial function and biogenesis in plantaris muscles of PEA and PEA + INU
old rats. (
A
) Mitochondrial function was assessed by measuring complex 1, citrate synthase, and
3-hydroxyacyl-CoA dehydrogenase (HAD) activity in plantaris muscles. Enzyme activities were
expressed as fold change vs. the value found for the PEA rat group. (
B
) Mitochondrial biogenesis was
determined by measuring PGC1
α
mRNA expression using quantitative RT-PCR and was expressed as
fold change vs. value found for the PEA rat group. Data are expressed as means
±
SEM.
* significantly
different from PEA rats at p< 0.05. ** significantly different from PEA rats at p< 0.01. (A.U.): arbitrary
units. PEA rat group, n = 14; PEA + INU rat group, n = 12.

Nutrients 2023,15, 3766 10 of 16
4. Discussion
This study demonstrates that pea protein combined with inulin improves muscle
anabolism and muscle mitochondrial activity, suggesting potential benefits for muscle
health in old rats. The weight of fast-twitch muscles, which are precisely the muscles
most damaged by aging, was higher in old rats following consumption of a diet based on
pea protein plus dietary fiber (inulin) compared with pea protein given without dietary
fiber. Mitochondria are the energy powerhouses of cells, including muscle cells, so as pea
protein with inulin improves mitochondrial function and efficiency, it could potentially also
enhance energy production, which is crucial for optimal muscle function and performance.
These findings align with the potential benefits of plant proteins and inulin separately
identified in previous specific studies [
9
,
16
,
30
]. Pea protein is a good source of essential
amino acids, which are crucial for muscle protein turnover. Inulin, acting as a prebiotic,
can positively influence gut microbiota, leading to reduced inflammation and metabolic
improvements.
The primary aim of this study was to evaluate the quality and impact of pea protein
enriched with inulin on protein retention in old rats compared to pea protein given without
inulin. The nutritional value of proteins is dependent on their amino acid composition
and how readily they can be digested, absorbed, and incorporated into body proteins [
4
].
As expected, pea protein and inulin lead to a higher fecal nitrogen excretion in old rats,
resulting in a higher ratio of fecal nitrogen-to-nitrogen intake. The presence of the dietary
fiber, e.g., inulin, could interfere with protein digestion and increase nitrogen excretion.
Dietary fiber has been shown to increase the secretion of nitrogenous substances into the
alimentary tract [
31
–
33
] and to increase the quantity of endogenous amino acids at the
terminal ileum of simple-stomached animals [
34
]. It has also been reported that nitrogenous
secretions including pancreatic juice [
31
,
32
] and mucus [
33
] are secreted in larger amounts
when experimental animals are fed purified diets supplemented with fiber. Furthermore,
Bergner et al. [
35
] reported that dietary fiber hindered the reabsorption of endogenous
amino acids from the small intestine. However, here, body weight and lean mass changes
were similar between old rats fed pea protein and pea protein plus inulin, despite the
higher fecal nitrogen-to-nitrogen intake ratio under the pea-protein-plus-inulin diet. In
addition, protein retention appeared to be similar or even improved when also considering
muscle mass in old rats fed the pea protein-plus-inulin diet, despite a higher fecal excretion
of nitrogen. Nitrogen balance tended to be better in the group fed pea protein-plus-inulin,
showing that the presence of dietary fiber enabled more efficient utilization of dietary
nitrogen.
One of the most remarkable results of the present work is the better maintenance of
type-2 muscle mass in old rats fed dietary fiber in addition to pea protein compared to rats
fed only pea protein. The effects of plant proteins and inulin on muscle in old rats can vary
depending on several factors. We previously reported that plant-source proteins, such as
pea, lentil or faba bean, can have beneficial effects on muscle health in older rats [
9
,
16
].
In particular, we demonstrated that pea proteins had a similar effect on nitrogen balance,
true digestibility, net protein utilization, body composition, tissue weight, skeletal muscle
protein synthesis or degradation, and muscle mitochondrial activity in old rats as compared
to a control group [
16
]. In addition, the inclusion of peas as a source of protein in the diets
of growing rats was shown to lead to similar fractional synthesis rates of liver and muscle
proteins to casein-fed controls [36].
The positive effect on muscle of pea protein consumed with inulin in old rats may
also be partially fiber-dependent. Inulin is a type of dietary fiber that is widely found
in many plants. Inulin acts as a prebiotic, meaning it serves as food for beneficial gut
bacteria [
30
]. While there is limited direct research on inulin and muscle health in old rats,
a healthy gut microbiota composition is associated with improved nutrient absorption,
reduced inflammation, and enhanced overall health, all of which indirectly supports muscle
health. In addition, inulin consumption has been linked to improved glucose metabolism
and insulin sensitivity [
37
]. Better metabolic control may also indirectly benefit muscle

Nutrients 2023,15, 3766 11 of 16
health, as insulin resistance and metabolic dysfunction can impair muscle function and
disrupt muscle protein turnover [
38
]. Here we showed that the consumption of inulin
can decrease certain markers of muscle proteolysis. Note that Wang et al. [
39
] previously
showed that inulin supplementation in pigs significantly decreased the expression level of
muscle-specific ubiquitin ligase MuRF-1, i.e., the same marker of proteolysis downregulated
here. Moreover, although the ingestion of dietary fiber had no specific additional effect
on muscle protein synthesis in old rats, there was a significant postprandial stimulation
of muscle protein synthesis in these animals after the consumption of pea proteins. As
already mentioned, the specific effect of plant proteins such as pea proteins on muscle
protein synthesis has already been demonstrated in rodents, particularly in old rats [9,16].
Here, in the pea protein-plus-inulin group, we posit that a positive effect of pea protein on
muscle protein synthesis rate coupled with a positive action of inulin on muscle proteolysis
may lead to increased protein anabolism and thus contribute to maintenance of muscle
mass. In addition, apart from a well-balanced amino acid content, pea protein can be
accompanied by plant bioactives, such as polyphenols, which have an antioxidant action.
It has previously been shown that antioxidants can stimulate muscle protein synthesis in
old rats [10].
Dietary fibers that are not digested by host enzymes but fermented by gut bacteria
could modulate the gut microbiome composition within a relatively short space of time.
These fermented non-digestible compounds favor the proliferation of health-promoting bac-
teria that may positively affect muscle health. In older adults affected by frailty syndrome,
the administration of a prebiotic significantly improved muscle-related frailty criteria, e.g.,
exhaustion and handgrip strength [
40
], compared with a placebo. Cani et al. reported
decreased levels of inflammation and increased muscle mass in obese mice supplemented
with oligofructose fiber [
41
], and the beneficial effect of prebiotic administration on gut
microbiota was further confirmed by increased levels of Lactobacillus and Bifidobacterium
spp. found in follow-up analysis [
42
]. Recently, Giron et al. showed that when one of these
bacteria, Lactobacillus, was ingested daily for 1 month, food-restricted 18-month-old rats
were able to preserve muscle protein mass by improving both muscle protein synthesis and
insulin sensitivity [
43
]. These findings suggest that Lactobacillus and Bifidobacterium may
influence gut–muscle communication and regulate muscle size. Intestinal Bifidobacterium
content decreases with age [
44
], and an age-related decrease in gut Bifidobacterium content
may be the mechanism underpinning the increase in circulating endotoxin that induces
muscle atrophy [
45
]. Interestingly, supplementation with galactooligosaccharides (GOS)
in middle-aged and older people was shown to attenuate the age-associated reduction in
gut Bifidobacteria [
46
]. In particular, the GOS treatment led to an increase in the number
of Bifidobacteria and Lactobacilli together with higher butyrate levels. In the same way,
inulin enhances the growth of indigenous Lactobacilli and Bifidobacteria by inducing colonic
production of short-chain fatty acids (SCFA), and these properties are related to decreased
mucosal lesion scores and diminished mucosal inflammation [
15
]. Thus, inulin, by increas-
ing the number of beneficial bacteria on the mucosal surface, may improve the gut mucosal
barrier and prevent gastrointestinal infections with enteric pathogens as well as systemic
inflammation from the translocation of gut bacteria and inflammatory by-products [
45
].
Inulin has shown anti-inflammatory properties by decreasing pro-inflammatory mark-
ers [
47
]. As chronic inflammation can negatively impact muscle health and is associated
with age-related muscle decline [
48
], reducing inflammation may support muscle function
and prevent age-related muscle decline. The administration of a symbiotic comprising the
probiotic Bifidobacterium longum and an inulin-based prebiotic component has also been
demonstrated to enhance butyrate production and blunt proinflammatory responses [
49
].
This and other studies based on inulin supplementation converge towards the hypoth-
esis that administration of the pea protein-plus-inulin mixture in advanced age might
positively affect the microbiota and slow the age-associated decline of muscle mass and
function. Taken together, these lines of evidence support the idea that pre- and/or probi-
otic supplementation may prevent age-related muscle loss by increasing the abundance

Nutrients 2023,15, 3766 12 of 16
of Bifidobacterium and butyrate producers in old individuals. In addition, plant proteins,
especially those containing some remaining bioactive compounds such as antioxidants and
polyphenols, may help reduce inflammation and further support muscle health.
Our work also shows that an additional dietary fiber intake in addition to plant
proteins improved muscle mitochondrial function in old rats compared to a pea protein
intake alone. The direct effects of dietary fiber on mitochondria in muscle have not been
extensively studied, but their indirect impact on muscle health and performance suggests
potential benefits for mitochondrial function. Emerging evidence suggests that the gut
microbiota may affect mitochondrial function in several tissues [
50
–
52
], including skeletal
muscle [
53
]. Gut dysbiosis increases the permeability of intestinal mucosa, promotes
systemic inflammation, subclinical immune activation and insulin resistance, and ultimately
leads to muscle mitochondria damage [
54
]. Studies have demonstrated that probiotic
intake or fecal microbial transplantation can regulate mitochondrial energy metabolism and
skeletal muscle functions in advanced age [
55
,
56
]. For example, Chen et al. showed that
Lactobacillus casei supplementation in old mice produces anti-inflammatory effects and leads
to maintained muscle mitochondrial functions [
57
]. Very recently, Zhang et al. reported that
insoluble dietary fiber intake in rats prevents obesity and improves the dyslipidemia and
hepatic steatosis caused by a high-fat diet by promoting hepatic mitochondrial fatty acid
oxidation [
58
]. Importantly, this intervention promotes medium- and long-chain fatty acid
oxidation in hepatic mitochondria by improving the contents of key mitochondrial enzymes
such as carnitine palmitoyl transferase-1, acyl-coenzyme A oxidase 1, acetyl coenzyme A
synthase, and acetyl coenzyme A carboxylase [
58
]. Further studies are needed to explore
the specific effect of dietary fiber on muscle mitochondria, but this action may also exist in
skeletal muscle. Here we show that the consumption of inulin specifically increases the
activity of enzymes that play a central role in the citric acid cycle, beta-oxidation, and the
respiratory chain, i.e., complex 1, citrate synthase, and 3-hydroxyacyl-CoA dehydrogenase
(HAD) activities, in plantaris muscle. Although the underlying mechanism needs to be
studied further, the existing evidence shows that certain gut bacteria produce metabolites
that can influence mitochondrial metabolism and energy production. Note that SCFA,
such as butyrate, are key intermediates affecting the gut–muscle axis [
59
]. SCFA can be
used for de novo synthesis of lipids and glucose, which are the main energy sources
for the host. In addition, two orphan G-protein-coupled receptors, GPR41 and GPR43,
were reported to be activated by SCFA. SCFA-mediated GPR43 activation suppressed
insulin signaling in adipose tissue, leading to inhibition of fat accumulation [
60
]. The study
found that unincorporated lipids and glucose were primarily utilized in muscles where
the expression levels of energy expenditure, glycolysis and
β
-oxidation-related genes was
increased [
60
]. In another study, the incorporation of butyrate in the diet induced higher
energy expenditure and oxygen consumption in mice, suggesting an increase in fatty acid
oxidation that was confirmed by monitoring 14C-labeled palmitic acid in butyrate-treated
mice [
61
]. The authors explained the observed effects by an activation of peroxisome
proliferator-activated receptor-
γ
coactivator-1
α
(PGC-1
α
), which is a master regulator
of mitochondrial biogenesis, in brown fat, skeletal muscle and liver [
61
]. Interestingly,
we also found that muscle PGC-1
α
was also upregulated here. Taken as a whole, SCFA
are good candidates to explain the biological effects of dietary fiber consumption on
muscle mitochondrial function found here, as they can serve as substrates for mitochondria
and regulate key mitochondrial functions [
58
]. By supporting a diverse and balanced
gut microbiota, dietary fiber may indirectly affect mitochondrial function in muscle. In
addition, the chronic low-grade inflammation observed during aging, i.e., Inflamm-aging,
can negatively impact mitochondrial function [
62
]. Dietary fibers, particularly those with
anti-inflammatory properties such as inulin, can help reduce systemic inflammation [
47
].
By lowering inflammation levels, dietary fibers may also contribute to better mitochondrial
function in muscle.
This study has potential limitations. It would have been interesting to evaluate the
composition of the microbiota of rats, in particular of those having consumed inulin. This

Nutrients 2023,15, 3766 13 of 16
analysis would have made it possible to better understand the impact of changes in gut
bacterial abundance on the effect of inulin on skeletal muscle. Of note, the modification
of the gut microbiota induced by a diet enriched with inulin has already been described
in rats in different conditions, e.g., standard condition, obesity [
63
,
64
]. Inulin is usually
regarded as a type of prebiotic, favorably stimulating the growth of Bifidobacteria lactobacilli
and actinobacteria and inhibiting the growth of bacteroidetes. In addition, in this work
we were unable to measure inflammatory markers for technical reasons. Inulin intake has
been shown to reduce inflammation in various situations [
15
,
45
,
47
]. Age-related muscle
loss is partly explained by an increased inflammatory state, i.e., increased production of
pro-inflammatory cytokines. It therefore remains to be determined whether the intake of
inulin in older individuals is able to reduce muscle loss by limiting inflammation.
In conclusion, pea protein is a plant-based source of high-quality protein that includes
all the essential amino acids required to sustain muscle protein turnover. Furthermore, pea
protein is generally well-tolerated and digestible, making it a suitable protein source for
older people. Inulin is a type of dietary fiber found in various plants and is considered
a prebiotic, which means that it provides nourishment to beneficial gut bacteria. The
direct effects of inulin on muscle in old rats have been under-researched, but the evidence
suggests it can promote a healthy gut microbiota by stimulating the growth of beneficial
bacteria, and thus stave off systemic inflammation. Inulin has also been shown to improve
glucose metabolism and insulin sensitivity. Dietary fiber intakes are generally too low
in the general population and especially in older people, which may contribute to the
main metabolic syndromes that accompany aging, such as sarcopenia. Note that caution is
warranted when attempting to translate these findings from rats to humans, as there may be
species-specific differences. Nonetheless, these findings provide promising insights into the
potential benefits of combining pea protein with inulin for muscle health in older people.
While more research is needed to fully understand the additional or even synergistic effects
of plant-derived compounds such as proteins and fibers on muscle health during aging,
there is already enough evidence to recommend combining intake of a high-quality protein
with a sufficient fiber intake to protect muscle mass and function, and thus extend the
autonomy of older people.
Author Contributions:
Conceptualization: J.S., C.M.-F., Y.B., H.J. and S.W.; Data curation: J.S. and
S.W.; Formal analysis: J.S., C.M.-F., H.J. and S.W.; Funding acquisition: H.J. and S.W.; Investigation:
J.S., M.G., V.P., C.M.-F., C.G. (Christelle Guillet), O.L.B., C.G. (Christophe Giraudet), P.S., M.-L.C.,
J.H., C.P., Y.B., H.J. and S.W.; Methodology: J.S., C.M.-F., Y.B., H.J. and S.W.; Project administration:
J.S., C.M.-F., Y.B., H.J. and S.W.; Resources: H.J. and S.W.; Supervision: H.J. and S.W.; Validation: J.S.,
C.M.-F., H.J. and S.W.; Visualization: J.S., C.M.-F., H.J. and S.W.; Writing—Original draft: J.S. and S.W.;
Writing—Review and editing: J.S., H.J. and S.W. All authors have read and agreed to the published
version of the manuscript.
Funding:
This study was partly supported by a grant from COSUCRA Groupe Warcoing SA,
Warcoing, Belgium.
Institutional Review Board Statement:
The study was approved by the local institutional animal
care and use committee (Comitéd’Ethique en Matière d’Expérimentation Animale Auvergne: C2EA-
02) (protocol code: APAFIS#5329-2016051115541284 v2 approved on 23 January 2017).
Data Availability Statement: Not applicable.
Acknowledgments:
The authors thank all the members of the animal facility at the INRAE center
in Theix (France) for their valuable assistance in conducting this study, including Philippe Denis,
Philippe Lhoste, Mehdi Djelloul-Mazouz, Yoann Delorme, Yves Guerin and Benoit Cohade. Protein
synthesis measurements were performed within the MetaboHUB French infrastructure (ANR-INBS-
0010).
Conflicts of Interest:
C.M.-F. and H.J. are employees of COSUCRA-Groupe Warcoing SA (Belgium).

Nutrients 2023,15, 3766 14 of 16
References
1.
Floret, C.; Monnet, A.F.; Micard, V.; Walrand, S.; Michon, C. Replacement of animal proteins in food: How to take advantage of
nutritional and gelling properties of alternative protein sources. Crit. Rev. Food Sci. Nutr.
2023
,63, 920–946. [CrossRef] [PubMed]
2.
FAO. The state of food and agriculture. In Livestock in the Balance; Food and Agriculture Organization of the United Nations:
Rome, Italy, 2009; Available online: http://www.fao.org/3/a-i0680e.pdf (accessed on 1 May 2023).
3.
FAO. World agriculture: Towards 2030/2050. In ESA Working Paper No. 12-03, 160; Food and Agriculture Organization of the
United Nations: Rome, Italy, 2012; Available online: http://www.fao.org/fileadmin/templates/esa/Global_persepctives/world_
ag_2030_50_2012_rev.pdf (accessed on 1 May 2023).
4.
Berrazaga, I.; Micard, V.; Gueugneau, M.; Walrand, S. The Role of the Anabolic Properties of Plant-versus Animal-Based Protein
Sources in Supporting Muscle Mass Maintenance: A Critical Review. Nutrients 2019,11, 1825. [CrossRef] [PubMed]
5.
Ferrari, L.; Panaite, S.A.; Bertazzo, A.; Visioli, F. Animal- and Plant-Based Protein Sources: A Scoping Review of Human Health
Outcomes and Environmental Impact. Nutrients 2022,14, 5115. [CrossRef] [PubMed]
6. Walrand, S.; Boirie, Y. Optimizing protein intake in aging. Curr. Opin. Clin. Nutr. Metab. Care 2005,8, 89–94. [CrossRef]
7.
Daily, J.W.; Park, S. Sarcopenia Is a Cause and Consequence of Metabolic Dysregulation in Aging Humans: Effects of Gut
Dysbiosis, Glucose Dysregulation, Diet and Lifestyle. Cells 2022,11, 338. [CrossRef] [PubMed]
8.
Yuan, S.; Larsson, S.C. Epidemiology of sarcopenia: Prevalence, risk factors, and consequences. Metabolism
2023
,144, 155533.
[CrossRef]
9.
Berrazaga, I.; Salles, J.; Laleg, K.; Guillet, C.; Patrac, V.; Giraudet, C.; Le Bacquer, O.; Gueugneau, M.; Denis, P.; Pouyet, C.; et al.
Anabolic Properties of Mixed Wheat-Legume Pasta Products in Old Rats: Impact on Whole-Body Protein Retention and Skeletal
Muscle Protein Synthesis. Nutrients 2020,12, 1596. [CrossRef]
10.
Marzani, B.; Balage, M.; Vénien, A.; Astruc, T.; Papet, I.; Dardevet, D.; Mosoni, L. Antioxidant supplementation restores defective
leucine stimulation of protein synthesis in skeletal muscle from old rats. J. Nutr. 2008,138, 2205–2211. [CrossRef]
11.
El Hajj, C.; Fares, S.; Chardigny, J.M.; Boirie, Y.; Walrand, S. Vitamin D supplementation and muscle strength in pre-sarcopenic
elderly Lebanese people: A randomized controlled trial. Arch. Osteoporos. 2018,14, 4. [CrossRef]
12.
Chanet, A.; Salles, J.; Guillet, C.; Giraudet, C.; Berry, A.; Patrac, V.; Domingues-Faria, C.; Tagliaferri, C.; Bouton, K.; Bertrand-
Michel, J.; et al. Vitamin D supplementation restores the blunted muscle protein synthesis response in deficient old rats through
an impact on ectopic fat deposition. J. Nutr. Biochem. 2017,46, 30–38. [CrossRef]
13.
Walrand, S. Effect of vitamin D on skeletal muscle. Geriatr. Psychol. Neuropsychiatr. Vieil.
2016
,14, 127–134. [CrossRef] [PubMed]
14.
Tominaga, K.; Tsuchiya, A.; Nakano, O.; Kuroki, Y.; Oka, K.; Minemura, A.; Matsumoto, A.; Takahashi, M.; Kadota, Y.; Tochio, T.;
et al. Increase in muscle mass associated with the prebiotic effects of 1-kestose in super-elderly patients with sarcopenia. Biosci.
Microbiota Food Health 2021,40, 150–155. [CrossRef] [PubMed]
15.
Akram, W.; Garud, N.; Joshi, R. Role of inulin as prebiotics on inflammatory bowel disease. Drug Discov. Ther.
2019
,13, 1–8.
[CrossRef] [PubMed]
16.
Salles, J.; Guillet, C.; Le Bacquer, O.; Malnero-Fernandez, C.; Giraudet, C.; Patrac, V.; Berry, A.; Denis, P.; Pouyet, C.; Gueugneau,
M.; et al. Pea Proteins Have Anabolic Effects Comparable to Milk Proteins on Whole Body Protein Retention and Muscle Protein
Metabolism in Old Rats. Nutrients 2021,13, 4234. [CrossRef] [PubMed]
17.
Anthony, J.C.; Reiter, A.K.; Anthony, T.G.; Crozier, S.J.; Lang, C.H.; MacLean, D.A.; Kimball, S.R.; Jefferson, L.S. Orally
administered leucine enhances protein synthesis in skeletal muscle of diabetic rats in the absence of increases in 4E-BP1 or S6K1
phosphorylation. Diabetes 2002,51, 928–936. [CrossRef]
18.
Balage, M.; Dupont, J.; Mothe-Satney, I.; Tesseraud, S.; Mosoni, L.; Dardevet, D. Leucine supplementation in rats induced a delay
in muscle IR/PI3K signaling pathway associated with overall impaired glucose tolerance. J. Nutr. Biochem.
2011
,22, 219–226.
[CrossRef] [PubMed]
19.
Dijk, F.J.; van Dijk, M.; Walrand, S.; van Loon, L.J.C.; van Norren, K.; Luiking, Y.C. Differential effects of leucine and leucine-
enriched whey protein on skeletal muscle protein synthesis in aged mice. Clin. Nutr. ESPEN 2018,24, 127–133. [CrossRef]
20.
Dumas, A. [N-Determination According to Dumas]. Stickstoffbestimmung Nach Dumas. Die Praxis des Org. Chemikers, 41st ed.; Schrag:
Nuremberg, Germany, 1962.
21.
Proll, J.; Petzke, K.J.; Ezeagu, I.E.; Metges, C.C. Low nutritional quality of unconventional tropical crop seeds in rats. J. Nutr.
1998
,
128, 2014–2022. [CrossRef]
22.
Zangarelli, A.; Chanseaume, E.; Morio, B.; Brugère, C.; Mosoni, L.; Rousset, P.; Giraudet, C.; Patrac, V.; Gachon, P.; Boirie, Y.; et al.
Synergistic effects of caloric restriction with maintained protein intake on skeletal muscle performance in 21-month-old rats: A
mitochondria-mediated pathway. FASEB J. 2006,20, 2439–2450. [CrossRef]
23.
Salles, J.; Chanet, A.; Berry, A.; Giraudet, C.; Patrac, V.; Domingues-Faria, C.; Rocher, C.; Guillet, C.; Denis, P.; Pouyet, C.; et al. Fast
digestive, leucine-rich, soluble milk proteins improve muscle protein anabolism, and mitochondrial function in undernourished
old rats. Mol. Nutr. Food Res. 2017,61, 1700287. [CrossRef]
24.
Salles, J.; Cardinault, N.; Patrac, V.; Berry, A.; Giraudet, C.; Collin, M.-L.; Chanet, A.; Tagliaferri, C.; Denis, P.; Pouyet, C.; et al. Bee
pollen improves muscle protein and energy metabolism in malnourished old rats through interfering with the Mtor signaling
pathway and mitochondrial activity. Nutrients 2014,6, 5500–5516. [CrossRef] [PubMed]

Nutrients 2023,15, 3766 15 of 16
25.
Salles, J.; Chanet, A.; Guillet, C.; Vaes, A.M.; Brouwer-Brolsma, E.M.; Rocher, C.; Giraudet, C.; Patrac, V.; Meugnier, E.; Montaurier,
C.; et al. Vitamin D status modulates mitochondrial oxidative capacities in skeletal muscle: Role in sarcopenia. Commun. Biol.
2022,5, 1288. [CrossRef] [PubMed]
26.
Salles, J.; Chanet, A.; Giraudet, C.; Patrac, V.; Pierre, P.; Jourdan, M.; Luiking, Y.C.; Verlaan, S.; Migné, C.; Boirie, Y.; et al. 1,
25(OH)2-vitamin D3 enhances the stimulating effect of leucine and insulin on protein synthesis rate through Akt/PKB and mTOR
mediated pathways in murine C2C12 skeletal myotubes. Mol. Nutr. Food Res. 2013,57, 2137–2146. [CrossRef]
27.
Medja, F.; Allouche, S.; Frachon, P.; Jardel, C.; Malgat, M.; Mousson de Camaret, B.; Slama, A.; Lunardi, J.; Mazat, J.P.;
Lombès, A. Development and implementation of standardized respiratory chain spectrophotometric assays for clinical diagnosis.
Mitochondrion 2009,9, 331–339. [CrossRef] [PubMed]
28.
Cortés, N.G.; Pertuiset, C.; Dumon, E.; Börlin, M.; Hebert-Chatelain, E.; Pierron, D.; Feldmann, D.; Jonard, L.; Marlin, S.; Letellier,
T.; et al. Novel mitochondrial DNA mutations responsible for maternally inherited nonsyndromic hearing loss. Hum. Mutat.
2012,33, 681–689. [CrossRef] [PubMed]
29.
Tardif, N.; Salles, J.; Guillet, C.; Tordjman, J.; Reggio, S.; Landrier, J.F.; Giraudet, C.; Patrac, V.; Bertrand-Michel, J.; Migne, C.; et al.
Muscle ectopic fat deposition contributes to anabolic resistance in obese sarcopenic old rats through eIF2alpha activation. Aging
Cell 2014,13, 1001–1011. [CrossRef]
30.
Du, M.; Cheng, X.; Qian, L.; Huo, A.; Chen, J.; Sun, Y. Extraction, Physicochemical Properties, Functional Activities and
Applications of Inulin Polysaccharide: A Review. Plant Foods Hum. Nutr. 2023,78, 243–252. [CrossRef]
31.
Ikegami, S.; Tsuchihashi, F.; Harada, H.; Tsuchihashi, N.; Nishide, E.; Innami, S. Effect of viscous indigestible polysaccharides on
pancreatic-biliary secretion and digestive organs in rats. J. Nutr. 1990,120, 353–360. [CrossRef]
32.
Partridge, I.G.; Low, A.G.; Sambrook, I.E.; Corring, T. The influence of diet on the exocrine pancreatic secretion of growing pigs.
Br. J. Nutr. 1982,48, 137–145. [CrossRef]
33.
Cassidy, M.M.; Lightfoot, F.G.; Grau, L.E.; Story, J.A.; Kritchevsky, D.; Vahouny, G.V. Effect of chronic intake of dietary fibers
on the ultrastructural topography of rat jejunum and colon: A scanning electron microscopy study. Am. J. Clin. Nutr.
1981
,34,
218–228. [CrossRef]
34.
Libao-Mercado, A.J.; Yin, Y.; van Eys, J.; de Lange, C.F. True ileal amino acid digestibility and endogenous ileal amino acid losses
in growing pigs fed wheat shorts- or casein-based diets. J. Anim. Sci. 2006,84, 1351–1361. [CrossRef] [PubMed]
35.
Bergner, H.; Simon, O.; Zimmer, M. [Influence of crude fibers in the diet of rats on the absorption of amino acids]. Einfluss des
Gehaltes nativer Rohfaser in Diaten von Ratten auf die Aminosaurenresorption. Arch Tierernahr.
1975
,25, 95–104. [CrossRef]
[PubMed]
36.
Martinez, J.A.; Marcos, R.; Macarulla, M.T.; Larralde, J. Growth, hormonal status and protein turnover in rats fed on a diet
containing peas (Pisum sativum L.) as the source of protein. Plant Foods Hum. Nutr. 1995,47, 211–220. [CrossRef] [PubMed]
37.
Nakajima, H.; Nakanishi, N.; Miyoshi, T.; Okamura, T.; Hashimoto, Y.; Senmaru, T.; Majima, S.; Ushigome, E.; Asano, M.;
Yamaguchi, M.; et al. Inulin reduces visceral adipose tissue mass and improves glucose tolerance through altering gut metabolites.
Nutr. Metab. 2022,19, 50. [CrossRef]
38.
Guillet, C.; Masgrau, A.; Walrand, S.; Boirie, Y. Impaired protein metabolism: Interlinks between obesity, insulin resistance and
inflammation. Obes. Rev. 2012,13 (Suppl. S2), 51–57. [CrossRef] [PubMed]
39.
Wang, W.; Chen, D.; Yu, B.; Huang, Z.; Luo, Y.; Zheng, P.; Mao, X.; Yu, J.; Luo, J.; He, J. Effect of Dietary Inulin Supplementation
on Growth Performance, Carcass Traits, and Meat Quality in Growing-Finishing Pigs. Animals
2019
,9, 840. [CrossRef] [PubMed]
40.
Buigues, C.; Fernández-Garrido, J.; Pruimboom, L.; Hoogland, A.J.; Navarro-Martínez, R.; Martínez-Martínez, M.; Verdejo, Y.;
Carmen Mascarós, M.; Peris, C.; Cauli, O. Effect of a Prebiotic Formulation on Frailty Syndrome: A Randomized, Double-Blind
Clinical Trial. Int. J. Mol. Sci. 2016,17, 932. [CrossRef]
41.
Cani, P.D.; Possemiers, S.; Van de Wiele, T.; Guiot, Y.; Everard, A.; Rottier, O.; Geurts, L.; Naslain, D.; Neyrinck, A.; Lambert, D.M.;
et al. Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement
of gut permeability. Gut 2009,58, 1091–1103. [CrossRef]
42.
Everard, A.; Lazarevic, V.; Derrien, M.; Girard, M.; Muccioli, G.G.; Neyrinck, A.M.; Possemiers, S.; Van Holle, A.; François, P.; de
Vos, W.M.; et al. Responses of gut microbiota and glucose and lipid metabolism to prebiotics in genetic obese and diet-induced
leptin-resistant mice. Diabetes 2011,60, 2775–2786. [CrossRef]
43.
Giron, M.; Thomas, M.; Jarzaguet, M.; Mayeur, C.; Ferrere, G.; Noordine, M.L.; Bornes, S.; Dardevet, D.; Chassard, C.; Savary-
Auzeloux, I. Lacticaseibacillus casei CNCM I-5663 supplementation maintained muscle mass in a model of frail rodents. Front.
Nutr. 2022,9, 928798. [CrossRef]
44.
Enck, P.; Zimmermann, K.; Rusch, K.; Schwiertz, A.; Klosterhalfen, S.; Frick, J.S. The effects of maturation on the colonic microflora
in infancy and childhood. Gastroenterol. Res. Pract. 2009,2009, 752401. [CrossRef] [PubMed]
45.
Morales, M.G.; Olguin, H.; Di Capua, G.; Brandan, E.; Simon, F.; Cabello-Verrugio, C. Endotoxin-induced skeletal muscle wasting
is prevented by angiotensin-(1-7) through a p38 MAPK-dependent mechanism. Clin. Sci.
2015
,129, 461–476. [CrossRef] [PubMed]
46.
Picca, A.; Fanelli, F.; Calvani, R.; Mulè, G.; Pesce, V.; Sisto, A.; Pantanelli, C.; Bernabei, R.; Landi, F.; Marzetti, E. Gut Dysbiosis and
Muscle Aging: Searching for Novel Targets against Sarcopenia. Mediat. Inflamm. 2018,2018, 7026198. [CrossRef]
47.
Schaafsma, G.; Slavin, J.L. Significance of Inulin Fructans in the Human Diet. Compr. Rev. Food Sci. Food Saf.
2015
,14, 37–47.
[CrossRef] [PubMed]

Nutrients 2023,15, 3766 16 of 16
48.
Byrne, T.; Cooke, J.; Bambrick, P.; McNeela, E.; Harrison, M. Circulating inflammatory biomarker responses in intervention trials
in frail and sarcopenic older adults: A systematic review and meta-analysis. Exp. Gerontol. 2023,177, 112199. [CrossRef]
49.
Macfarlane, S.; Cleary, S.; Bahrami, B.; Reynolds, N.; Macfarlane, G.T. Synbiotic consumption changes the metabolism and
composition of the gut microbiota in older people and modifies inflammatory processes: A randomised, double-blind, placebo-
controlled crossover study. Aliment Pharmacol. Ther. 2013,38, 804–816. [CrossRef]
50.
Bellanti, F.; Lo Buglio, A.; Vendemiale, G. Hepatic Mitochondria-Gut Microbiota Interactions in Metabolism-Associated Fatty
Liver Disease. Metabolites 2023,13, 322. [CrossRef]
51.
Colangeli, L.; Escobar Marcillo, D.I.; Simonelli, V.; Iorio, E.; Rinaldi, T.; Sbraccia, P.; Fortini, P.; Guglielmi, V. The Crosstalk between
Gut Microbiota and White Adipose Tissue Mitochondria in Obesity. Nutrients 2023,15, 1723. [CrossRef]
52.
Righetto, I.; Gasparotto, M.; Casalino, L.; Vacca, M.; Filippini, F. Exogenous Players in Mitochondria-Related CNS Disorders:
Viral Pathogens and Unbalanced Microbiota in the Gut-Brain Axis. Biomolecules 2023,13, 169. [CrossRef]
53.
Gu, X.; Wang, W.; Yang, Y.; Lei, Y.; Liu, D.; Wang, X.; Wu, T. The Effect of Metabolites on Mitochondrial Functions in the
Pathogenesis of Skeletal Muscle Aging. Clin. Interv. Aging 2022,17, 1275–1295. [CrossRef]
54.
Murphy, E.A.; Velazquez, K.T.; Herbert, K.M. Influence of high-fat diet on gut microbiota: A driving force for chronic disease risk.
Curr. Opin. Clin. Nutr. Metab. Care 2015,18, 515–520. [CrossRef] [PubMed]
55.
van Krimpen, S.J.; Jansen, F.A.C.; Ottenheim, V.L.; Belzer, C.; van der Ende, M.; van Norren, K. The Effects of Pro-, Pre-, and
Synbiotics on Muscle Wasting, a Systematic Review-Gut Permeability as Potential Treatment Target. Nutrients
2021
,13, 1115.
[CrossRef] [PubMed]
56.
Vrieze, A.; Van Nood, E.; Holleman, F.; Salojärvi, J.; Kootte, R.S.; Bartelsman, J.F.; Dallinga-Thie, G.M.; Ackermans, M.T.; Serlie,
M.J.; Oozeer, R.; et al. Transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic
syndrome. Gastroenterology 2012,143, 913–916.e7. [CrossRef] [PubMed]
57.
Chen, L.; Chang, S.; Chang, H.; Wu, C.; Pan, C.; Chang, C.; Chan, C.; Huang, H. Probiotic supplementation attenuates age-related
sarcopenia via the gut-muscle axis in SAMP8 mice. J. Cachexia Sarcopenia Muscle 2022,13, 515–531. [CrossRef] [PubMed]
58.
Zhang, J.; Wang, S.; Wang, J.; Liu, W.; Gong, H.; Zhang, Z.; Lyu, B.; Yu, H. Insoluble Dietary Fiber from Soybean Residue (Okara)
Exerts Anti-Obesity Effects by Promoting Hepatic Mitochondrial Fatty Acid Oxidation. Foods
2023
,12, 2081. [CrossRef] [PubMed]
59.
Sridharan, G.V.; Choi, K.; Klemashevich, C.; Wu, C.; Prabakaran, D.; Pan, L.B.; Steinmeyer, S.; Mueller, C.; Yousofshahi, M.;
Alaniz, R.C.; et al. Prediction and quantification of bioactive microbiota metabolites in the mouse gut. Nat. Commun.
2014
,5, 5492.
[CrossRef]
60.
Kimura, I.; Inoue, D.; Hirano, K.; Tsujimoto, G. The SCFA Receptor GPR43 and Energy Metabolism. Front. Endocrinol.
2014
,5, 85.
[CrossRef]
61.
Gao, Z.; Yin, J.; Zhang, J.; Ward, R.E.; Martin, R.J.; Lefevre, M.; Cefalu, W.T.; Ye, J. Butyrate improves insulin sensitivity and
increases energy expenditure in mice. Diabetes 2009,58, 1509–1517. [CrossRef]
62.
Nucci, R.A.B.; Filho, V.A.N.; Jacob-Filho, W.; Otoch, J.P.; Pessoa, A.F.M. Role of Nutritional Supplements on Gut-Muscle Axis
Across Age: A Mini-Review. Cell. Physiol. Biochem. 2023,57, 161–168.
63.
Mao, B.; Li, D.; Zhao, J.; Liu, X.; Gu, Z.; Chen, Y.Q.; Zhang, H.; Chen, W. Metagenomic insights into the effects of fructo-
oligosaccharides (FOS) on the composition of fecal microbiota in mice. J. Agric. Food Chem. 2015,63, 856–863. [CrossRef]
64.
Miralles-Pérez, B.; Nogués, M.R.; Sánchez-Martos, V.; Fortuño-Mar, À.; Ramos-Romero, S.; Torres, J.L.; Ponomarenko, J.;
Amézqueta, S.; Zhang, X.; Romeu, M. Influence of Dietary Inulin on Fecal Microbiota, Cardiometabolic Risk Factors, Eicosanoids,
and Oxidative Stress in Rats Fed a High-Fat Diet. Foods 2022,11, 4072. [CrossRef] [PubMed]
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