# Response of adult stem cell populations to a high-fat/high-fiber diet in skeletal muscle and adipose tissue of growing pigs divergently selected for feed efficiency

Marie-Hélène Perruchot, Frederic Dessauge, Florence Gondret, Isabelle Louveau

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Marie-Hélène Perruchot, Frederic Dessauge, Florence Gondret, Isabelle Louveau. Response of adult stem cell populations to a high-fat/high-fiber diet in skeletal muscle and adipose tissue of growing pigs divergently selected for feed efficiency. European Journal of Nutrition, 2021, 60, pp.2397-2408. 10.1007/s00394-020-02418-7. hal-02986493

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<table><tr><td>Manuscript Number:</td><td>Response of adult stem cell populations to a high fat high fiber diet in skeletal muscle and adipose tissue of growing pigs</td></tr><tr><td>Full Title:</td><td>Original Contribution</td></tr><tr><td>Article Type:</td><td>Adult stem cells. Feed efficiency. Growth. Adipose tissue. Skeletal muscle</td></tr><tr><td>Keywords:</td><td>Marie-Hélène Perruchot<br>INRA Centre de Rennes<br>Saint Gilles, Bretagne FRANCE</td></tr><tr><td>Corresponding Author:</td><td>Marie-Hélène Perruchot<br>Frédéric Dessauge<br>Florence Gondret<br>Isabelle Louveau</td></tr><tr><td>Corresponding Author Secondary Information:</td><td></td></tr><tr><td>Corresponding Author's Institution:</td><td>INRA Centre de Rennes</td></tr><tr><td>Corresponding Author's Secondary Institution:</td><td></td></tr><tr><td>First Author:</td><td>Marie-Hélène Perruchot</td></tr><tr><td>First Author Secondary Information:</td><td></td></tr><tr><td>Order of Authors:</td><td>Marie-Hélène Perruchot<br>Frédéric Dessauge<br>Florence Gondret<br>Isabelle Louveau</td></tr><tr><td>Order of Authors Secondary Information:</td><td></td></tr><tr><td>Funding Information:</td><td>ANR<br>(ANR-11-BSV7-004)</td><td>Dr Florence Gondret</td></tr><tr><td>Abstract:</td><td>Abstract<br>Purpose The control of body composition by dietary nutrients is of the upmost importance for both human and animal physiology. Adult stem cells (aSC) may represent a relevant level of tissue adaptation. The purpose of this study was to determine the impact of macronutrient composition on aSC populations isolated from adipose tissue or muscle in growing pigs.<br>Methods Pigs from two lines divergently selected for feed efficiency were fed ad libitum either a low-fat high-starch (LF) diet or a high-fat high-fiber (HF) diet (n = 6 per line and diet) from 74 to 132 days of age. Stroma-vascular cells were isolated from adipose tissue and muscle and characterized with cell surface markers.<br>Results Pigs fed the HF diet exhibited a reduced adiposity (P < 0.001) compared with pigs fed the LF diet whatever the line. In the four groups, CD90 and PDGFRa markers were predominantly expressed in adipose cells, whereas CD90 and CD56 markers were highly expressed in muscle cells. In adipose tissue, the proportion of CD56+/PDGFRa+ cells, and the proportion of CD90+/PDGFRa+ were lower (P < 0.05) in HF pigs than in LF pigs. On the opposite, in muscle, these proportions were higher (P < 0.001) in HF pigs.<br>Conclusion This study indicates that dietary nutrients affected the relative proportions of aSC populations in pigs, with opposite effects on CD90+/PDGFRa+, CD56+/PDGFRa+ cell populations between muscle and adipose tissue. Therefore, the control of these cells may be a strategy to modulate body composition.</td></tr></table>
Suggested Reviewers:

Jean Charles Gabillard
jean-charles.gabillard@inrae.fr

Christophe Praud
christophe.praud@inrae.fr

louis casteilla
louis.casteilla@inserm.fr

louis lefaucheur
louis.lefaucheur@inrae.fr
<figure><img src="image_2.png" /></figure>

1 **Response of adult stem cell populations to a high fat high fiber diet in skeletal muscle**

2 **and adipose tissue of growing pigs**

3 Marie-Hélène Perruchot, Frédéric Dessauge, Florence Gondret, Isabelle Louveau

4

5 INRAE, PEGASE, AGROCAMPUS OUEST, 35590, SAINT-GILLES, FRANCE

6 Corresponding author, e-mail: marie-helene.perruchot@inrae.fr

7 ORCID 0000-0002-6608-9445

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9

10 Acknowledgments

11 The authors' responsibilities were as follows—MHP, FG and IL designed the research and had primary
12 responsibility for the final content; MHP conducted the research; MHP and FD analyzed the data; MHP, FD, FG
13 and IL wrote the paper; and all authors read and approved the final manuscript. The authors thank H. Gilbert
14 (INRAE, GenPhySE 31326 Castanet-Tolosan, FRANCE), and Y. Billon and A. Priet (INRAE, GenESI, 17700
15 Surgères, FRANCE) for line selection. They are also grateful to the technical staff of PEGASE, INRAE: P.

16 Roger and J. Delamarre for animal care, G. Guillemois for diet preparation, J. Liger and J.F. Rouault for animal slaughter procedures, and F. Mayeur and C. Tréfeu for their help in sample collection and/or laboratory analyzes.

18 The current study was granted by the French National Research Agency (Agence Nationale de la Recherche, ANR-11-SVSE7004 FatInteger).

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21 Word count for the entire manuscript (introduction through discussion);

22 Number of figures: 5

23 Number of tables: 5

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**Abstract**

**Purpose** The control of body composition by dietary nutrients is of the upmost importance for both human and animal physiology. Adult stem cells (aSC) may represent a relevant level of tissue adaptation. The purpose of this study was to determine the impact of macronutrient composition on aSC populations isolated from adipose tissue or muscle in growing pigs.

**Methods** Pigs from two lines divergently selected for feed efficiency were fed ad libitum either a low-fat high-starch (LF) diet or a high-fat high-fiber (HF) diet (n = 6 per line and diet) from 74 to 132 days of age. Stromal-vascular cells were isolated from adipose tissue and muscle and characterized with cell surface markers.

**Results** Pigs fed the HF diet exhibited a reduced adiposity (*P* < 0.001) compared with pigs fed the LF diet whatever the line. In the four groups, CD90 and PDGFRα markers were predominantly expressed in adipose cells, whereas CD90 and CD56 markers were highly expressed in muscle cells. In adipose tissue, the proportion of CD56+/PDGFRα+ cells, and the proportion of CD90+/PDGFRα+ were lower (*P* < 0.05) in HF pigs than in LF pigs. On the opposite, in muscle, these proportions were higher (*P* < 0.001) in HF pigs.

**Conclusion** This study indicates that dietary nutrients affected the relative proportions of aSC populations in pigs, with opposite effects on CD90+/PDGFRα+, CD56+/PDGFRα+ cell populations between muscle and adipose tissue. Therefore, the control of these cells may be a strategy to modulate body composition.

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**Keywords** Adult stem cells. Feed efficiency. Growth. Adipose tissue. Skeletal muscle
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# Introduction

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The control of body composition by nutrition is a topic of importance for both humans and farm animals. In humans, the increase in the prevalence of obesity throughout the world (OECD obesity update 2017; https://www.oecd.org/els/health-systems/Obesity-Update-2017.pdf) highlights the need to prevent and/or treat this pathology that is associated with an increased adiposity and a decrease in muscle mass [1]. In animals, the lean-to-fat ratio and the intramuscular fat content are of special interest for the economy of production and meat quality traits [2]. The development of adipose tissue and skeletal muscle is basically triggered by variations in the number and size of constitutive cells. These cells are recruited from various populations of multipotent mesenchymal stem cells (MSC) resident in tissues, which are also named adult stem cells (aSC) [3]. They are characterized by their ability to self-renew and to differentiate into cell types of different lineages and by the expression of cell surface markers [21].

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In adults, it has been shown that different degrees of adiposity mass are associated with changes in the biology of aSC in lean and fat tissues [4]. For instance, aSC from adipose tissue of obese subjects have impaired differentiation and migration properties compared with aSC from non-obese patients [5,6]. Other studies have described the responses of aSC from skeletal muscle to dietary changes, such as short-term calorie restriction or dietary lipid composition [7,8]. We hypothesized that the responses of aSC populations to genetics and nutritional challenges may differ between tissues.

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# Materials and methods

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## Ethics statement

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The care and use of pigs were performed in compliance with the European Union legislation (directive 2010/63/EU). The protocol was approved by the local Ethics Committee in Animal Experiment of Rennes, France (Comité Rennais d'Ethique en matière d'Expérimentation Animale, CREEA,

http://ethique.ipbs.fr/creeapresent.html; agreement N°R-2012-07). All animals were reared and killed in compliance with national regulations, and according to procedures approved by the French Veterinary Services.

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## Animals, diets and slaughtering

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The present study was part of a larger work whose results related to growth performance, body composition and tissue metabolic pathways have been published [11,12]. In brief, a total of 24 pigs (purebred French Large White barrows) from two genetic lines divergently selected over eight generations of residual feed intake (RFI), a measure of feed efficiency corresponding to the net use of nutrients by tissues [13], were considered as a representative subset (n = 6 per line and per diet) of the full design. From 74 days of age to 132 days of age, pigs were housed individually (full slatted floor) allowing individual feed intake recording (offers minus refusals).

Within each line, pigs were randomly assigned to one of the two dietary groups, and fed ad libitum either a low-fat, low-fiber (LF) diet or a high-fat, high-fiber (HF) diet. The diets were formulated in two formulas distributed during the growing and finishing periods, respectively, and for which nutritional composition (protein content and energy values) followed recommendations for optimal growth during these two periods. The HF and LF diets included similar crude protein contents (17% and 13% for growing and finishing formulas, respectively) and digestible essential amino acids (lysine, methionine and threonine). Starch derived from cereal grains (wheat and barley) in the LF diet was partially replaced by rapeseed and soybean oils in the HF diet and crushed wheat straw (insoluble fiber) was included as a diluent of dietary energy in this diet, so that LF and HF diets were formulated on an isocaloric basis (12.9 MJ/kg metabolizable energy). This strategy resulted in large variations between HF and LF diets in their nutritional composition (as fed basis): respectively, 33.5% vs. 45.5% of starch, 7.2% vs. 2.2% of fat, 18% vs. 12.1% of neutral-detergent fiber and 8.2% vs. 3.6% of acid-detergent fiber.

Detailed diet composition was given in Table 1. Pigs were fed experimental diets during 58.5 ± 0.5 days. All pigs were killed at the same age (132.5 ± 0.5 days) at an average BW of 75.3 ± 1.2 kg. They were euthanized by electronarcosis followed by jugular exsanguination 2 h after their first morning meal, at the INRA experimental facilities (35590 Saint-Gilles, France).
103 Tissue sampling

104 Samples of subcutaneous dorsal adipose tissue (SCAT) and of skeletal muscle (longissimus lumborum) were
105 rapidly excised. Slices of adipose tissue (15 g) were immediately placed in 37°C Krebs-Ringer bicarbonate
106 buffer, whereas slices of muscle (15 g) were placed in ice-cold Dulbecco's Phosphate Buffer Saline (DPBS,
107 Dutscher, Brumath, France) containing 1% glucose (D-Glucose; Sigma, Saint-Quentin Fallavier, France), and
108 further used for cell isolation. Other SCAT and muscle (oriented according to myofiber longitudinal axis)
109 samples (0.5 x 0.5 x 1 cm) were fixed with pins on flat sticks, frozen in 2-methylbutane (isopentane) chilled with
110 liquid nitrogen, and stored at -70°C until histological procedures.

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112 Immunohistochemistry

113 Thick transverse cross-sections (8 µm) of tissues were obtained using a cryostat (2800 FrigocutN, Reichert Jung,
114 Heidelberg, Germany) equilibrated at -30 and -20°C for adipose tissue and muscle, respectively. Sections were
115 mounted on silane-coated slides for enhanced adhesion. Immunocyto-localization of PDGFRα marker was
116 carried out using a standard procedure. Briefly, after incubation in 2% bovine serum albumin (BSA; Sigma,
117 Saint-Quentin Fallavier, France) to prevent non-specific binding, sections were incubated in PBS/0.1% BSA for
118 45 min at room temperature with the following primary mouse antibodies: anti-PDGFRα and anti-laminin
119 complex (Interchim, Montluçon, France). Nuclei were counterstained with 4'-6-diamidino-2-phenylindole
120 (DAPI; Dako, Trappes, France). No significant staining was detected in slides incubated with control serum
121 and/or goat IgG in the absence of the primary antibody. A Nikon DS-Ri1 epifluorescence microscope was used
122 to acquire digital images using an Eclipse E400 digital camera and NIS-Elements software version 3.0 (Nikon).
123 Qualitative and quantitative analyses of stained cells were performed using a self-developed plugin for ImageJ
124 (ImageJ 1.43, National Institutes of Health, Bethesda, MD, USA).

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126 Cells isolation

127 Cells were isolated from adipose tissue by collagenase digestion according to De Clercq et al. [14]. Adipose
tissue was minced and digested in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid phosphate buffer
128 (HEPES, 2 mL/g of tissue) containing 2% BSA and 2 mg/mL collagenase II and XI (800 U/mg; Sigma) in a
129 shaking water bath for 45 min at 37°C. A fivefold excess of collagenase buffer free was added before cells were
130 centrifuged at 400 × g for 10 min to separate floating adipocytes from the pellet of stroma vascular (SV) cells.
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132 After re-suspension in Dulbecco's Modified Eagle Medium (DMEM), SV cells were successively filtered
through a 200- and 25-µm nylon mesh (Nytex, Dutscher Brumath, France). Cells were isolated from skeletal
muscle according to Perruchot et al. [15]. Muscle was digested for one hour in a water bath in a mixture of
trypsin 0.25% (Invitrogen, Cergy Pontoise, France)/Collagenase de type II 1.5 mg (PAA Les Mureaux,
France)/DNAse 0.1% Sigma Saint-Quentin Fallavier, France). After centrifugation, cell pellet was suspended in
DMEM and filtered through a 200 µm Nylon membrane and then a 50 µm Nylon membrane (Nytex, Dutscher
Brumath, France). The SV cell sub-fractions isolated from adipose tissue or skeletal muscle were placed in 90%
Fetal Calf Serum (FCS) and 10% dimethyl sulfoxide (1 to 2 million cells per mL) and frozen at -150°C.

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141 Flow cytometry

142 Viable cells in SV sub-fractions were thawed and counted with the Vi-CellTM XR analyzer using the trypan-blue
143 exclusion test (Beckman Coulter, Paris, France). Cells with viability greater than 90% were further analyzed for
144 cell surface markers using human or porcine cross-reactive antibodies (Table 1). About 800, 000 cells were
145 placed in PBS containing 0.5% FCS and incubated with labeled monoclonal antibodies (mAb) coupled to
146 different fluorochromes [Phycoerythrin-cyanine 7 (PE-Cy7), R-Phycoerythrin (RPE), phycoerythrin (PE),
147 fluorescein isothiocyanate (FITC), Allophycocyanine (APC)] or with a non-labeled anti-PDGFRα antibody
148 which was detected using an appropriate FITC-conjugated secondary antibody. All data were compared to
149 isotype-matched negative controls. Labeled cells were then analyzed using a MACSQuant® flow cytometer and
150 software (Miltenyi Biotec, Paris, France). A minimum of 20,000 events was acquired for each sample.

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152 Cell selection using magnetic microbeads

153 Frozen cells were thawed and were first counted using a Malassez hemocytometer. Cells were then sorted using
154 antibodies against CD56, PDGFRα, and CD45 cell surface markers using magnetic activated cell sorting (MACS
155 ® technology) (Miltenyi Biotec, Bergisch Gladbach, Germany). The cell suspension was first treated with an anti-
156 pig CD45 mouse monoclonal antibody (Table 2). After antibody binding, CD45 positive cells were removed
157 through indirect anti-FITC IgG microbead sorting (Miltenyi Biotec), leaving the CD45- fraction. From this CD45-
158 fraction, the PDGFRα+ or CD56+ cells were enriched through incubation with a polyclonal anti-PDGFRα
159 antibody followed by incubation with an appropriate PE-conjugated secondary antibody or with a monoclonal
160 anti-pig CD56 antibody (Table 2) followed by anti-PE IgG microbead sorting (Miltenyi Biotec) or anti-APC IgG
161 microbead sorting, respectively. Cells were purified using MACS. The purity of the preparation was assessed by
162 flow cytometry.

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164 Cell culture

165 Cells isolated from adipose tissue and muscle were thawed and counted with the Vi-CellTM XR analyzer using
166 the trypan-blue exclusion test (Beckman Coulter). After centrifugation, some cells were maintained in the
167 control medium (DMEM supplemented with 10% FCS, 0.25 µg/mL amphotericin D and 20 µg/mL gentamicin)
168 for 9 days to confirm the absence of spontaneous differentiation. Other cells were placed in a medium allowing
169 adipogenic or myogenic differentiation. For both conditions, cells were seeded at a density of 7 x 10⁴ cells/cm² in
170 six-well plates (9.6 cm²/plate) coated with growth factor reduced Matrigel (1/50 v/v, BD Biosciences, Le Pont de
171 Claix, France) and grown at 37°C under 95% air and 5% CO₂.

172 Adipogenic and myogenic differentiations were assessed as previously described [16]. In adipogenic condition,
173 on the first day of culture (day 1), cells were placed in a medium with 10% swine serum. On day 3 of culture, the
174 medium was switched to 2.5% swine serum. After 5 days of culture, cells achieved 80% confluence and were
175 induced to differentiate in DMEM Glutamax High Glucose supplemented with 25 µM IBMX (3-Isobutyl-1-
176 methylxanthine), 20 µg/mL gentamicin, 0.25 µg/mL fungizone, 8 nM insulin, 1 µM dexamethasone, 10 µM
177 troglitazone, 0.2 nM Triiodothyronine (T3), 10 µg/mL transferrin and 0.1 nM ascorbic acid. Lipid accumulation
178 in cells was estimated using ImageJ (3 wells per animal and 3 random fields per well containing approximately
179 900 cells). Results were expressed as the surface of lipid droplets divided by the total area of cells in the same
180 field. In myogenic condition, the fusion index was estimated at 3 wells per animal and 3 random fields per well
181 and expressed as the number of DAPI stained nuclei inside myotubes that were stained with MF-20 antibody
182 (Developmental Studies Hybridoma Bank, Iowa) when divided by the total number of nuclei in the same field. A
183 Nikon DS-Ri1 epifluorescence microscope was used as previously described for immunohistochemistry.
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185 Statistical analysis

186 Data were analyzed by ANOVA considering the effects of genetic lines (RFI-/RFI+), diet (HF/LF) and their
187 interaction (line x diet) using the R software version 3.0.2 (R Development Core Team, 2008) followed by
188 Turkey's multiple comparison test. Differences were considered significant at *p* ≤ 0.05, whereas 0.05 < *p* < 0.10
189 was considered as a trend.

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191 **Results**

192 **Body composition of investigated pigs**

193 Growth performance and body composition of pigs have been previously described for the full experimental
194 design [12]. Data are summarized for the subset of pigs investigated in the current study (Table 3). Importantly,
195 there was no significant interaction between diet and line on performance and body composition. Irrespective of
196 RFI line, pigs fed a high-fat high-fiber (HF) diet ate less (P = 0.01) than pigs fed the low-fat low-fiber starch-
197 based (LF) during the experimental period. Compared with the LF diet, the HF diet reduced the backfat
198 proportion in the carcass in both lines (P < 0.001) and increased the proportion of lean mass (P < 0.05). The
199 loin/backfat ratio was increased in both lines of pigs receiving the HF diet compared with pigs receiving the LF
200 diet (+25% on average, P < 0.001).

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202 **In situ localization of PDGFRα in adipose tissue and skeletal muscle**

203 In SCAT, immunohistochemical staining with antibodies against PDGFRα and laminin revealed that
204 PDGFRα+ cells were mostly present in the stroma in close vicinity to mature adipocytes and capillaries (Fig.
205 1A). In muscle, PDGFRα+ cells were detected in the perimysium and the interstitial space (endomysium)
206 between muscle fibers but not in the satellite position (Fig. 1B).

207 **Expression of individual cell surface markers in SV cells from SCAT and skeletal muscle**

208 Flow cytometry analysis revealed significant expressions of CD34, CD56, CD90 and PDGFRα markers on the
209 surface of SCAT cells (Fig. 2A). Whereas the proportion of cells expressing the CD34 marker was less than 2%
210 and did not differ between groups, proportions of cells expressing CD56+, CD90+ and PDGFRα+ markers were
211 highly dependent on the groups. The proportion of CD56+ cells was similar between pig lines, but it was lower
212 in HF pigs than in LF pigs (-40% on average, P < 0.001). The proportion of CD90+ cells was lower in RFI+ than
213 in RFI- pigs, but it was not affected by the diet. For PDGFRα+ cells, there was a lie x diet interaction (P =
214 0.001); indeed, the proportion of PDGFRα+ cells was lower in RFI- pigs fed HF diet than in RFI- pigs fed LF
215 diet (P < 0.05), but was not affected by diet in RFI+ pigs.

216 In skeletal muscle, CD56+ and CD90+ cells were the most represented subtypes, with 22-25% and 17-28%
217 of the cells, respectively (Fig. 2B). The proportion of CD56+ cells did not differ between groups. The proportion
218 of CD34+ cells was affected by diet and line with a lower proportion of cells in HF than in LF pigs (-25% on average, P < 0.01) and a greater proportion of cells in RFI- than in RFI+ pigs (P < 0.001). The proportion of CD90+ cells was not affected by the diet and was lower in RFI- pigs than in RFI+ pigs (P < 0.001). Compared with the LF diet, the HF diet increased the proportion of PDGFRα+ in both lines (P < 0.001); it also tended to be lower in RFI+ pigs than in RFI- pigs (P < 0.07)..

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224 **Expression of combined cell surface markers in SV cells from SCAT and skeletal muscle**

225 In SCAT (Fig. 3A), the HF diet reduced the proportion of CD90+/PDGFRα+ cells (P < 0.05) compared with the LF diet. (suppl. Table 1). On the opposite, in muscle (Fig. 3B), the proportions of CD90+/PDGFRα+ cells were increased (P < 0.001) by the HF diet compared with the LF diet. In SCAT, the proportion of CD56+/PDGFRα- cells was lower in HF pigs than in LF pigs (P < 0.05) whereas in muscle, the proportion of CD56+/PDGFRα+ cells was higher (P < 0.001) in HF than in LF pigs.

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231 **Relationships between the percentage of cell populations in SCAT and skeletal muscle**
232 **and tissue weights**

233 Positive correlations (r > 0.50; P < 0.05) were observed between SCAT relative proportion of the body, used as a surrogate of adiposity, and proportions of CD34-/PDGFRα+, CD56+/PDGFRα+ and CD90+/PDGFRα+ cells in SCAR, whereas a negative correlation (P < 0.05) was observed between these cells populations in muscle and the percentage of the loin (Table 4). There was also a positive correlation between loin mass and proportion of CD34+/PDGFRα- cells (P < 0.001) in muscle.

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239 **Adipogenic and myogenic differentiation potentials of sorted cells**

240 Cells selected using the combination of CD56 and PDGFRα markers (Fig. 4) were further analyzed for developmental features in adipogenic or myogenic differentiation media cells isolated from SCAT and placed in myogenic conditions did not exhibit any significant expression of MF-20 as indicative of myotubes fusion. In adipogenic conditions, these cells differentiated into multilocular adipocytes (Fig. 5A); however, the percentage of differentiation reached 72.5 ± 7% for CD56-/PDGFRα+, 26.5 ± 7% for CD56+/PDGFRα+ and was below 5% for CD56+/PDGFRα- cells, respectively.

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246 In muscle, all cells formed long multinucleated myotubes when cultured in a myogenic medium. The fusion
247 index reached 61 ± 5%, 72 ± 6% and 21 ± 4%, for CD56+/PDGFRα+, CD56+/PDGFRα- α and CD56-
248 /PDGFRα+, respectively. In adipogenic conditions, CD56-/PDGFRα+ cells were filled with lipid droplets and
249 42 ± 4% of these cells differentiated into mature multilocular adipocytes (Fig. 5B). The rate of differentiation
250 into adipocytes was lower than 5% for CD56+/PDGFRα+ and CD56+/PDGFRα- cells.

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252

Discussion

253 The current study further documents the diversity of aSC in adipose tissue and skeletal muscle of pigs. It also
254 clearly indicates that the diet-induced changes in body composition are associated with changes in the relative
255 proportions of several aSC populations in adipose tissue and skeletal muscle.

256 Diversity of aSC in adipose tissue and muscle

257 In agreement with our previous work [16], this study shows that cells expressing either CD90, a well-known
258 mesenchymal marker, CD34, an hematopoietic stem cell marker, or CD56, a myogenic marker, were represented
259 at significant levels in both adipose and muscle tissues. It further demonstrates the presence of cells expressing
260 the PDGFRα marker at significant levels in both tissues. PDGFRα is considered as a useful marker to
261 differentiate adipogenic from non-adipogenic cells among interstitial cells with myogenic potential (PW1+
262 interstitial cells) in mice and in human muscle [25, 26]. PDGFRα is also known to be a marker of mesenchymal
263 progenitors distinct from myogenic cells [30, 27]. Among muscle-resident cells, a population of PDGFRα+
264 mesenchymal cells has been identified as progenitors of fibroblasts and adipocytes (fibro/adipogenic progenitors,
265 FAPs) [17,18]. In the current study, cells positive for PDGFRα were more frequent in the perimysium than in
266 the endomysium of muscle, an observation that fitted data reported in humans and mice [17,18]. As reported in
267 white adipose tissue of adult mice [19] PDGFRα+ mesenchymal progenitors were also detected in the stroma of
268 subcutaneous adipose tissue. Indeed, Lee et al. (2012) [20] demonstrated that PDGFRα+ progenitors contributed
269 to white adipogenesis in adipose tissue of mice [20,21]. We further show that the proportions of cell populations
270 expressing PDGFRα and CD56 or PDGFRα and CD90 markers were significant in both tissues as already
271 reported in human muscle [18] and adipose tissue [21] with high expression of CD90+ and PDGFRα+ cells in
272 adipose tissue and high expression of CD90+ and CD56+ cells in skeletal muscle. Importantly, we showed that
273 CD56-/PDGFRα+ cells were mainly adipogenic whatever they originated from adipose tissue and muscle,
whereas they had a capacity for myogenic differentiation only when harvested from muscle. Similarly, CD56+/PDGFRα+ and CD56+/PDGFRα- cells also exhibited a tissue-specific differentiation potential. In agreement with our data, it has been shown that (PDGFRα-/CD56+ cells isolated from human muscle biopsies only differentiated into myotubes whereas PDGFRα+/CD56- cells differentiated into adipocytes or fibroblast-like cells [22]. Moreover, both CD56+/PDGFR- and CD56+/PDGFR+ cells exhibited a high myogenic potential in skeletal muscle of adult human donors [23]. Taken together, we suggest that PDGFRα could be a relevant marker to sort cells with predominant adipogenic properties in adipose tissue, as it has been already demonstrated [24], but cannot be a relevant marker to differentiate adipogenic from myogenic cells in skeletal muscle. Moreover, CD56, a marker of myogenic commitment in skeletal muscle [16,25], appeared as the best marker to discriminate mesenchymal markers from myogenic cells in pig skeletal muscle. However, CD56 cannot be considered as a marker of myogenic commitment in adipose tissue. Indeed, cells positive for CD56also displayed adipogenic capacities in adipose tissue, in agreement with previous observations [17]. Moreover, the presence of CD56+ in adipose cells was not associated with a significant capacity for myogenic differentiation. Other authors have shown that CD56+cells from adipose tissue in adult human were prone to enter in the myogenic program but could not undergo terminal differentiation [32]. Changes in aSC due to diet remain scarce and even more in early growth stages. We provided evidence that the reduction of body weight and fatness induced by the HF diet compared with the LF diet [26] was associated with changes in aSC populations in adipose tissue and muscle of growing pigs. This suggests that aSC may be a relevant target to influence later body composition. With the role of programming events in the etiology of obesity, it is important to determine how early nutrition can affect long-term tissue function and systemic metabolism [25,26]. The dietary-induced changes in the proportion of several cell populations are consistent with studies demonstrating that the composition of the aSC compartment of adipose tissue or skeletal muscle is affected in obese adults [27,28,29]. The current study reveals that two aSC populations, CD56+/PDGFRα+ and CD90+/PDGFRα+ cells, were found to be affected by the diet in both adipose tissue and skeletal muscle but in an opposite manner. In adipose tissue, the proportions of these two populations declined with the decrease in backfat percentage induced by the HF diet. In muscle, their proportions increased with the lean mass. The decreased proportion of these cells in adipose tissue may result from a release of these cells as suggested very recently in mice [25]. These types of cells, as well as their activity undergo a modulation related to the variation of body composition during growth. Indeed, there is a strong link between stem cells, preadipocyte differentiation and hyperplasia. This process gives a protection of adipose tissue even in
304 obesity case. The regulation of hyperplastic adipocytes may exert beneficial effects against adipocyte

305 hypertrophy and subsequent insulin resistance [26]. So we can suggest that in our case hyperplasia is regulated at

306 the stem cell level like it has been previously reported in mice [27].

307 In conclusion, the current study clearly shows the link between nutrition, stem cells and the final body

308 composition of animals. It suggests that these cell populations, specifically cells that have opposite regulation in

309 skeletal muscle and adipose tissue (CD90+/PDGFRα+, CD56+/PDGFRα+), can be targeted to modulate body

310 composition.

311

312
315 1. Hilton TN, Tuttle LJ, Bohnert KL, Mueller MJ, Sinacore DR (2008) Excessive adipose tissue infiltration in skeletal muscle in individuals with obesity, diabetes mellitus, and peripheral neuropathy: association with performance and function. Physical therapy 88 (11):1336-1344.
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325 5. Onate B, Vilahur G, Ferrer-Lorente R, Ybarra J, Diez-Caballero A, Ballesta-Lopez C, Moscatiello F, Herrero J, Badimon L (2012) The subcutaneous adipose tissue reservoir of functionally active stem cells is reduced in obese patients. Faseb J 26 (10):4327-4336. doi:10.1096/fj.12-207217

328 6. Perez LM, Bernal A, de Lucas B, San Martin N, Mastrangelo A, Garcia A, Barbas C, Galvez BG (2015) Altered metabolic and stemness capacity of adipose tissue-derived stem cells from obese mouse and human. PLoS One 10 (4):e0123397. doi:10.1371/journal.pone.0123397

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335 9. Yan H, Potu R, Lu H, Vezzoni de Almeida V, Stewart T, Ragland D, Armstrong A, Adeola O, Nakatsu CH, Ajuwon KM (2013) Dietary fat content and fiber type modulate hind gut microbial community and metabolic markers in the pig. PLoS One 8 (4):e59581. doi:10.1371/journal.pone.0059581

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346 12. Gondret F, Louveau I, Mourot J, Duclos MJ, Lagarrigue S, Gilbert H, van Milgen J (2014) Dietary energy sources affect the partition of body lipids and the hierarchy of energy metabolic pathways in growing pigs differing in feed efficiency. J Anim Sci 92 (11):4865-4877. doi:10.2527/jas.2014-7995

349 13. Gilbert H, Bidanel JP, Gruand J, Caritez JC, Billon Y, Guillouet P, Lagant H, Noblet J, Sellier P (2007) Genetic parameters for residual feed intake in growing pigs, with emphasis on genetic relationships with carcass and meat quality traits. J Anim Sci 85 (12):3182-3188. doi:10.2527/jas.2006-590

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355 15. Perruchot MH, Ecolan P, Sorensen IL, Oksbjerg N, Lefaucheur L (2012) In vitro characterization of proliferation and differentiation of pig satellite cells. Differentiation; research in biological diversity
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364 18. Uezumi A, Fukada S, Yamamoto N, Ikemoto-Uezumi M, Nakatani M, Morita M, Yamaguchi A,
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370 20. Lee YH, Petkova AP, Mottillo EP, Granneman JG (2012) In vivo identification of bipotential
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376 22. Arrighi N, Moratal C, Clement N, Giorgetti-Peraldi S, Peraldi P, Loubat A, Kurzenne JY, Dani C,
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394 doi:10.1038/srep35884

395

396

397 FIGURE LEGENDS

398 FIGURE 1. Representative immunohistochemical pictures showing in situ localization of stroma vascular cells

399 in subcutaneous adipose tissue (A) and skeletal muscle (B) of pigs. Cross-sections were stained in red for

400 PDGFRα cell surface marker with a blue DAPI counterstaining to localize nuclei. An antibody recognizing

401 laminin (green) was also used to stain the basal membrane. In subcutaneous adipose tissue, white arrows
402 indicated stromal and perivascular structures and grey arrows indicate unstained satellite cells in skeletal muscle.

403 Scale bar 20 µm.

404

405 FIGURE 2. Expression of CD34, CD56, CD90 and PDGFRα markers in stromal vascular cells isolated from
406 subcutaneous adipose tissue (SCAT, A) and skeletal muscle (B) of pigs with low (RFI-) or high residual feed
407 intake (RFI+) fed a high-fat, high-fiber (HF) diet and low-fat, low-fiber (LF)

408 After cell isolation, percentages of positive cells for CD34 Phycoerythrin (PE), CD56 Phycoerythrin-Cyanine 7(PE-Cy7), CD90 Allophycocyanin (APC), and PDGFRα Fluorescein isothiocyanate (FITC) were determined
409 by flow cytometry. Percentages of positive cells are expressed as means ± SE (n=5 animals/ group). For each
410 marker, means with different letters differed, P < 0.05.
411

412

413 FIGURE 3. Expression of CD34 Phycoerythrin (PE), CD56 Phycoerythrin-Cyanine 7(PE-Cy7), CD90
414 Allophycocyanin (APC) and PDGFRα Fluorescein isothiocyanate (FITC) markers in stromal vascular cells
415 isolated from subcutaneous adipose tissue (SCAT, A) and skeletal muscle (B) of pigs with low (RFI-) (white
416 bars) or high residual feed intake (RFI+) (black bars) fed a high-fat, high-fiber (HF) diet and low-fat, low-fiber
417 (LF).

418 After cell isolation, percentages of cells expressing phenotypes : CD34+/PDGFRα+, CD90+/PDGFRα+,
419 CD56+/PDGFRα-, CD56+/PDGFRα+, and CD56-/PDGFRα+ of total isolated cells were determined by flow
420 cytometry. Percentages of cell populations are expressed as means ± SE (n=5 animals/ group). For each marker,
421 means with different letters differed, P < 0.05.

422

423 FIGURE 4. Representative flow cytometry analyses of cells isolated from subcutaneous adipose tissue (A)
424 before and (B) after cell sorting for CD56-PE Cy. Representative flow cytometry analyses of cells isolated from
425 skeletal muscle tissue before (C) and after (D) cell sorting for CD56-PE Cy. Each representation is illustrated by
426 the isotype control (up) associated to its corresponding positive staining (down).

427

428 FIGURE 5. Cells isolated from adipose tissue and muscle of RFI- pigs fed the LF diet were considered as
429 controls. (A) Typical morphology of stromal vascular (SV) cells originating from (A) subcutaneous adipose
430 tissue (SCAT) and (B) skeletal muscle of RFI- pigs fed the LF diet after nine days of culture. Cells were cultured
431 for 9 days in adipogenic or myogenic media and day 0 corresponded to cell seeding. Adipogenic differentiation
432 was evaluated with Oil Red O staining and myogenic differentiation with desmin and myosin heavy chain

433 (MF20) immunostaining. Cell morphology was further examined using phase contrast on D9 and arrows indicate

434 lipid droplets. Scale bar 10 µm.

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<table><caption>447 **Table 1** Ingredients, chemical and nutritional compositions of the experimental diets¹</caption><thead><tr><th rowspan="2">Items</th><th colspan="2">Growing</th><th colspan="2">Finishing</th></tr><tr><th>HF</th><th>LF</th><th>HF</th><th>LF</th></tr></thead><tbody><tr><td>Ingredients, g/kg (as fed basis)</td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><tbody><tr><td>Wheat</td><td>269.5</td><td>362.2</td><td>330.9</td><td>417.2</td></tr><tr><td>Barley</td><td>269.5</td><td>362.2</td><td>330.9</td><td>417.2</td></tr><tr><td>Soybean meal</td><td>233.5</td><td>199.9</td><td>116.1</td><td>84.9</td></tr><tr><td>Corn starch</td><td>0</td><td>27.5</td><td>0</td><td>35.7</td></tr><tr><td>Crushed wheat straw</td><td>114.5</td><td>0</td><td>112.6</td><td>0</td></tr><tr><td>Rapeseed oil</td><td>25.1</td><td>3.4</td><td>25.1</td><td>3.5</td></tr><tr><td>Soybean oil</td><td>50.1</td><td>6.8</td><td>50.3</td><td>6.7</td></tr><tr><td>L-Lysine HCl</td><td>2.8</td><td>3.2</td><td>2.9</td><td>3.3</td></tr><tr><td>L-Threonine</td><td>1.17</td><td>1.20</td><td>10.3</td><td>10.5</td></tr><tr><td>L-Tryptophane</td><td>0.28</td><td>0.26</td><td>0.22</td><td>0.19</td></tr><tr><td>DL-Methionine</td><td>1.04</td><td>0.76</td><td>0.46</td><td>0.38</td></tr><tr><td>Salt</td><td>4.5</td><td>4.50</td><td>4.50</td><td>4.50</td></tr><tr><td>Calcium carbonate</td><td>10.5</td><td>10.5</td><td>10.0</td><td>10.0</td></tr><tr><td>Dicalcium phosphate</td><td>12.5</td><td>12.5</td><td>10.0</td><td>10.0</td></tr><tr><td>Vitamins and minerals premix</td><td>5.0</td><td>5.0</td><td>5.0</td><td>5.0</td></tr><tr><td>DM, g/100 g</td><td>88.0</td><td>87.4</td><td>87.2</td><td>86.9</td></tr><tr><td colspan="5">Chemical composition, % (as fed basis)</td></tr><tr><td>Ash</td><td>5.4</td><td>5.0</td><td>4.7</td><td>4.3</td></tr></tbody></table>
<table><tr><td>OM</td><td>82.6</td><td>82.4</td><td>82.5</td><td>82.6</td></tr><tr><td>CP</td><td>17.3</td><td>17.4</td><td>13.1</td><td>13.4</td></tr><tr><td>Fat</td><td>7.0</td><td>2.1</td><td>7.4</td><td>2.2</td></tr><tr><td>Starch</td><td>30.8</td><td>42.6</td><td>36.2</td><td>48.5</td></tr><tr><td>NDF</td><td>17.7</td><td>12.8</td><td>18.3</td><td>11.5</td></tr><tr><td>ADF</td><td>8.5</td><td>3.8</td><td>7.9</td><td>3.5</td></tr><tr><td>GE, MJ/kg</td><td>17.5</td><td>15.9</td><td>17.3</td><td>15.8</td></tr></table>

Nutritional values

<table><tr><td>ME, MJ/kg</td><td>12.9</td><td>12.9</td><td>12.9</td><td>12.9</td></tr><tr><td>NE, MJ/kg</td><td>9.8</td><td>9.6</td><td>10.0</td><td>9.9</td></tr><tr><td>Digestible lysine, g/kg</td><td>9.5</td><td>9.5</td><td>7.0</td><td>7.0</td></tr><tr><td>Digestible threonine, g/kg</td><td>0.62</td><td>0.62</td><td>0.46</td><td>0.46</td></tr><tr><td>Digestible methionine, g/kg</td><td>0.32</td><td>0.30</td><td>0.21</td><td>0.21</td></tr></table>

Fatty acid (FA) composition, % extracted FA

<table><tr><td>C18:2 n-6</td><td>41.0</td><td>45.3</td><td>41.6</td><td>44.6</td></tr><tr><td>C18:3 n-3</td><td>5.6</td><td>5.0</td><td>5.7</td><td>4.7</td></tr><tr><td>Saturated FA</td><td>13.9</td><td>18.5</td><td>13.8</td><td>19.0</td></tr><tr><td>Mono-unsaturated FA</td><td>36.2</td><td>27.0</td><td>36.4</td><td>25.9</td></tr></table>
<table><tr><td>Poly-unsaturated FA</td><td>54.6</td><td>49.8</td><td>50.0</td><td>55.1</td></tr><tr><td>Ratio n-6: n-3</td><td>5.0</td><td>5.3</td><td>5.4</td><td>4.8</td></tr></table>

¹A low fat low fiber diet (LF) and a high fat high fiber diet (HF) were formulated at the same protein and metabolizable energy (ME) contents. For each dietary group, a growing diet was distributed to pigs from 76 d of age onwards for 6 weeks, and a finishing diet which differed for the protein and amino-acid contents, was distributed thereafter and until slaughter.

<caption>**Table 2** List of antibodies used for flow cytometry (FACS) analysis or immunohistochemistry</caption>
<table><thead><tr><th>Marker</th><th>Isotype</th><th>Fluorochrome¹</th><th>Clone</th><th>Supplier</th><th></th></tr></thead><tbody><tr><td>CD11b</td><td>mouse IgG1</td><td>PE-Cy7</td><td>ICRF44</td><td>Biolegend</td><td>466</td></tr><tr><td>CD14</td><td>mouse IgG2a</td><td>RPE-alexa647</td><td>TUK4</td><td>AbD sérotec</td><td>467</td></tr><tr><td>CD31</td><td>mouse IgG1</td><td>RPE</td><td>LCI-4</td><td>AbD sérotec</td><td>468</td></tr><tr><td>CD34</td><td>mouse IgG1</td><td>PE</td><td>563</td><td>BD Biosciences</td><td>469</td></tr><tr><td>CD45</td><td>mouse IgG1</td><td>FITC</td><td>K252-1E4</td><td>AbD sérotec</td><td>470</td></tr><tr><td>CD56</td><td>mouse IgG2b</td><td>PE-Cy7</td><td>NCAM 16.2</td><td>BD Biosciences</td><td>471</td></tr><tr><td>CD56</td><td>mouse IgG2b</td><td>APC</td><td>NCAM 16.2</td><td>BD Biosciences</td><td>472</td></tr><tr><td>CD90</td><td>mouse IgG1</td><td>APC</td><td>5E10</td><td>BD Biosciences</td><td>473</td></tr><tr><td>PDGFRα</td><td>Goat IgG</td><td>Unconjugated</td><td>Polyclonal</td><td>Santa Cruz</td><td>474</td></tr><tr><td>Laminin</td><td>Rabbit</td><td>Unconjugated</td><td>Polyclonal</td><td>Sigma</td><td>475</td></tr><tr><td colspan="5"></td><td>476</td></tr></tbody></table>

¹ Fluorochromes for FACS analysis, APC,

Allophycocyanine, BD, Becton Dickinson; CD, Cluster of Differentiation; Cy7, Cyanine 7; FITC, fluorescein isothiocyanate; IgG, Immunoglobulin G; PE, PhycoErythrin; RPE, R-PhycoErythrin.
482

**Table 3** Performance of pigs with low (RFI-) or high residual feed intake (RFI+) fed a low-fat, high-fat, high-fiber (HF) or low-fiber (LF) diet

483

484

<table><thead><tr><th rowspan="2">Diet</th><th colspan="2">HF</th><th colspan="2">LF</th><th colspan="3">Effects</th></tr><tr><th>RFI-</th><th>RFI+</th><th>RFI-</th><th>RFI+</th><th>RSD</th><th>Diet</th><th>Line</th></tr></thead><tbody><tr><td>Genotype</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>n</td><td>6</td><td>6</td><td>6</td><td>6</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Final live weight, kg</td><td>70.6</td><td>69.7</td><td>81.7</td><td>69.2</td><td>4.2</td><td>&lt;0.001</td><td>0.007</td></tr><tr><td>ADG, g/d</td><td>826.6</td><td>739.5</td><td>990.9</td><td>856.4</td><td>71</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>ADFI, g/d</td><td>2134</td><td>2121</td><td>2550</td><td>2373</td><td>602</td><td>0.01</td><td>0.01</td></tr><tr><td colspan="8">Composition, % of chilled carcass weight</td></tr><tr><td>Backfat</td><td>5.7</td><td>6.2</td><td>8.0</td><td>8.5</td><td>0.85</td><td>&lt;0.001</td><td>0.51</td></tr><tr><td>Loin</td><td>28.7</td><td>28.3</td><td>28.3</td><td>27.6</td><td>0.41</td><td>&lt;0.05</td><td>0.07</td></tr><tr><td>Loin/Backfat</td><td>4.83</td><td>4.85</td><td>3.88</td><td>3.42</td><td>0.8</td><td>&lt;0.001</td><td>0.57</td></tr></tbody></table>

485

¹Value are least squares means for the effects of line and diet. There was no interaction between RFI line and diet for any analyzed traits. RSD, Relative Standard Deviation.

486

487

²There was no interaction (P > 0.10) between genetic line (high or low residual feed intake) and diet (HF: high fiber high fat; LF: low fiber low fat) for any analyzed traits. Lsmeans for the effects of Line and of Diet are shown. Average daily gain (ADG), average daily feed intake (ADFI) of growing pigs.

488

489

490
Table 4 Correlations between the percentage of cell populations in subcutaneous adipose tissue (SCAT) relative to the backfat weight, and between the cell populations in skeletal muscle and the loin weight.

<table><thead><tr><th rowspan="2">Cell populations</th><th colspan="2">Backfat weight</th><th colspan="2">Loin weight</th></tr><tr><th>r<sup>a</sup></th><th>P value</th><th>r<sup>a</sup></th><th>P value</th></tr></thead><tbody><tr><td>CD34+/PDGFRα-</td><td>-0.37</td><td>0.10</td><td>0.69</td><td>&lt;0.001</td></tr><tr><td>CD34-/PDGFRα+</td><td>0.53</td><td>&lt;0.05</td><td>-0.68</td><td>&lt;0.05</td></tr><tr><td>CD56+/PDGFRα+</td><td>0.59</td><td>&lt;0.05</td><td>-0.55</td><td>&lt;0.05</td></tr><tr><td>CD90+/PDGFRα+</td><td>0.62</td><td>&lt;0.05</td><td>-0.57</td><td>&lt;0.05</td></tr></tbody></table>

rª square root of R² obtained for linear regression analysis

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504

505
<table><caption>**Suppl Table 1** Percentage of cell populations in subcutaneous adipose tissue (SCAT) and skeletal muscle of pigs with low (RFI-) or high residual feed intake (RFI+) fed a low-fat, low-fiber (LF) or high-fat, high-fiber (HF) diet.</caption><thead><tr><th rowspan="2"></th><th colspan="2">HF</th><th colspan="2">LF</th><th rowspan="2">RSD</th><th rowspan="2">Diet</th><th rowspan="2">Line</th><th rowspan="2">Diet * Line</th></tr><tr><th>RFI-</th><th>RFI+</th><th>RFI-</th><th>RFI+</th></tr></thead><tbody><tr><td colspan="9"><strong>SCAT</strong></td></tr><tr><td>CD34+/PDGFRα-</td><td>0.33</td><td>0.60</td><td>0.10</td><td>0.19</td><td>0.08</td><td>0.09</td><td>0.47</td><td>0.17</td></tr><tr><td>CD34+/PDGFRα+</td><td>0.86</td><td>0.80</td><td>0.65</td><td>0.83</td><td>0.09</td><td>0.18</td><td>0.41</td><td>0.28</td></tr><tr><td>CD34-/PDGFRα+</td><td>21.8</td><td>29.4</td><td>32.8</td><td>31.1</td><td>4.61</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>CD56+/PDGFRα-</td><td>0.5</td><td>0.6</td><td>0.4</td><td>0.4</td><td>0.11</td><td>0.33</td><td>0.22</td><td>0.86</td></tr><tr><td>CD56+/PDGFRα+</td><td>7.0</td><td>8.9</td><td>14.4</td><td>12.6</td><td>6.73</td><td>&lt;0.001</td><td>0.96</td><td>0.13</td></tr><tr><td>CD56-/PDGFRα+</td><td>15.6</td><td>21.4</td><td>17.8</td><td>19.4</td><td>13.94</td><td>0.95</td><td>&lt;0.05</td><td>0.23</td></tr><tr><td>CD90+/PDGFRα-</td><td>16.1</td><td>8.3</td><td>6.1</td><td>4.3</td><td>21.86</td><td>&lt;0.05</td><td>&lt;0.05</td><td>0.18</td></tr><tr><td>CD90+/PDGFRα+</td><td>19.7</td><td>21.2</td><td>23.8</td><td>26.8</td><td>16.97</td><td>&lt;0.05</td><td>0.24</td><td>0.70</td></tr><tr><td>CD90-/PDGFRα+</td><td>2.9</td><td>9.0</td><td>8.4</td><td>5.2</td><td>14.49</td><td>0.64</td><td>0.42</td><td>&lt;0.05</td></tr><tr><td colspan="9"><strong>Muscle</strong></td></tr><tr><td>CD34+/PDGFRα-</td><td>4.0</td><td>1.4</td><td>11.0</td><td>2.7</td><td>4.92</td><td>&lt;0.05</td><td>&lt;0.05</td><td>0.05</td></tr><tr><td>CD34+/PDGFRα+</td><td>8.4</td><td>5.0</td><td>6.7</td><td>5.5</td><td>2.37</td><td>0.34</td><td>&lt;0.05</td><td>0.11</td></tr><tr><td>CD34-/PDGFRα+</td><td>4.6</td><td>5.6</td><td>1.7</td><td>1.0</td><td>2.31</td><td>&lt;0.05</td><td>0.37</td><td>0.61</td></tr><tr><td>CD56+/PDGFRα-</td><td>12</td><td>15.09</td><td>18.9</td><td>17.9</td><td>12.33</td><td>&lt;0.05</td><td>0.54</td><td>0.22</td></tr><tr><td>CD56+/PDGFRα+</td><td>11.2</td><td>8.4</td><td>6.4</td><td>6.4</td><td>2.36</td><td>&lt;0.001</td><td>0.06</td><td>0.57</td></tr><tr><td>CD56-/PDGFRα+</td><td>1.8</td><td>2.2</td><td>1.7</td><td>1.0</td><td>0.39</td><td>&lt;0.05</td><td>0.67</td><td>0.06</td></tr><tr><td>CD90+/PDGFRα-</td><td>9.6</td><td>20.0</td><td>8.4</td><td>21.3</td><td>13.94</td><td>0.26</td><td>&lt;0.001</td><td>0.70</td></tr><tr><td>CD90+/PDGFRα+</td><td>11.3</td><td>9.9</td><td>7.5</td><td>6.9</td><td>2.05</td><td>&lt;0.001</td><td>0.15</td><td>0.57</td></tr><tr><td>CD90-/PDGFRα+</td><td>0.48</td><td>1.70</td><td>0.6</td><td>0.5</td><td>0.7</td><td>&lt;0.05</td><td>0.11</td><td>0.17</td></tr></tbody></table>

<footnote n="1">¹Values are means expressed as a percentage of cell phenotyping (n=6 animals/age group).</footnote>
A **Subcutaneous adipose tissue**

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

B

<figure><img src="image_4.png" /></figure>

Laminin

<figure><img src="image_5.png" /></figure>

DAPI

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

PDGFRα

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

Laminin

PDGFRα

DAPI

<figure><img src="image_10.png" /></figure>

FIGURE 1
<figure><img src="image_11.png" /></figure>

<table><thead><tr><th rowspan="2">Marker</th><th colspan="3">Subcutaneous adipose tissue</th><th colspan="3">Skeletal muscle</th></tr><tr><th>Diet</th><th>Line</th><th>Diet X Line</th><th>Diet</th><th>Line</th><th>Diet X Line</th></tr></thead><tbody><tr><td>CD34</td><td>0.26</td><td>0.19</td><td>0.78</td><td>0.003</td><td>P &lt; 0.0001</td><td>0.24</td></tr><tr><td>CD56</td><td>0.0003</td><td>0.96</td><td>0.13</td><td>0.33</td><td>0.79</td><td>0.68</td></tr><tr><td>CD90</td><td>0.18</td><td>0.02</td><td>0.14</td><td>0.45</td><td>P &lt; 0.0001</td><td>0.88</td></tr><tr><td>PDGFRα</td><td>P &lt; 0.0001</td><td>0.001</td><td>0.001</td><td>0.0001</td><td>0.07</td><td>0.30</td></tr></tbody></table>

FIGURE 2
A. Subcutaneous adipose tissue

<figure><img src="image_12.png" /></figure>

B. Skeletal muscle

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>FIGURE 3</figcaption></figure>
A

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

B

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

C

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

D

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

FIGURE 4
<figure><img src="image_27.png" /><figcaption>FIGURE 5</figcaption></figure>