Characterization of adipocytes derived from fibro/adipogenic progenitors resident in human skeletal muscle

Abstract

A population of fibro/adipogenic but non-myogenic progenitors located between skeletal muscle fibers was recently discovered. The aim of this study was to determine the extent to which these progenitors differentiate into fully functional adipocytes. The characterization of muscle progenitor-derived adipocytes is a central issue in understanding muscle homeostasis. They are considered as being the cellular origin of intermuscular adipose tissue that develops in several pathophysiological situations. Here fibro/adipogenic progenitors were isolated from a panel of 15 human muscle biopsies on the basis of the specific cell-surface immunophenotype CD15+/PDGFRα+CD56-. This allowed investigations of their differentiation into adipocytes and the cellular functions of terminally differentiated adipocytes. Adipogenic differentiation was found to be regulated by the same effectors as those regulating differentiation of progenitors derived from white subcutaneous adipose tissue. Similarly, basic adipocyte functions, such as triglyceride synthesis and lipolysis occurred at levels similar to those observed with subcutaneous adipose tissue progenitor-derived adipocytes. However, muscle progenitor-derived adipocytes were found to be insensitive to insulin-induced glucose uptake, in association with the impairment of phosphorylation of key insulin-signaling effectors. Our findings indicate that muscle adipogenic progenitors give rise to bona fide white adipocytes that have the unexpected feature of being insulin-resistant.

Figure 1

CD15 and PDGFRα are specific markers of human FAPs. (a) Representative results from flow cytometry analysis of adherent human muscle cells. The CD56 negative cells (left upper panel) were labeled with both anti-CD15 and anti-PDGFRα (CD140a; right upper panel). The lower panels represent the respective isotype negative labelings. Percentages of the fractions are indicated. (b) Kinetic expression of PDGFRα and CD15 shown for a representative muscle biopsy (FAP biopsy 7). PDGFRα expression was measured by quantitative RT-PCR and CD15 expression was measured by flow cytometry. At day D0, the cells were confluent and proliferation medium was replaced by differentiation medium. Measurements were done every 2 days until day D8. (c) After confluence (D0), cells were put under differentiation conditions. Myotubes were labeled with antimyosin heavy-chain antibodies and muscle creatine kinase (MCK) expression was compared with quantitative RT-PCR between days D0 and D5. Adipocytes were stained with oil red O and FABP4 expression was compared with quantitative RT-PCR between days D0 and D11. Fibroblast-like cells were labeled with anti-α smooth muscle actin and its expression was compared with quantitative RT-PCR between days D0 and D5. Quantitative RT-PCR results are mean±S.E. of the mean (n=3; FAP biopsies 2, 9, and 10). **P<0.01. FITC, fluorescein isothiocyanate; PE, phycoerythrin; SSC, side scatter detector

Figure 2

Adipogenic differentiation. (a) FAPs and ASCs were grown to confluence in adjacent culture wells and then treated with differentiation-inducing medium. Cells were fixed and assayed for intracellular lipid droplets with oil red O staining from day 0 (start of differentiation induction) to day 30, as indicated. Cell cultures were counterstained with crystal violet. Pictures were visualized by light microscopy with × 200 magnification. Fields representative of whole cell culture wells are shown and were obtained with ASC biopsy 4 and FAP biopsy 6. Scale bar=50 μm. (b) Adipocyte and lipid droplet sizes (μm²) were analyzed using ImageJ software in cultures after 2 weeks of adipogenic differentiation. Lipid droplet quantity was estimated by the intracellular droplet number counted per adipocyte. ASC-As are represented in gray bars (n=3; ASCs biopsies 1, 3, and 4) and FAP-As in black bars (n=3; FAP biopsies 4, 6, and 7). Results are mean±S.E. of the mean for three independent measurements. *P<0.05, **P<0.01. (c) FAP-As mRNA content of classical early (PPARγ and CEBPβ) and late (FABP4, CD36, adipsin, adiponectin, leptin, and LPL) adipogenic markers was quantified by quantitative RT-PCR. The results are expressed as ratios (%) to respective ASC-As mRNA contents, which are represented by the horizontal line. FAP-As (n=3; FAP biopsies 4, 6, and 7) and ASC-As (n=3; ASC biopsies 1, 2, and 4) were strictly maintained under the same culture conditions. Results are mean±S.E. of the mean for three independent ratio measurements *P<0.05. (d) UCP1 expression was measured with quantitative RT-PCR in FAP-As, which were prepared from a panel of biopsies (n=10; FAP biopsies 1, 4, 6, 7, 8, 9, 11, 12, 14, and 15) in comparison with ASCs-As from biopsies 1, 3, and 4. *P<0.05, **P<0.01

Figure 3

Triglyceride synthesis, storage, and lysis. Biochemical activities were measured in FAP-As and ASC-As prepared from age-matching donors (<9 years). (a) Triglyceride synthesis was assessed with GPDH activity. (b) Lipolysis was assayed by glycerol released. (c) Lipid storage was estimated through triglyceride content measurement. ASC-As values are represented with gray bars (n=3; ASC biopsies 1, 2, and 4), FAP-As values in black bars (n=3; FAP biopsies 4, 6, and 7). Results are mean±S.E. of the mean for three independent measurements. No significant differences were found between ASC-As and FAP-As

Figure 4

Impaired glucose transport and alterations in insulin signaling. (a) Insulin-stimulated glucose transport was determined using nonmetabolizable [³H]2-deoxy-d-glucose. The results are expressed as a percentage of glucose transport measured in the absence of insulin separately for ASC-As and FAP-As. ASC-As values are represented with gray bars (n=4; ASC biopsies 1, 2, 3, and 4) and FAP-As values with black bars (n=8; FAP biopsies 3, 4, 5, 6, 7, 13, 14, and 15). (b) mRNA relative expression of Glut4 and IR measured by quantitative RT-PCR. n=3 for ASC-As (ASC biopsies 1, 3, and 4) and FAP-As (FAP biopsies 4, 6, and 7). The results are mean±S.E. of the mean for three independent measurements. (c) Phosphorylations were compared in adipocytes without insulin treatment and with 100 nM insulin stimulation, for 20 min. Cell lysates were subjected to immunoblotting with specific antibodies. Representative blots are shown. (d) Immunoblot signals were quantified and normalized with tubulin signals. Phosphorylated-form contents are expressed as fold change induced by insulin relative to the absence of insulin treatment. The phosphorylations concern IR tyrosines, IRS-1 tyrosines, Akt-Thr³⁰⁸, 42 MAPK-Thr²⁰², and 44 MAPK-Tyr²⁰⁴. ASC-As values (n=3; ASC biopsies 1, 2, and 4) are represented with gray bars and FAP-As values in black bars (n=6; FAP biopsies 1, 2, 8, 9, 10, and 12; except for 42 and 44 MAPK where n=4; FAP biopsies 1, 2, 8, and 9). Data are expressed as mean±S.E. of the mean. (e and f) Relative mRNA contents of PTP1B and SHP2 were measured by quantitative RT-PCR and expressed as a percentage of values found in ASC-As. For ASC-As, n=5 (ASC biopsies 1, 2, 3, 4, and 5); for FAP-As, n=7 (FAP biopsies 2, 4, 6, 8, 9, 10, and 12). The results are mean±S.E. of the mean for three independent measurements. *P<0.05 and **P<0.01