Metabolic Rescue of Obese Adipose-Derived Stem Cells by Lin28/Let7 Pathway

Glucose and lipid metabolism during ASC differentiation. A: Insulin-stimulated glucose uptake by cASC and oASC cultures at different stages of differentiation. Cells were stimulated with 10 nmol/L insulin (Ins) for 30 min. Results are expressed as pmol/mg protein (prot) per 10 min (n = 6), *P < 0.01. B: GLUT4 expression in 7-day differentiated cASCs and oASCs. Cells were stimulated for 20 min with 10 nmol/L insulin and lysed or fractioned. Plasma and internal membrane proteins, together with whole lysates, were analyzed by Western blot with anti–GLUT4 antibody; anti-caveolin (Cav)1, anti-PI3K, or anti-tubulin antibodies were used as controls. Densitometric bar graph quantification of three independent membranes is shown. O.D., optical density. *P < 0.05. C: Glycerol release from 7-day differentiated cASCs and oASCs. Cells were treated with 10 nmol/L insulin for 1 h before stimulation with 1 μmol/L isoproterenol (Iso) for 15 min (n = 6). *P < 0.03. D: NEFA release in 7-day differentiated ASCs treated as in C (n = 5). *P < 0.05. E: Activation of IRS1: 7-day differentiated ASCs were stimulated with 10 nmol/L insulin for 10 min and lysed. Total protein was analyzed by Western blot with anti-phospho- and anti-total IRS1, together with antitubulin antibodies. Densitometric bar graph quantification of three independent membranes is shown. *P < 0.05.

The altered cytokine profile of oASCs transfers insulin resistance to cASC-derived adipocytes. A–C: cASCs and oASCs were differentiated for up to 7 days and cultured overnight in serum-free medium. Adiponectin, MPC-1, and TNF-α content were measured in conditioned medium (pg/mL) (n = 4). *P < 0.01. D and E: Effect of adipocyte-conditioned medium on insulin-stimulated glucose uptake. cASC- or oASC-derived 7-day adipocytes were cultured in the presence of conditioned medium from cCM or oCM for 24 h (n = 8). Cells were then maintained for 2 h in serum-free, low-glucose medium before stimulating with insulin (100 nmol/L, 30 min). Data are expressed as the percentage of the induced glucose uptake (stimulated – basal) in nontreated cells at 7-day differentiation (n = 8). *P < 0.01. F: oASC- and cASC-derived 7-day adipocytes were pretreated (24 h) with cCM or oCM of day 7, stimulated with 100 nmol/L Ins for 5 min, and lysates analyzed by Western blot with antibodies against phosphorylated and total IRS1. Densitometric bar graph quantification of three independent membranes is shown. O.D., optical density. *P < 0.05. G: Differentiation into mature adipocytes by cASC in the presence or absence of 1 ng/mL TNF-α or 0.2 ng/mL MCP-1 cytokines and by oASC in the presence or absence of 10 ng/mL anti–TNF-α or anti–MCP-1 antibodies. Oil Red O quantification of five independent experiments is shown. H: Insulin-stimulated glucose uptake by cASC-derived 7-day adipocytes in the presence or absence of 1 ng/mL TNF-α or 0.2 ng/mL MCP-1 cytokines and by oASC-derived 7-day adipocytes in the presence or absence of 10 ng/mL anti–TNF-α or anti–MCP-1 antibodies. Cells were stimulated with 10 nmol/L insulin (Ins) for 30 min. Results are expressed as pmol glucose/mg protein (prot)/10 min (n = 5). *P < 0.03, +P < 0.05. I: Gene expression profile at day 7 of differentiation of adipocytes derived from cASCs in the presence or absence of 1 ng/mL TNF-α or 0.2 ng/mL MCP-1 cytokines or from oASCs in the presence or absence of 10 ng/mL anti–TNF-α or anti–MCP-1 antibodies (Abs) (n = 5). *P < 0.04. Ctrl, control; Densi, densitometric; d, days.

Isolation and delivery of ASC subcellular fractions. A: Scheme for the isolation of cASC subcellular fractions and their transfer into oASCs. B: NFs and CFs were analyzed by agarose gel electrophoresis and Western blot for GADPH (cytosol marker) and SP1 (nuclear marker). A representative of three experiments is shown. C: Representative images of oASCs after transfer of NF or CF, called mASC, are shown (n = 8). Bar, 50 μm. D: Light microscopy and Oil Red O staining in adipocyte cultures differentiated (7 days) from oASCs and mASCs (CF transferred). Representative images are shown (n = 8). Bar, 30 μm. E: Gene expression profile after delivering of mASC (n = 5). *P < 0.02. F: Cell number (left) and cell size (right) analyzed by flow cytometry in oASCs and mASCs (n = 5).

Transfer of cASC cytosol to oASCs restores nonobese metabolic parameters. A: Insulin-stimulated glucose uptake by adipocytes differentiated (7 days) from cASCs, oASCs, and mASCs. Adipocyte cultures were maintained overnight in serum-free, low-glucose medium and then stimulated with 10 nmol/L insulin (30 min; n = 6). *P < 0.01 between mASCs and oASCs. B and C: GLUT4 expression and IRS phosphorylation are restored in 7-day differentiated mASCs. cASCs, oASCs, and mASCs were stimulated with 10 nmol/L insulin for 10 min, and membrane fractions or cell lysates were probed by Western blot with anti–GLUT4 or anti-IRS1 antibodies. Densitometric quantification of three independent membranes is shown. *P < 0.03. D: Adiponectin, MCP-1, and TNF-α production by oASCs and mASCs. Cells were cultured overnight in serum-free medium, and adiponectin, MCP-1, and TNF-α were detected by immunoassay. Results are expressed as the percentage of detection comparing undifferentiated ASC at day 0 to differentiated ASC at day 7 (n = 6). *P < 0.02 between mASCs and oASCs. Densi, densitometric; Ins, insulin; prot, protein.

Analysis of the CF content and its action mechanism. A and B: CFs, inactivated by proteinase-k (CFp) or by RNase cocktail (CFr), were delivered into oASC. Glucose uptake and the cell size-to-cell number ratio were measured in mASCs (n = 5). *P < 0.05. C: Analysis of Lin28 protein expression by Western blot in oASC-derived or cASC-derived CFs or in whole ASC lysates to note the restoration of Lin28 levels after cASC-derived CF (cCF) delivery into oASC (mASC). One representative image of three independent experiments is shown. D: Let7 microRNA expression was analyzed by RT-PCR (n = 5). *P < 0.04. E: Insulin-stimulated glucose uptake by adipocytes differentiated (7 days) from modified oASCs or oASC delivered with Lin28 protein. Adipocyte cultures were maintained overnight in serum-free, low-glucose medium and then stimulated with 10 nmol/L insulin (30 min; n = 5). *P < 0.03 between oASCs and mASCs or oASCs+Lin28. F: Representative images of Oil Red O–stained oASCs and oASC delivered with Lin28 protein after 7 days of differentiation into mature adipocytes (n = 5). Bar, 30 μm. G: Adiponectin, MCP-1, and TNF-α production by oASC-derived adipocytes (7 days) transferred or not with Lin28 and detected by immunoassay. Results are expressed as the percentage of detection comparing undifferentiated oASCs at day 0 to differentiated oASCs at day 7 (n = 5). *P < 0.02. H: Let7 microRNA and Lin28 expression were analyzed by RT-PCR in cASC-derived adipocytes (7 days) in the presence or absence of 1 ng/mL TNF-α or 0.2 ng/mL MCP-1 cytokines and in oASC-derived adipocytes in the presence or absence of 10 ng/mL anti–TNF-α or anti–MCP-1 antibodies (Abs) (n = 5). *P < 0.05 and **P < 0.02. d, days; Fibrob, fibroblast.