Christian Dani Team “Stem Cells and Differentiation” Faculty of Medicine, iBV, CNRS/Inserm, Nice, France Human Adipocyte Progenitors: Proliferation and Differentiation Diabetic Medications Las Vegas USA

The Energy Intake and Energy Expenditure Balance Obesity Type 2 Diabetes … Factors regulating adipocyte generation in an obese context?

Stromal-vascular fraction Endothelial cells, immune cells, preadipocytes, multipotent stem cells (adipocyte progenitors, adipose-derived stem cells). Human Adipose Stem Cells hMADS Cells (Self-renewal) Collagenase A digestion Adipocyte fraction Adipocytes Chondrocytes Osteoblasts Brown AdipocytesWhite Adipocytes Therapeutic Potential: mdx mice (Rodriguez et al. 2005, J Ex Med)

Stromal fraction of human adipose tissue Adherent cells plated at clonal density Efficiency of colony formation: 12% No adipocyte/osteoblast 24% Osteoblasts only 4% Adipocytes only 24% Adipocytes+Osteoblasts 48% Human Adipose Stem Cells Adipocyte+Osteoblast differentiations

Adipose Stem Cells miRNA 30, C/EBPs, PPAR ,.. Adipose Stem Cells Adipocytes Osteoblats

Role of miRNA 30 in Commitment of Adipose Stem Cells towards the Adipogenic Lineage

Differential Expression of miR30 in Adipogenesis and Osteogenesis Day 8 Adipo Day 8 Osteo

Anti-miR control Anti-miR 30 Effects of miR30 Knock-down on Adipocyte Differentiation

Effects of miR30 knock-down on Adipogenic/Osteogenic Differentiation Anti miR control Anti-miR control Anti-miR 30

PremiR ctr PremiR 30a PremiR 30d Runx2 Tubulin Expression of RunX2 in Adipocyte Progenitors overexpressing miR30

RunX2  actin AdipogenesisOsteogenesis PPAR  Days Expression of Runx2 During Adipocyte Progenitor Differentiation miR30

Adipose Stem Cells miRNA 30, C/EBPs, PPAR ,.. Adipose Stem Cells Adipocytes ? Osteoblats

Adipocyte Progenitor Proliferation is Required for Adipocyte Renewal Growth of white adipose tissue in obesity is the result of an increase in number adipocyte number. Given that adipocytes do not divide, this increase depends on the proliferation of a pool of adipocyte progenitors. A pool of adipocyte progenitors remains present in adipose tissue during adult life. This pool is responsible for regeneration of adipocytes and the potential of this tissue to expand in response to chronic energy excess. However, factors controlling proliferation of human adipocyte progenitors in obesity are largely unknown. Identification of regulators of self-renewal could provide a means to understand the mechanisms that lead to obesity and type 2 diabetes.

Regulators of human adipocyte progenitor proliferation : FGF2, Activin A and IER3.

Regulators of hMADS Cell Proliferation Control FGF2, Activin A We analyzed the transcriptome of hMADS cells and we screened for genes differentially expressed in undifferentiated and differentiated cells: FGF2, Activin A, IER3

FGF2

Expression of FGF2 and its receptor in undifferentiated hMADS cells FGFR-1 FGFR-3 FGFR-2 FGFR-4  -Actin Secretion of FGF fg FGF2/cell Hypothesis : autocrine FGF signalling participates in proliferation Tubulin FGF2 WB RT-PCR

Effect of FGF2 on hMADS cell Propagation number of passages doubling time (days) + FGF2 (2 ng/ml) No effect of: EGF, PDGF, FGF10 Tubulin FGF2

Inhibition of FGF signalling during proliferation Impairs subsequent adipocyte differentiation Control PD a specific inhibitor of FGFR

FGF2 Autocrine/Paracrine Loop Regulating Proliferation and Differentiation of human Adipose-derived Stem Cells FGF2 ERK1/2 Proliferation, Adipogenesis FGFR1 Human adipose tissue is a source of FGF ( Mapping of the fibroblast growth factors in human white adipose tissue. Mejhert et al. J Clin Endocrinol Metab 2010). Excess of FGF2 in adipose tissue of obese people could contribute to increase the size of adipose precursor pool. Factors regulating FGF2 expression?

Activin A

d0d+3 Activin A secreted levels compared to d-3 d+15 Expression of Activin A during Human Adipocyte Differentiation Expression in hMADS cells Days relative to adipogenic induction INHBA expression (A.U) Progenitors Adip. Expression in Adipose Tissue

Activin A Promotes Proliferation and Inhibits Adipocyte Differentiation Number of cells per well (x1000) Control * Activin A  -Activin A * Activin A : 0 Activin A: 100 ng/ml

38 kD CEBP  Tubulin - + Activin A Log2 C/EBP  C/EBP  PPAR  SREBP GPDH FABP4 FABP5 GLUT4 CD36 LPL FASN HSL PEPCK UCP2 PAI1 IL6 QPCR GFP CEBP  FABP4 tubulin Suppression of Anti-Adipogenic Effects of Activin A by C/EBP  Activin A

Differentiation Activin A ActRII ActRI Proliferation Smad2 C/EBP  Activin A is a Critical Regulator of Human Adipocyte Progenitor Proliferation and Differentiation (Zaragosi et al. Diabetes, 2010) EX

Activin A, FGF2 Lean Subjects Obese Patients Macrophages Lean * Activin A expression (A.U.) Obese Activin A, FGF2

CD14+ Macrophages Conditioned Media (ATM-CM) Activin A and FGF2 are Regulated by Macrophage-Secreted Factors hMADS cells Ctr ATM-CM 6 * ** Relative gene expression FGF2 inhba control

Regulation of Adipocyte Progenitor Proliferation and Differentiation by Macrophage Factors. Macrophages Activin A, FGF2 Adipocyte Progenitors TGF  1

IER3

IER3 is Preferentially Expressed in Adipocyte Progenitors and is Increased by an Obese Microenvironment

IER3 Expression is Regulated by Actvin A, Hypoxia and Macrophages Factors

IER3 mediates Hypoxic and Activin-A-induced proliferation

IER3 ACTIVIN A FGF2 Hypoxia HIF1 PROLIFERATION ADIPOCYTE PROGENITOR MACROPHAGES Activin A, FGF2 and Macrophages support Adipocyte Progenitor Expansion through IER3 expression ERK1/2 Smad2

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