Glucagon modulates proliferation and differentiation of human adipose precursors

in Journal of Molecular Endocrinology
Correspondence should be addressed to G Cantini or M Luconi: giulia.cantini@unifi.it or michaela.luconi@unifi.it
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Glucagon-like peptide 1 receptor agonists (GLP-1RAs), which are currently used for the treatment of type 2 diabetes, have recently been proposed as anti-obesity drugs, due to their relevant effects on weight loss. Furthermore, dual agonists for both GLP-1R and glucagon receptor (GCGR) are under investigation for their promising action on adiposity, although underlying mechanisms still need to be clarified. We have recently demonstrated that GLP-1 and liraglutide interfere with the proliferation and differentiation of human adipose precursors, supporting the hypothesis of a peripheral action of GLP-1RA on weight. Here, we investigated glucagon activity in an in vitro model of primary human adipose-derived stem cells (ASCs). Glucagon significantly inhibited ASC proliferation in a dose- and time-dependent manner, as evaluated by cell count and thymidine incorporation. When added during in vitro-induced adipogenesis, glucagon significantly reduced adipocyte differentiation, as demonstrated by the evaluation of intracellular fat content and quantitative expression of early and mature adipocyte markers (PPARγ and FABP4, HSL). Notably, the inhibitory effect of glucagon on cell proliferation and adipogenesis was reversed by specific GLP-1R (exendin-9) and GCGR (des-His1-Glu9-glucagon(1–29)) antagonists. The presence of both receptors was demonstrated by Western blot, immunofluorescence and cytofluorimetric analysis of ASCs. In conclusion, we demonstrated a direct inhibitory action of glucagon on the proliferation and differentiation of human adipose precursors, which seems to involve both GLP-1R and GCGR. These findings suggest that the adipose stem compartment is a novel target of glucagon, possibly contributing to the weight loss obtained in vivo with dual GLP-1R/glucagon agonists.

 

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    The anti-proliferative action of glucagon in adipose precursors. ASCs were untreated (CTRL) or treated for 1, 2 or 3 days with increasing concentration (1-10-100 nM) of glucagon. (A) Haemocytometer cell-counting results are expressed as mean ± s.e. (B) Representative scatter plots of cytofluorimetric evaluation of cell viability are shown; (C) bar-graphs represent mean ± s.e. of cell percentages. (D) Data represent mean ± s.e. cpm after a 4-h pulse of [3H]-thymidine in ASCs untreated/treated for 24–48 h with 10-100 nM glucagon. (E) Representative scatter plots of DNA content index are presented; (F) data obtained from cell cycle analysis are indicated as mean ± s.e. of cell percentage in ASCs untreated/treated for 24–48 h with 10-100 nM glucagon. Statistical analysis was performed with ANOVA (F and P value are indicated) followed by Dunnett’s post hoc test: *P < 0.05, **P < 0.01, §P < 0.000 vs respective Ctrl, n = 5 independent experiments.

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    Glucagon stimulation does not affect apoptosis in adipose precursors. ASCs were untreated (CTRL) or treated for 1, 2 or 3 days with increasing concentration (10-100 nM) of glucagon. (A) Representative scatter plots of cytofluorimetric evaluation of annexin V are shown. (B) Bar graphs represent mean ± s.e. of cell percentages. Statistical analysis was performed with ANOVA resulting in no statistically significant differences, n = 3 independent experiments. A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0095.

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    Glucagon interferes with the in vitro-induced adipose differentiation. ASCs were untreated (ASC) or in vitro induced to differentiate towards the adipose phenotype with the specific inductive media (ADIPO) alone or in the presence of increasing concentration (1-10-100 nM) of glucagon. Representative images of in vitro-derived adipocytes stained with AdipoRed fluorescence reagent (A–H) or Oil Red O staining (I–L), captured by epifluorescence or optical microscopy, respectively. Any Oil Red O or AdipoRed staining was present in undifferentiated ASC controls (not shown). Magnification (20× A–D, 40× E–L). (M) Quantitative evaluation of intracellular triglyceride depots expressed as mean ± s.e. absorbance fold increase (FI) vs ASC after spectrofluorometric measurement of AdipoRed staining. Percentages of glucagon inhibitory effect in lipid content are indicated. Statistical analysis was performed with ANOVA (F = 5.915; P = 0.001) followed by Dunnett’s post hoc test: §P < 0.000 vs undifferentiated ASCs, *P < 0.005, **P < 0.000 vs ADIPO, n = 5 independent experiments. (N) TaqMan qRT-PCR evaluation of adipose marker gene expression in in-vitro-differentiated adipocytes alone or treated with increasing concentration (1-10-100nM) of glucagon: bar-graph represents mean ± s.e. gene expression fold increase (FI) vs ADIPO. Statistical analysis was performed with ANOVA followed by Dunnett’s post hoc test: *P < 0.05, **P < 0.001 vs ADIPO, n = 5 independent experiments.

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    GCGR and GLP-1R expression in human adipose precursor cells. (A) ASCs were subjected to an immunocytochemistry using the different antibodies for GCGR and GLP-1R, as specified in the methods section. Representative images of cells incubated with GCGR primary antibody (panels b and c), monoclonal (panels e-f) and polyclonal (panels h-i) primary GLP-1R antibodies and visualized on epifluorescence microscopy are shown. Negative controls (panels a-d-g) avoiding the primary antibody are shown. Nuclear counterstaining with DAPI is presented (blue signal). Magnification is 20×. (B) Cytofluorimetric evaluation of GCGR and GLP-1R on cell surface. Representative scatter plots are shown: data is expressed as the mean ± s.d. of positive cells in n = 3 independent experiments. (C) Western blot analysis of GCGR and GLP-1R expression in different ASC populations compared to the positive controls (AT, adipose tissue and HT, heart samples). Protein ladder bands are shown in the left.

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    GCGR and GLP-1R involvement underlying the anti-proliferative effect of glucagon in human adipose precursor cells. (A) ASCs were untreated (CTRL) or treated for 1, 2 or 3 days with 10 nM glucagon alone or combined with an equimolar concentration (10 nM) of the GCGR antagonist, des-His1-Glu9-glucagon(1–29) (GI) or the GLP-1R antagonist exendin 9-39, (EX): results are expressed as mean ± s.e. of direct cell count. (B) ASCs were untreated (CTRL) or treated for 1, 2 or 3 days with 10 nM GLP-1 alone or combined with an equimolar concentration (10 nM) of the GCGR antagonist, des-His1-Glu9-glucagon(1–29) (GI): quantitative results are expressed as mean ± s.e. of direct cell count. (C) ASCs were untreated (CTRL) or treated for 1, 2 or 3 days with 10 nM glucagon (GLUC) alone or combined with an equimolar concentration (10 nM) of GLP-1: results are expressed as mean ± s.e. of direct cell count. Statistical analysis was performed with Student’s t test: *P < 0.05, **P < 0.01, n = 3 independent experiments.

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    GCGR and GLP-1R involvement underlying the anti-adipogenic effect of glucagon in human adipose precursor cells. ASCs were untreated (ASC) or in vitro induced to differentiate towards the adipose phenotype with the specific inductive media (ADIPO), with 10 nM glucagon (GLUC) or combined with an equimolar concentration (10 nM) of GCGR (GI) or GLP-1R (EX) antagonists. (A) Quantitative evaluation of intracellular triglyceride depots expressed as mean ± s.e. absorbance fold increase (FI) vs ADIPO after spectrofluorometric measurement of AdipoRed staining. Statistical analysis was performed with Student’s t test: P < 0.000 vs undifferentiated ASC, §P < 0.001 vs ADIPO, *P < 0.05, **P < 0.001 vs Gluc10, n = 7 independent experiments. (B) qRT-PCR TaqMan evaluation of adipose marker gene expression in in-vitro-differentiated adipocytes alone (ADIPO), treated with 10 nM glucagon (GLUC) or combined with an equimolar concentration (10 nM) of GCGR (GI) or GLP-1R (EX) antagonists. Bar-graph represents mean ± s.e. gene expression fold increase (FI) vs ADIPO. Statistical analysis was performed with Student’s t test: P < 0.000 vs undifferentiated ASC, §P < 0.001 vs ADIPO, *P < 0.05, **P < 0.001 vs GLUC, n = 6 independent experiments. (C) qRT-PCR TaqMan evaluation of the adipose marker gene expression (FABP4) in in-vitro-differentiated adipocytes treated with 10 nM glucagon (GLUC) stimulated with an equimolar concentration (10 nM) of GCGR (GI) or GLP-1R (EX) antagonists alone or combined. Statistical analysis was performed with Student’s t test: P < 0.000 vs undifferentiated ASC and P < 0.001 vs ADIPO (not shown), *P < 0.05, **P < 0.001 vs GLUC, #P < 0.05 vs GI + EX combined, n = 4 independent experiments from two ASC populations.

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