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. 2019 Nov 12:10:768.
doi: 10.3389/fendo.2019.00768. eCollection 2019.

Biological Effects of Ciliary Neurotrophic Factor on hMADS Adipocytes

Jessica Perugini  1 , Eleonora Di Mercurio  1 , Giovanni Tossetta  1 , Ilenia Severi  1 , Federica Monaco  2 , Marcella Reguzzoni  3 , Marco Tomasetti  2 , Christian Dani  4 , Saverio Cinti  1   5 , Antonio Giordano  1
Affiliations

Biological Effects of Ciliary Neurotrophic Factor on hMADS Adipocytes

Jessica Perugini (V体育2025版) et al. Front Endocrinol (Lausanne). .

"VSports在线直播" Abstract

Administration of ciliary neurotrophic factor (CNTF) to experimental animals exerts anti-obesity effects by acting on multiple targets. In white adipose tissue CNTF reduces lipid content, promotes fatty acid (FA) oxidation and improves insulin sensitivity. This study was performed to establish whether CNTF exerts similar effects on human white adipocytes. To this end, adipose differentiation was induced in vitro in human multipotent adipose-derived stem (hMADS) cells. CNTF receptor α (CNTFRα) expression was assessed in hMADS cells and adipocytes by qRT-PCR, Western blotting, and immunocytochemistry. After administration of human recombinant CNTF, signaling pathways and gene expression were evaluated by Western blotting and qRT-PCR VSports手机版. Glucose uptake was assessed by measuring 2-nitrobenzodeoxyglucose uptake with a fluorescence plate reader. Lastly, CNTF-induced anti-inflammatory responses were evaluated in hMADS adipocytes stressed with tumor necrosis factor α (TNFα) for 24 h. Results showed that CNTFRα protein expression was higher in undifferentiated hMADS cells than in hMADS adipocytes, where it was however clearly detectable. In hMADS adipocytes, 1 nM CNTF strongly activated the JAK-STAT3 (Janus kinase-signaling transducer and activator of transcription 3) pathway and acutely and transiently activated the AMPK (AMP-activated protein kinase) and AKT (protein kinase B) pathways. Acute CNTF treatment for 20 min significantly increased basal glucose uptake and was associated with increased AKT phosphorylation. Longer-term (24 and 48 h) treatment reduced the expression of lipogenic markers (FA synthase and sterol regulatory element-binding protein-1) and increased the expression of lipolytic [hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL)] and mitochondrial (peroxisome proliferator-activated receptor γ coactivator-1α and carnitine palmitoyltransferase 1) markers. In TNFα-treated hMADS adipocytes, CNTF significantly reduced the expression of monocyte chemoattractant protein 1 and TNFα-induced AKT inhibition. Collectively, these findings demonstrate for the first time that CNTF plays a role also in human adipocytes, driving their metabolism toward a less lipid-storing and more energy-consuming phenotype. .

Keywords: JAK STAT pathway; adipocytes; ciliary neurotrophic factor; diabetes; inflammation; lipolysis; mitochondria; obesity. V体育安卓版.

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Figures

Figure 1
Figure 1
CNTFRα expression in undifferentiated cells and differentiated hMADS adipocytes. (A) Representative pictures from Oil Red O-stained confluent hMADS cells (day 0, left panel) and adipocytes (day 13, right panel). Data were analyzed using Student's t-test. (B) qRT-PCR analysis of PPARγ, FABP4, perilipin, and adiponectin expression in hMADS cells (white bars) and adipocytes (gray bars). Data (n = 3) are mean ± SEM, *p < 0.05 compared with hMADS cells. Data were analyzed using Student's t-test. qRT-PCR (C) and representative immunoblot and quantification (D) of CNTFRα expression in hMADS cells (white bars) and adipocytes (gray bars). Data (n = 3) are mean ± SEM, *p < 0.05 compared with hMADS cells. Data were analyzed using Student's t-test. (E) hMADS adipocytes: double immunostaining and confocal microscopy images showing perilipin staining (left, green) around lipid droplets and CNTFRα staining (middle, red) in the cytoplasm and on some tracts of the plasma membrane (arrowheads).
Figure 2
Figure 2
JAK-STAT3 pathway activation in hMADS adipocytes by CNTF. (A) Representative immunoblot and quantification of the dose-dependent increase in 705-tyrosine STAT3 phosphorylation in hMADS adipocytes treated with increasing concentrations of human recombinant CNTF for 10 min. Data (n = 3) are mean ± SEM, *p < 0.05 compared with untreated cells (NT). Data were analyzed using one-way ANOVA. (B) Representative immunoblot and quantification of the time-dependent STAT3 phosphorylation in hMADS adipocytes treated with 1 nM CNTF. Data (n = 3) are mean ± SEM, *p < 0.05 compared with untreated cells (NT). Data were analyzed using one-way ANOVA. (C) Representative immunoblot and quantification of STAT3 phosphorylation as a response to 1 nM CNTF treatment for 10 min, detected in hMADS adipocytes pretreated with 10 μM curcumin for 24 h. Data (n = 3) are mean ± SEM, *p < 0.05 compared with untreated cells (NT), $p < 0.05 compared with cells treated with curcumin, #p < 0.05 compared with cells treated with CNTF. Data were analyzed using one-way ANOVA. (D) Time-dependent SOCS3 mRNA induction in hMADS adipocytes by 1 nM CNTF treatment. Data (n = 3) are mean ± SEM, *p < 0.05 compared with untreated cells (NT). Data were analyzed using one-way ANOVA.
Figure 3
Figure 3
AMPK and AKT pathway activation in hMADS adipocytes by CNTF. (A) Representative immunoblot and quantification of time-dependent 172-threonine AMPK phosphorylation in hMADS adipocytes treated with 1 nM CNTF. Data (n = 3) are mean ± SEM, *p < 0.05 compared with untreated cells (NT). Data were analyzed using one-way ANOVA. (B) Representative immunoblot and quantification of 473-serine AKT phosphorylation in hMADS adipocytes treated with 1 nM CNTF for 20 min (CNTF); with CNTF and 100 nM insulin for 20 min (INS); and with insulin alone. Data (n = 3) are mean ± SEM, *p < 0.05 compared with untreated cells (NT), #p < 0.05 compared with cells treated with insulin alone. Data were analyzed using one-way ANOVA. (C) Representative immunoblot and quantification of 473-serine AKT phosphorylation detected in hMADS adipocytes treated with 100 nM insulin for 20 min (INS); with 1 nM CNTF for 24 h (CNTF 24 h + INS), or 48 h (CNTF 48 h + INS); only with CNTF for 24 (CNTF 24 h) or 48 h (CNTF 48 h). Data (n = 3) are mean ± SEM, *p < 0.05 compared with untreated cells (NT). Data were analyzed using one-way ANOVA. (D) 2-NBDG uptake, expressed as mean fluorescent intensity (MFI), detected in hMADS adipocytes treated with 1 nM CNTF alone applied for different periods (white bars) or else with 100 nM insulin for 10 min (black bars). Data (n = 3) are from three independent experiments and are mean ± SEM, *p < 0.05 compared with untreated cells (NT). Data were analyzed using Student's t-test.
Figure 4
Figure 4
Transcriptional changes induced by long-term CNTF treatment in hMADS adipocytes. mRNA levels of SREBP-1 and FAS lipogenic markers (A) and HSL and ATGL lipolytic enzymes (B) in hMADS adipocytes treated with 1 nM CNTF for 24 or 48 h. Data (n = 3) are mean ± SEM, *p < 0.05 compared with untreated cells (NT). Data were analyzed using one-way ANOVA. Representative immunoblots and quantitative analyses of FAS (C) and ATGL (D) protein expression in hMADS adipocytes treated with 1 nM CNTF for 24 or 48 h. Data (n = 3) are mean ± SEM, *p < 0.05 compared with untreated cells (NT). Data were analyzed using Student's t-test. (E) PGC-1α mRNA and representative immunoblot and protein quantification in hMADS adipocytes treated with 1 nM CNTF for 24 or 48 h. Data (n = 3) are mean ± SEM, *p < 0.05 compared with untreated cells (NT). Data were analyzed using one-way ANOVA. (F) CPT1 mRNA and representative immunoblot and protein quantification in hMADS adipocytes treated with 1 nM CNTF for 24 or 48 h. Data (n = 3) are mean ± SEM, *p < 0.05 compared with untreated cells (NT). Data were analyzed using one-way ANOVA.
Figure 5
Figure 5
Anti-inflammatory effects of CNTF in hMADS adipocytes. (A) MCP1, IL-6, IL-1β, and PAI1 mRNA levels in hMADS adipocytes treated for 24 h with 10 ng/ml TNFα alone or administered with 1 nM CNTF for 24 or 48 h. Data (n = 3) are mean ± SEM, *p < 0.05 compared with untreated cells (NT), #p < 0.05 compared with cells treated with TNFα. Data were analyzed using one-way ANOVA. 2-NBDG uptake, expressed as mean fluorescent intensity (MFI), (B) and representative immunoblot and quantification of 473-serine AKT phosphorylation (C) in hMADS adipocytes treated with 10 ng/ml TNFα for 24 h; with 100 nM insulin for 20 min; with TNFα and insulin; or with TNFα, insulin and 1 nM CNTF for 24 or 48 h. Data (n = 3) are mean ± SEM, *p < 0.05 compared with untreated cells (NT), $p < 0.05 compared with cells treated with insulin, #p < 0.05 compared with cells treated with insulin and TNFα. Data were analyzed using one-way ANOVA.
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