Cinnamaldehyde induces fat cell-autonomous thermogenesis and metabolic reprogramming

Abstract

Objective: Cinnamaldehyde (CA) is a food compound that has previously been observed to be protective against obesity and hyperglycemia in mouse models. In this study, we aimed to elucidate the mechanisms behind this protective effect by assessing the cell-autonomous response of primary adipocytes to CA treatment.

Methods: Primary murine adipocytes were treated with CA and thermogenic and metabolic responses were assessed after both acute and chronic treatments. Human adipose stem cells were differentiated and treated with CA to assess whether the CA-mediated signaling is conserved in humans.

Results: CA significantly activated PKA signaling, increased expression levels of thermogenic genes and induced phosphorylation of HSL and PLIN1 in murine primary adipocytes. Inhibition of PKA or p38 MAPK enzymatic activity markedly inhibited the CA-induced thermogenic response. In addition, chronic CA treatment regulates metabolic reprogramming, which was partially diminished in FGF21KO adipocytes. Importantly, both acute and chronic effects of CA were observed in human adipose stem cells isolated from multiple donors of different ethnicities and ages and with a variety of body mass indexes (BMI).

Conclusions: CA activates thermogenic and metabolic responses in mouse and human primary subcutaneous adipocytes in a cell-autonomous manner, giving a mechanistic explanation for the anti-obesity effects of CA observed previously and further supporting its potential metabolic benefits on humans. Given the wide usage of cinnamon in the food industry, the notion that this popular food additive, instead of a drug, may activate thermogenesis, could ultimately lead to therapeutic strategies against obesity that are much better adhered to by participants.

Keywords: Cinnamaldehyde; Obesity; Subcutaneous adipocytes; Thermogenesis.

Fig. 1

Acute CA treatment upregulates thermogenic gene expression and activates PKA signaling in murine subcutaneous fat cells. (A) Real-time qPCR analyses of thermogenic genes (Fgf21 and Ucp1) and adipogenic markers (Pparg and Adipoq) in differentiated murine inguinal adipocytes (n=4) after 400 μM CA treatment for 4 h. It has been reported that CA treatment in 3T3-L1 preadipocytes negatively influences adipogenesis [7]. We observed similar levels of adipogenesis and equal pan-fat marker expression (Pparg and Adipoq) in control and CA treated groups. (B) Representative immunoblots of PKA substrate phosphorylation, p38 MAPK phosphorylation, HSL phosphorylation (Ser660) and PLIN1 phosphorylation (Ser517) in differentiated inguinal adipocytes exposed to 400 μM CA for the indicated time or 10 μM Iso for 10 min as a positive control. (C) Representative immunofluorescence staining of PLIN1 phosphorylation (red) in differentiated inguinal adipocytes treated with 400 μM CA for 1 h or 10 μM Iso for 1 h as a positive control. BODIPY was used to stain lipid droplets (green). Scale bar = 20 μm. (D) Real-time qPCR analyses of thermogenic genes (Fgf21 and Ucp1) and adipogenic markers (Pparg and Adipoq) in differentiated WT or TRPA1KO inguinal adipocytes following vehicle control or 400 μM CA treatment for 4 h (n=6). (E) Representative immunoblots of p38 MAPK phosphorylation, HSL phosphorylation (Ser660) and PLIN1 phosphorylation (Ser517) in differentiated TRPA1KO inguinal adipocytes treated with 400 μM CA for the indicated time or 10 μM Iso for 10 min as a control. (F) Representative immunoblots of phosphorylated PKA substrates, phosphorylated p38 MAPK, phosphorylated HSL (Ser660) and phosphorylated PLIN1 (Ser517) in differentiated inguinal adipocytes treated with 50 μM H-89 for 30 min and then 400 μM CA for 1 h or 10 μM Iso for 10 min as a positive control. (G and H) Real-time qPCR analyses of thermogenic markers (Fgf21, Ucp1) in differentiated inguinal adipocytes treated with 50 μM H-89 (G, n=3) or 10 μM SB203580 (SB, H, n=3) for 1 h and then 400 μM CA for 4 h. All data in A, D, G, and H are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

Fig. 2

CA activates a thermogenic response and regulates lipid metabolic gene profiles in human subcutaneous fat cells. (A) Locations of the human subcutaneous fat biopsy sites used in this study and representative Oil Red O staining of differentiated hASCs. Scale bar = 50 μm. (B) Real-time qPCR analyses of thermogenic genes (FGF21 and UCP1) and adipogenic markers (PPARG and ADIPOQ) in differentiated hASCs after 200 μM CA treatment for 4 h (n=4). (C and D) Representative immunoblots of phosphorylated p38 MAPK, phosphorylated HSL (Ser660) and phosphorylated PLIN1 (Ser522) in differentiated hASCs treated with 200 μM CA for the indicated time (C) or 50 μM H-89 for 30 min and then 200 μM CA for 2 h (D). (E and F) Real-time qPCR analyses of thermogenic markers in differentiated hASCs treated with 50 μM H-89 (E, n=3) or 10 μM SB (F, n=3) for 1 h and then 200 μM CA for 4 h. (G and H) Real-time qPCR analyses of thermogenic genes (Fgf21 and Ucp1), FGF21-target genes (Egr1, cFos) and the adipogenic marker Pparg in differentiated inguinal adipocytes of WT mice (G, n=4) or FGF21KO mice (H, n=4) after 400 μM CA treatment for 4 h. (I) Representative immunoblots of phosphorylated p38 MAPK, phosphorylated HSL (Ser660) and phosphorylated PLIN1 (Ser517) in differentiated inguinal adipocytes from FGF21KO mice treated with 400 μM CA for the indicated time. (J–M) Real-time qPCR analyses of thermogenic and metabolic genes in differentiated WT (J, n=4) or FGF21KO (K, n=4) murine inguinal adipocytes after 200 μM CA treatment for 48 h, or differentiated hASCs stimulated with 200 μM CA for 24 h (L and M, n=4). All data in B, E–H, and J–M are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.