Nutr Res Pract.  2023 Dec;17(6):1043-1055. 10.4162/nrp.2023.17.6.1043.

Cydonia oblonga Miller fruit extract exerts an anti-obesity effect in 3T3-L1 adipocytes by activating the AMPK signaling pathway

• Lee HS
• Jung JI
• Hwang JS
• Hwang MO
• Kim EJ

• KMID: 2548863
• DOI: http://doi.org/10.4162/nrp.2023.17.6.1043

Abstract

BACKGROUND/OBJECTIVES
The fruit of Cydonia oblonga Miller (COM) is used traditionally in Mediterranean region medicine to prevent or treat obesity, but its mechanism of action is still unclear. Beyond a demonstrated anti-obesity effect, the fruit was tested for the mechanism of adipogenesis in 3T3-L1 preadipocytes.
MATERIALS/METHODS
3T3-L1 preadipocytes were cultured for 8 days with COM fruit extract (COME) at different concentrations (0–600 µg/mL) with adipocyte differentiation medium. The cell viability was measured using an MTT assay; triglyceride (TG) was stained with Oil Red O. The expression levels of the adipogenesis-related genes and protein expression were analyzed by reverse transcription polymerase chain reaction and Western blotting, respectively.
RESULTS
COME inhibited intracellular TG accumulation during adipogenesis. A COME treatment in 3T3-L1 cells induced upregulation of the adenosine monophosphateactivated protein kinase (AMPK)α phosphorylation and downregulation of the adipogenic transcription factors, such as sterol regulatory element-binding protein 1c, peroxisome proliferator-activated receptor γ, and CCAAT/enhancer binding protein α. The COME treatment reduced the mRNA expression of fatty acyl synthetase, adenosine triphosphatecitrate lyase, adipocyte protein 2, and lipoprotein lipase. It increased the mRNA expression of hormone-sensitive lipase and carnitine palmitoyltransferase I in 3T3-L1 cells.
CONCLUSIONS
COME inhibits adipogenesis via the AMPK signaling pathways. COME may be used to prevent and treat obesity.

Keyword

3T3-L1 cells; AMP-activated protein kinase; adipogenesis; lipogenesis; lipolysis

Figure

• 

  Fig. 1 Effects of COME on the cell viability in 3T3-L1 preadipocyte. 3T3-L1 cells were plated at 3 × 104 cells/well and incubated for 24 h. After 24 h incubation, the cells were incubated for 72 h in a medium containing COME concentrations ranging from 0 to 800 μg/mL. The cell viability was measured using a MTT assay. The values are expressed as the mean ± SEM. Means with different letters are significantly different (P < 0.05).COME, Cydonia oblonga Miller fruit extract.

• 

  Fig. 2 Effects of COME on lipid accumulation, cellular TG content, and glycerol contents in the 3T3-L1 cells. 3T3-L1 preadipocytes were incubated in an ADM in the absence or presence of various COME concentrations for 8 days. (A) Differentiated 3T3-L1 cells were stained with Oil-red O solution and observed by optical microscopy. Scale bar, 50 µm. (B) Accumulated lipid droplets in stained 3T3-L1 adipocytes with Oil red O solution were quantified. (C) 3T3-L1 cells were differentiated and treated with COME for 8 days. The intracellular TG accumulation in differentiated 3T3-L1 adipocytes was measured. (D) Cells were differentiated and treated with COME for 8 days. The 24-h conditioned media were collected, and the free glycerol contents in the 24-h conditioned media were measured. The values are expressed as mean ± SEM. The means with different letters are significantly different (P < 0.05).ADM, adipocyte differentiation medium; COME, Cydonia oblonga Miller fruit extract.***P < 0.001 significantly different from that of non-ADM-treated (undifferentiated) group.

• 

  Fig. 3 Effects of COME on the expression of adipogenic transcription factors in 3T3-L1 cells. 3T3-L1 preadipocytes were incubated in an ADM in the absence or presence of various concentrations of COME for 8 days. The total RNA in 3T3-L1 cells was isolated. Real-time RT-PCR of (A) SREBP-1c, (B) PPARγ, (C) C/EBP-α was conducted. The values are expressed as the mean ± SEM. Means with different letters are significantly different (P < 0.05).COME, Cydonia oblonga Miller fruit extract; SREBP-1c, sterol regulatory element-binding protein 1c; PPAR-γ, peroxisome proliferator-activated receptor γ; C/EBP-α, CCAAT/enhancer binding protein α; ADM, adipocyte differentiation medium.**P < 0.01, ***P < 0.001 significantly different from that of non-ADM-treated (undifferentiated) group.

• 

  Fig. 4 Effects of COME on the expression of adipogenesis and lipogenesis-related genes in 3T3-L1 cells. Eight days after the induction of differentiation and treatment with COME, the total RNA in 3T3-L1 cells was isolated. Real-time reverse transcription polymerase chain reaction of (A) ACL, (B) FAS, (C) aP2, and (D) LPL was performed. The values are expressed as mean ± SEM. Means with different letters are significantly different (P < 0.05).COME, Cydonia oblonga Miller fruit extract; ACL, ATP-citrate lyase; FAS, fatty acid synthetase; aP2, adipocyte protein 2; LPL, lipoprotein lipase; ADM, adipocyte differentiation medium.*P < 0.05, ***P < 0.001 significantly different from that of non-ADM-treated (undifferentiated) group.

• 

  Fig. 5 Effects of COME on the expression of lipolysis-related genes in 3T3-L1 cells. Eight days after the induction of differentiation and treatment with COME, the total RNA in 3T3-L1 cells was isolated. Real-time reverse transcription polymerase chain reaction of (A) HSL and (B) CPT1 was performed. The values are expressed as the mean ± SEM. Means with different letters are significantly different (P < 0.05).HSL, hormone-sensitive lipase; CPT1, carnitine palmitoyltransferase 1; ADM, adipocyte differentiation medium; COME, Cydonia oblonga Miller fruit extract.**P < 0.01 significantly different from that of the non-ADM-treated (undifferentiated) group.

• 

  Fig. 6 Effects of COME on the expression of AMPK and ACC in 3T3-L1 cells. 3T3-L1 preadipocytes were incubated in an ADM in the absence or presence of various concentrations of COME for 8 days. The total lysates were prepared and analyzed by Western blotting using the indicated antibodies. (A, C) Images of Western blots representative of 3 independent experiments are shown. (B, D) Quantitative analysis of Western blot results. The protein expression levels were normalized to β-actin and are expressed relative to those of the non-ADM-treated (undifferentiated) group. Means with different letters are significantly different (P < 0.05).ADM, adipocyte differentiation medium; COME, Cydonia oblonga Miller fruit extract; AMPK, adenosine monophosphate-activated protein kinase; ACC, acetyl coenzyme A carboxylase.*P < 0.05, **P < 0.01 significantly different from that of non-ADM-treated (undifferentiated) cells.

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