Phloretin enhances adipocyte differentiation and adiponectin expression in 3T3-L1 cells

doi:10.1016/j.bbrc.2007.07.021

Biochemical and Biophysical Research Communications

Volume 361, Issue 1, 14 September 2007, Pages 208-213

https://doi.org/10.1016/j.bbrc.2007.07.021 Get rights and content

Abstract

Adipocyte dysfunction is strongly associated with the development of cardiovascular risk factors and diabetes. It is accepted that the regulation of adipogenesis or adipokines expression, notably adiponectin, is able to prevent these disorders. In this report, we show that phloretin, a dietary flavonoid, enhances 3T3-L1 adipocyte differentiation as evidenced by increased triglyceride accumulation and GPDH activity. At a molecular level, mRNA expression levels of both PPARγ and C/EBPα, the master adipogenic transcription factors, are markedly increased by phloretin. Moreover, mRNA levels of PPARγ target genes such as LPL, aP2, CD36 and LXRα are up-regulated by phloretin. We also show that phloretin enhances the expression and secretion of adiponectin. Co-transfection studies suggest the induction of PPARγ transcriptional activity as a possible mechanism underlying the phloretin-mediated effects. Taken together, these results suggest that phloretin may be beneficial for reducing insulin resistance through its potency to regulate adipocyte differentiation and function.

Section snippets

Materials and methods

Reagents. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Invitrogen (Carlsbad, CA), fetal bovine serum (FBS) was obtained from PAA laboratories (Pasching, Austria) and charcoal-stripped FBS was from Biowest (Nuaille, France). Phloretin, isobutylmethylxanthine, dexamethasone, and insulin were purchased from Sigma–Aldrich (St. Louis, MO). TRIzol reagent, random primers and Moloney murine leukemia virus reverse transcriptase were obtained from Invitrogen (Carlsbad, CA). SYBR Green

Phloretin enhances 3T3-L1 adipocyte differentiation

Two-day postconfluent 3T3-L1 preadipocytes (day 0) were treated with phloretin at 50 μM every 2 days for 12 days. No significant effect has been observed with phloretin concentrations under 50 μM (1, 2, 10, and 20 μM; data not shown). When preadipocytes differentiated into adipocytes, morphological alterations were observed due to the accumulation of lipid droplets in the cytoplasm. As evidenced by Oil Red O staining, phloretin significantly increased lipid accumulation compared with control cells

Discussion

In the present study, we demonstrate that phloretin enhances 3T3-L1 adipocyte differentiation by increasing adipogenic gene expression. The cytosolic enzyme GPDH occupies a central position in the pathway of triglyceride synthesis and is linked to the characteristic changes of adipose conversion [14]. Here we show that phloretin significantly increases both GPDH activity and triglyceride content.

At the molecular level, adipogenesis is driven by a complex transcriptional cascade involving the

Acknowledgments

The present work was financially supported by ANRT (French Research Ministry) and the Andros Company. The authors thank Christiane Malezet-Desmoulins for her technical help in co-transfection studies.

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In silico docking was performed to check the effect of PTN and PZN on PPARγ S273-Cdk5 interactions. The docking results were further validated in preclinical settings by utilizing a mice model of high fat diet-induced T2DM.
Computational docking and further MD-simulation data revealed that PTN and PZN inhibited the activation of Cdk5, thereby blocking the phosphorylation of PPARγ. Our in vivo results further demonstrated that PTN and PZN administration significantly improved the secretory functions of adipocytes by increasing adiponectin and reducing inflammatory cytokine levels, which ultimately reduced the hyperglycaemic index. Additionally, combined treatment of PTN and PZN decreased in vivo adipocyte expansion and increased Glut4 expression in adipose tissues. Furthermore, PTN and PZN treatment reduced hepatic insulin resistance by modulating lipid metabolism and inflammatory markers.
In summary, our findings strongly imply that PTN and PZN are candidates as nutraceuticals in the management of comorbidities related to diabetes and its complications.
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The current study sought to evaluate the acute effects of phloretin (PH) on metabolic pathways involved in the maintenance of glycemia, specifically gluconeogenesis and glycogenolysis, in the perfused rat liver. The acute effects of PH on energy metabolism and toxicity parameters in isolated hepatocytes and mitochondria, as well as its effects on the activity of a few key enzymes, were also evaluated. PH inhibited gluconeogenesis from different substrates, stimulated glycogenolysis and glycolysis, and altered oxygen consumption. The citric acid cycle activity was inhibited by PH under gluconeogenic conditions. Similarly, PH reduced the cellular ATP/ADP and ATP/AMP ratios under gluconeogenic and glycogenolytic conditions. In isolated mitochondria, PH inhibited the electron transport chain and the F_oF₁–ATP synthase complex as well as acted as an uncoupler of oxidative phosphorylation, inhibiting the synthesis of ATP. PH also decreased the activities of malate dehydrogenase, glutamate dehydrogenase, glucose 6-phosphatase, and glucose 6-phosphate dehydrogenase. Part of the bioenergetic effects observed in isolated mitochondria was shown in isolated hepatocytes, in which PH inhibited mitochondrial respiration and decreased ATP levels. An aggravating aspect might be the finding that PH promotes the net oxidation of NADH, which contradicts the conventional belief that the compound operates as an antioxidant. Although trypan blue hepatocyte viability tests revealed substantial losses in cell viability over 120 min of incubation, PH did not promote extensive enzyme leakage from injured cells. In line with this effect, only after a lengthy period of infusion did PH considerably stimulate the release of enzymes into the effluent perfusate of livers. In conclusion, the increased glucose release caused by enhanced glycogenolysis, along with suppression of gluconeogenesis, is the opposite of what is predicted for antihyperglycemic agents. These effects were caused in part by disruption of mitochondrial bioenergetics, a result that should be considered when using PH for therapeutic purposes, particularly over long periods and in large doses.
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Phloretin enhances adipocyte differentiation and adiponectin expression in 3T3-L1 cells

Abstract

Section snippets

Materials and methods

Phloretin enhances 3T3-L1 adipocyte differentiation

Discussion

Acknowledgments

Drug Discov. Today

Cell Metab

Am. J. Clin. Nutr.

J. Biol. Chem.

Free Radic Biol. Med.

J. Nutr.

J. Biol. Chem.

Methods

J. Biol. Chem.

J. Lipid Res.

Biochem. Biophys. Res. Commun.

J. Nutr.