Fridamycin A, a Microbial Natural Product, Stimulates Glucose Uptake without Inducing Adipogenesis

Abstract

Type 2 diabetes is a complex, heterogeneous, and polygenic disease. Currently, available drugs for treating type 2 diabetes predominantly include sulfonylureas, α-glucosidase inhibitors, and biguanides. However, long-term treatment with these therapeutic drugs is often accompanied by undesirable side effects, which have driven interest in the development of more effective and safer antidiabetic agents. To address the urgent need for new chemical solutions, we focused on the analysis of structurally novel and/or biologically new metabolites produced by insect-associated microbes as they have recently been recognized as a rich source of natural products. Comparative LC/MS-based analysis of Actinomadura sp. RB99, isolated from a fungus-growing termite, led to the identification of the type II polyketide synthase-derived fridamycin A. The structure of fridamycin A was confirmed by ¹H NMR data and LC/MS analysis. The natural microbial product, fridamycin A, was examined for its antidiabetic properties in 3T3-L1 adipocytes, which demonstrated that fridamycin A induced glucose uptake in 3T3-L1 cells by activating the AMP-activated protein kinase (AMPK) signaling pathway but did not affect adipocyte differentiation, suggesting that the glucose uptake took place through activation of the AMPK signaling pathway without inducing adipogenesis. Our results suggest that fridamycin A has potential to induce fewer side effects such as weight gain compared to rosiglitazone, a commonly used antidiabetic drug, and that fridamycin A could be a novel potential therapeutic candidate for the management of type 2 diabetes.

Keywords: 3T3-L1 cells; Actinomadura sp. RB99; fridamycin A; glucose uptake; type 2 diabetes.

Figure 1

(A) LC/MS analysis of the EtOAc fraction (detection wavelength was set at 254 nm) and extracted ion chromatogram (EIC) for m/z 485.1 in negative ESI-MS mode. (B) Comparison of UV data of the peak at retention time 30.8 min with our in-house UV library. (C) Negative ion-mode ESI-MS data of the peak and the chemical structure of fridamycin A.

Figure 2

Fridamycin A increased glucose uptake in 3T3-L1 adipocytes. (A) 3T3-L1 adipocytes were incubated with the indicated concentrations of fridamycin A for 48 h and cell viability was determined using an EZ-Cytox assay kit. (B) Mature 3T3-L1 adipocytes were treated with control (Con; 0.05% dimethyl sulfoxide), 10 μM fridamycin A or 2 μM rosiglitazone for 1 h and then incubated with the fluorescent glucose indicator, 2-NBDG, for 30 min. Fluorescence intensity was measured using a fluorescence microplate reader. 3T3-L1 preadipocytes (C) or mature adipocytes (D) were incubated with fridamycin A or rosiglitazone for 3 days and analyzed by Western blot. (E) Quantification of phospho-AMP-activated protein kinase (AMPK) and total-AMPK was performed using ATTO image analysis software. Results are expressed as the mean ± the standard error of the mean (SEM). Data were analyzed using two-tailed unpaired t-tests. *** p < 0.001, ** p < 0.01 compared to the control group.

Figure 3

Fridamycin A did not affect adipocyte differentiation. (A) 3T3-L1 preadipocytes were differentiated in the presence of fridamycin A or rosiglitazone and the extent of differentiation was assessed by Oil Red O staining on day 6 of differentiation. (B) For the quantification of lipid accumulation, the absorbance of Oil Red O dye was measured using a microplate reader. Results are expressed as the mean ± SEM. Data were analyzed using two-tailed unpaired t-tests. * p < 0.05 compared to the control group. Scale bar: 200 μm.