Volume 35, Issue 2, Pages 414-421 (April 2016) Licochalcone F alleviates glucose tolerance and chronic inflammation in diet-induced obese mice through Akt and p38 MAPK  Eun-Jung Bak, Kyung-Chul Choi, Sungil Jang, Gye-Hyeong Woo, Ho-Geun Yoon, Younghwa Na, Yun-Jung Yoo, Youngseok Lee, Yangsik Jeong, Jeong-Heon Cha  Clinical Nutrition  Volume 35, Issue 2, Pages 414-421 (April 2016) DOI: 10.1016/j.clnu.2015.03.005 Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions

Fig. 1 Structure of lico F and effect of lico F on the TNFα-induced NF-κB-dependent inflammatory response in vitro. (A) Synthetic method and structure of lico F. Lico F was synthesized by condensation of 1-(4-(tetrahydro-2H-pyran-2-yloxy)phenyl)ethanone (1) with 2-methoxy-3-(3-methylbut-3-en-2-yl)-4-(tetrahydro-2H-pyran-2-yloxy)benzaldehyde (2) in EtOH with NaOH. (B) TNFα-induced NF-κB-dependent promoter activity of lico F. HEK293 cells were transfected with a NF-κB binding site driven luciferase reporter plasmid and treated with TNFα or various concentrations of lico F. Whole cell extracts were used in the luciferase assay. Results are presented as the means of two independent experiments performed in triplicate. (C) The mRNA level of pro-inflammatory cytokines in HEK293 cells. The mRNA expression levels were evaluated via quantitative RT-PCR. lico F, licochalcone F. Clinical Nutrition 2016 35, 414-421DOI: (10.1016/j.clnu.2015.03.005) Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions

Fig. 2 Glucose tolerance test after 3 week vehicle or lico F administration. Lico F dissolved in vehicle (10% alcohol, 10% tween 80, and 80% saline) was orally administered for 3 weeks. The control group was given vehicle alone. Blood glucose levels before administration (□, ○, ▵) and after administration (■, ●, ▴), in the lico F group (A), the control group (B), and the lean group (B) are shown in the graph, respectively. (C) Blood glucose levels of the lico F group (■) vs control group (●). All values are expressed as means ± standard errors. The symbols, * and ** indicate significant differences at p < 0.05 and p < 0.01, respectively. lico F, licochalcone F. Clinical Nutrition 2016 35, 414-421DOI: (10.1016/j.clnu.2015.03.005) Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions

Fig. 3 Histological analysis in liver after 3 week vehicle or lico F administration. Histological sections were stained with hematoxylin and eosin. Microscopic pictures of liver obtained at × 100. (Bar = 100 μm). lico F, licochalcone F. Clinical Nutrition 2016 35, 414-421DOI: (10.1016/j.clnu.2015.03.005) Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions

Fig. 4 Histological analysis in EWAT after 3 week vehicle or lico F administration. (A) Histological sections were stained with hematoxylin and eosin. Black arrows represent the infiltration of adipose tissue macrophages. Microscopic pictures of EWAT obtained at × 100. (B) An average adipocyte cell size in EWAT of the control and lico F groups was quantified. The error bars represent the standard error of the mean. The symbol *** indicates a significant difference at p < 0.001. (Bar = 100 μm). lico F, licochalcone F. Clinical Nutrition 2016 35, 414-421DOI: (10.1016/j.clnu.2015.03.005) Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions

Fig. 5 The expression of CD68, TNFα, and MCP-1 genes in EWAT. mRNA expression levels of CD68 (A), TNFα (B), and MCP-1 (C) in EWAT were evaluated via RT-PCR. The relative mRNA expression levels of control (□) vs lico F (■) are shown in the graph. The expression value of the control group was set as 1 to permit comparison of the relative mRNA expression levels between the control and lico F groups. The error bars represent the standard error of the mean. The symbols, * and ** indicate significant differences at p < 0.05 and p < 0.01, respectively. lico F, licochalcone F. Clinical Nutrition 2016 35, 414-421DOI: (10.1016/j.clnu.2015.03.005) Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions

Fig. 6 Akt, p38, Erk1/2, and JNK activation in EWAT were detected by western blotting (top). The phosphorylation of signals was compared with the total levels of those in the loading controls (bottom). Band intensities were plotted as a relative fold increase. The relative protein levels of control (□) vs lico F (■) are shown in the graph. The error bars represent the standard error of the mean. The symbol * indicates significant difference at p < 0.05. lico F, licochalcone F; con, control. Clinical Nutrition 2016 35, 414-421DOI: (10.1016/j.clnu.2015.03.005) Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions