A propolis-derived small molecule ameliorates metabolic syndrome in obese mice by targeting the CREB/CRTC2 transcriptional complex

The molecular targets and mechanisms of propolis ameliorating metabolic syndrome are not fully understood. Here, we report that Brazilian green propolis reduces fasting blood glucose levels in obese mice by disrupting the formation of CREB/CRTC2 transcriptional complex, a key regulator of hepatic gluconeogenesis. Using a mammalian two-hybrid system based on CREB-CRTC2, we identify artepillin C (APC) from propolis as an inhibitor of CREB-CRTC2 interaction. Without apparent toxicity, APC protects mice from high fat diet-induced obesity, decreases fasting glucose levels, enhances insulin sensitivity and reduces lipid levels in the serum and liver by suppressing CREB/CRTC2-mediated both gluconeogenic and SREBP transcriptions. To develop more potential drugs from APC, we designed and found a novel compound, A57 that exhibits higher inhibitory activity on CREB-CRTC2 association and better capability of improving insulin sensitivity in obese animals, as compared with APC. In this work, our results indicate that CREB/CRTC2 is a suitable target for developing anti-metabolic syndrome drugs.

i) The mRNA levels of CREB target genes in different tissues of male wild C57BL6 mice, which 1 oral administrated one dose of APC (20 mg/kg) or vehicle after fasting 12-h and for another 6-h 2 fasting before being sacrificed. Data are represented as mean ± SEM (n=4 per group). ns, p>0.05; *, 3 P < 0.05; **, p < 0.01; p values were determined by unpaired two-tailed multiple t test with two-stage 4 linear step-up procedure, each gene was analyzed individually, without assuming a consistent SD. 5 Source data for this figure are provided as a Source data file. The relative SREBP1 (left) and SREBP2 (right) protein levels in the liver of Crtc2KO mice was 5 induced by a high fat diet for 8 weeks. Crtc2KO mice and wild type littermates were administrated 6 VEH (vehicle control), MET (metformin, 200 mg/kg) or APC (20 mg/kg) for 3 weeks, and fasted 8 h 7 before anesthesia. Data are represented as mean ± SEM (n=4). *, p < 0.05; **, p < 0.01; p values were 8 determined by two-way ANOVA followed Bonferroni's multiple comparisons test.

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b) Immunoprecipitation of SEC31A from HEK293 cell lysates with overexpressed HA-Sec13A or 1 FLAG-Crtc2-WT. APC (10 μM) was added to the IP mixture overnight. One of two independent 2 experiments is shown here. LXRa protein level normalized to GAPDH shown as a bar graph (b). Data are represented as 7 mean ± SEM (n=3 per treatment). *, p < 0.05; **, p < 0.01; p values were determined by two-way 8 ANOVA repeated measures followed Bonferroni's multiple comparisons test. 9 c) Luciferase reporter activity of PPRE-LUC (PPAR response element driven luciferase reporter) in 1 HEK293T cell transfected with expression plasmids for Ppara, Rxra or Lxra with 8-h incubation of 2 APC (10 µM). One of three independent experiments presented here. Data are represented as 3 mean ± SEM. (n=3 per treatment). *, p < 0.05; **, p < 0.01; p values were determined by two-way 4 ANOVA followed Bonferroni's multiple comparisons test. 5 d) Schematic of APC inhibiting SREBP1 expression via the CRE-LXRa-LXRE axis, and reducing 6 SREBP2 expression by blocking half-CRE directly. Source data for this figure are provided as a 7 Source data file. db/db mice were continuously i.p. injected synthesized APC (20 mg/kg) or control vehicle 2 (VEH) one time daily for 5 weeks (n = 5-7). 3 a) Quantitative PCR analysis of G6pc, Pck1, Pgc-1α and Pgc-1b gene expression in the liver of 4 treated db/db mice. Data are represented as mean ± SEM (n=5 per group). *, p < 0.05; **, p < 0.01; p 5 values were determined by unpaired two-tailed multiple t test with two-stage linear step-up 6 procedure, each gene was analyzed individually, without assuming a consistent SD. 7 b) Immunoblotting of endogenous protein levels of PCK1, PGC-1α, CREB and CRTC2 in the livers 8 of 16-h-fasted db/db mice i.p. administered APC (20 mg/kg) or vehicle (VEH) one time daily for 5 9 weeks. One presentative result from two experiments is shown here (left), and relative PCK1 and 10 PGC-1α normalized to TUBULIN levels is presented as a bar graph (right). Data are represented as 11 mean ± SEM (n=3 per treatment). *, p < 0.05; **, p < 0.01; p values were determined by two-way 12 ANOVA followed Bonferroni's multiple comparisons test. 13 c) Fasting blood glucose levels and (d) plasma insulin of treated db/db mice (d) (n=6 per group).
14 Data are represented as mean ± SEM. ns, p > 0.05; *, p < 0.05; **, p < 0.01; p values were determined 15 by unpaired two-tailed t test with Welch's correction. 16 e) Plasma glucagon of DIO mice as in Fig. 7(d) and (f) glucagon level in treated db/db mice. Data 17 are represented as mean ± SEM (n=6 per group). *, p < 0.05; **, p < 0.01; p values were determined by 18 one-way ANOVA followed Dunnett's test in (e), or by unpaired two-tailed t test with Welch's 19 correction in (f). 20 g) Immunoblotting for phosphorylated AKT and total AKT in tissues from the liver, white adipose 1 (WAT), brown fat (BAT) and muscle (Mus) of treated db/db mice. One presentative result from two 2 experiments is shown here (n=3). 3 h) Daily food intake and plasma leptin level (i) of DIO mice stimulated with high fat diet for 13 4 weeks and 2 weeks of APC injection (n=6 per group). One of two independent experiments is shown 5 here. Data are represented as mean ± SEM. *, p < 0.05; **, p < 0.01; p values were determined by one-6 way ANOVA followed Dunnett's test. each test curve. Data are represented as mean ± SEM (n=6-8 per group). *, p < 0.05; **, p < 0.01; p 10 values of curves were determined by two-way ANOVA followed Bonferroni's multiple comparisons 11 test, and p values of AUC were determined by unpaired two-tailed t test with Welch's correction. 12 Source data for this figure are provided as a Source data file. Lean mice (wild C57) were i.p. injected with APC (20 mg/kg) or vehicle (VEH) one time daily for 3 3 weeks (n=7). 4 a) Twenty-four hours respiratory exchange ratio (RER), and a time course of RER curves (left) was 5 analyzed by AUC analysis (right) for these lean mice. Data are represented as mean ± SEM. (n=4 per 6 group). ns, p > 0.05; *, p < 0.05; **, p < 0.01; p values were determined by two-way ANOVA followed 7 Bonferroni's multiple comparisons test, or by unpaired two tailed t-test with Welch's correction in AUC 8 analysis of RER. 9 b) Body weight curves and (c) Plasma NEFA in lean mice. One of two independent experiments is 10 shown here. Data are represented as mean ± SEM. (n=7 per group). ns, p > 0.05; *, p < 0.05; **, 11 p < 0.01; p determined by two-way ANOVA followed Bonferroni's multiple comparisons test for (b), 12

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interaction, compound A58 exhibits a different configuration. It is possible that the steric bulk of the 1 phenyl group in compound A58 hinder interaction between the compound A58 and the protein CREB, 2 which reduced the inhibitory activity.

a)
The inhibitory-activity of A57 as determined by two-hybrid reporter assay. HEK293T cells were 2 co-transfected with CREB/CRTC2 two-hybrid reporter system plasmids, following incubation with 3 indicated small molecules (10 µM) overnight before luciferase reporter assays (n=4 per treatment). 4 One of three independent experiments is shown here. Data are represented as mean ± SEM. *, 5 p < 0.05; **, p < 0.01; p values were determined by one-way ANOVA followed Dunnett's multiple 6 comparisons test. 7 b) Combinatorial analysis of the cell toxicity and inhibitory activity of A57. The CREB/CRTC2-two 8 hybrid reporter activity (as a normalized percentage) is the X-axis, and the cell activity tested by MTT 9 assay (as a normalized percentage) is the Y-axis. n=3. 10 c) Immunoblot of P-CREB and CRTC2 de-phosphorylation in primary hepatocytes incubated with 11 A57 and APC (10 µM) 1-h prior to glucagon (100 nM) stimulation for 30 min. One result from three 12 experiments is shown here (top), and relative P-CREB normalized by CREB is presented as a bar 13 graph (bottom, n=3). Data are represented as mean ± SEM. ns, p > 0.05; *, p < 0.05; **, p < 0.01; p 14 values were determined by two-way ANOVA followed Bonferroni's multiple comparisons test.

e)
Immunoblotting of plasma and liver FGF21 protein in Crtc2KO mice, which induced by a high 20 fat diet for 13 weeks then orally administered APC (20 mg/kg), A57 (20 mg/kg) or vehicle (VEH) for