Non-estrogenic Xanthohumol Derivatives Mitigate Insulin Resistance and Cognitive Impairment in High-Fat Diet-induced Obese Mice

Abstract

Xanthohumol (XN), a prenylated flavonoid from hops, improves dysfunctional glucose and lipid metabolism in animal models of metabolic syndrome (MetS). However, its metabolic transformation into the estrogenic metabolite, 8-prenylnaringenin (8-PN), poses a potential health concern for its use in humans. To address this concern, we evaluated two hydrogenated derivatives, α,β-dihydro-XN (DXN) and tetrahydro-XN (TXN), which showed negligible affinity for estrogen receptors α and β, and which cannot be metabolically converted into 8-PN. We compared their effects to those of XN by feeding C57BL/6J mice a high-fat diet (HFD) containing XN, DXN, or TXN for 13 weeks. DXN and TXN were present at higher concentrations than XN in plasma, liver and muscle. Mice administered XN, DXN or TXN showed improvements of impaired glucose tolerance compared to the controls. DXN and TXN treatment resulted in a decrease of HOMA-IR and plasma leptin. C2C12 embryonic muscle cells treated with DXN or TXN exhibited higher rates of uncoupled mitochondrial respiration compared to XN and the control. Finally, XN, DXN, or TXN treatment ameliorated HFD-induced deficits in spatial learning and memory. Taken together, DXN and TXN could ameliorate the neurocognitive-metabolic impairments associated with HFD-induced obesity without risk of liver injury and adverse estrogenic effects.

Figure 1

Structures of xanthohumol (XN) and its metabolites and derivatives. Steps: 1, spontaneous cyclization by intramolecular Michael-type addition; 2, hepatic or gut microbial O-demethylation; 3, chemical synthesis.

Figure 2

Cell viability/proliferation of MCF-7 cells after a 48-h exposure to 8-PN (A), XN (B), DXN (C), or TXN (D). Low concentrations (0.001 to 1 µM) of XN, DXN and TXN did not significantly increase cell viability/proliferation of MCF-7 cells whereas high concentrations (10 µM) of these compounds significantly decreased cell viability/proliferation. Data are represented as mean ± SEM of 6 replicate wells. Asterisk denotes significantly different from control, p < 0.05 by ANOVA.

Figure 3

XN, DXN and TXN do not induce progesterone receptor (PGR) expression in MCF-7 cells. Cells were treated with 0.1% ethanol (EtOH), estradiol (E2, 1 and 10 nM), 8-PN (0.2, 1 and 5 μM), or XN and its derivatives, DXN and TXN, for 20 h. PGR expression was determined by quantitative real-time PCR. A statistically significant induction of PGR expression was observed with both concentrations of estradiol and all three concentrations of 8-PN, but not by XN, DXN or TXN. Data are represented as mean ± SD of 3 replicate experiments. Asterisk denotes significantly different from control (EtOH), p < 0.05 by ANOVA.

Figure 4

Weekly body weights (A) and weekly food intake (B) of male mice fed a HFD with no XN/DXN/TXN (control), with 30 mg XN/kg body weight/day (XN), 30 mg DXN/kg body weight/day (DXN), or 30 mg TXN/kg body weight/day (TXN) for 13 weeks. Values are expressed as mean ± SEM of 12 mice per group (11 for the TXN group). Asterisk denotes significantly different from control group, p < 0.05 by ANOVA.

Figure 5

Liver, muscle and plasma concentrations of XN, DXN, and TXN. Concentrations of these compounds were examined by UPLC-MS/MS in the (A) liver, (B) muscle and (C) plasma of mice fed a HFD for 13 weeks. Values are mean ± SEM. *p < 0.05 compared to XN + IX, ANOVA, n = 11–12/treatment.

Figure 6

Effect of XN, DXN, and TXN supplementation on glucose tolerance in mice fed a HFD. Blood glucose levels were measured in mice after 4 (A,B) and 11 (C,D) weeks of feeding. The mice were fasted for 6 h before i.p. injection of glucose, 2 g/kg body weight, and then blood was analyzed for glucose at designated times. Bar graphs on the right represent areas under the curve (AUC) shown on the left. *p < 0.05 compared to control (ANOVA), n = 5/treatment.

Figure 7

Western blotting analyses of pAMPK, AMPK and β-actin (loading control) in liver (A,B) and skeletal muscle (C,D) of mice fed a HFD with or without treatment with XN, DXN and TXN. Protein bands were detected using a chemiluminescence detector (BIO-RAD ChemiDoc MP Imaging System). Band intensities were quantitated using Image J software. Data in bar graphs represent the ratios of pAMPK/AMPK from four samples per group ± SEM. *p < 0.05 compared to control (ANOVA). The represented bands were extracted from independent blots shown in Supplementary Figure 8.

Figure 8

Comparison of XN, DXN and TXN as mild mitochondrial uncouplers. DXN and TXN cause mitochondrial uncoupling in cells. C2C12 cells were sequentially treated with oligomycin (1 μM) and 5 μM of the test compounds while (A) the oxygen rate (OCR) and (B) the extracellular acidification rate (ECAR) was monitored. *p < 0.05 compared to control by ANOVA (n = 5).

Figure 9

XN and its derivatives improve spatial learning and memory in HFD-fed mice. (A) Learning curves in visible and hidden platform trials of the water maze. While there were no treatment effects on ability of the mice to locate the visible platform, treatment effects were seen when the platform was hidden. Mice treated with XN or TXN showed better performance than control mice. *p < 0.05 compared to vehicle, repeated measures ANOVA. P: probe trial (see C). (B) There were no effects of treatment on the mean swim speed (cm/s) during the first two visible platform trials. Swim speed was calculated during the water maze using a video tracking system. (C) Long term spatial memory retention was tested in a 60-s probe trial (P, no platform) 24 h following the completion of the final hidden platform trial. Time spent in the Target (T), Right (R), Left (L) and Opposite (O) quadrants during the 24-h memory probe trial is shown in panel C. The dotted line marks the expected percent time in each quadrant due to chance (25%). *p < 0.05 compared to time in spent in the R, L, and O quadrants, ANOVA, followed by Dunnett’s multiple comparisons test. (D) Latency to first crossing of the platform position during the 24-h memory probe trial. *p < 0.05 compared to control group, ANOVA. (E) Representative heat maps show the swim patterns of mice during the memory probe trial. The platform location is marked by a white circle (bottom right, Target quadrant). A–D, n = 11–12 mice/treatment.