Effects of pinto bean and resistant starch supplementation on bone parameters in a mouse model of estrogen deficiency

Field: Pharmacology, Toxicology and Pharmaceutics (Life Sciences)

Objective: Estrogen plays a role in regulating bone metabolism and a decrease in estrogen compromises bone health. Pinto beans (PB) are good sources of plant-derived estrogens (i.e., phytoestrogens) and fiber that can be fermented by the gut bacteria to produce short-chain fatty acids (SCFAs) that have been shown to play a role in bone health. This project aims to compare the effects of PB and isolated fiber (i.e.., retrograded resistant starch, RS3 and chemically modified, RS4) on bone parameters and to understand the role of the gut bacteria in maintaining bone health in a mouse model of estrogen deficiency. Methods: One hundred twenty 3m-old female C57BL/6 mice were made estrogen deficient by injecting with either vinyl cyclohexene diepoxide (VCD, 160 mg/kg bw in sesame oil) or sesame oil (vehicle) for 30 days. After confirmation of estrogen status with vaginal cytology, mice were assigned to one of eight treatment groups for 16 weeks in 4x2 factorial design with diet (Control [AIN-93M], 10% (wt/wt) PB, 5% (wt/wt) RS3 or 5% RS4) and estrogen status (sesame oil or VCD) as factors. The dose of RS is equivalent to the fiber content of the 10% PB. At the end of treatment, fecal samples were collected, body composition was assessed, and femur and L4 vertebrae were collected. Dual energy x-ray absorptiometry was used to assess whole body composition as well as bone mineral area (BMA), content (BMC) and density (BMD) of the femur and L4 vertebrae. Micro-computed (uCT) x-ray tomography was also used for microarchitectural analysis of the femur and L4 vertebrae. Fecal samples were analyzed for the concentrations of SCFAs acids using gas chromatography while the activity of the estrogen-deconjugating enzyme, β-glucuronidase, was assessed by colorimetric method. Cecal bacteria was analyzed via 16S rDNA sequencing to assess gut microbial diversity. Data were analyzed using 2-way ANOVA and P < 0.05 was considered statistically significant. Results: VCD has no effect on whole body BMA but significantly reduce whole body BMC (PVCD = 0.014) and BMD (PVCD = 0.021). However, there was a significant diet effect on whole body BMC (Pdiet = 0.021) with the RS groups having the highest BMC. Despite the effects of VCD on whole body bone parameters, it has no effect on the bone densitometric parameters of the isolated femur and L4 vertebra. Similar to the whole-body BMC, there was a significant dietary effect on L4 vertebral BMA (Pdiet = 0.003), BMC (Pdiet < 0.0001), and BMD (Pdiet < 0.0001) as well as femoral BMD (Pdiet = 0.007). There was a significant dietary effect on femoral Tb. Th and Tb. N (p = 0.0003, 0.0077 respectively), with RS treatment diets tending to have highest measurements. Mice that were on the RS diets also displayed a significant dietary effect, regardless of estrogen status, on the L4 BV/TV (p = 0.0001), Tb. Sp (p = 0.029), Tb. Th (p = 0.0008) and Th. N (p = 0.0112). Mice that received the RS4 diet had the highest vertebral and femoral BMD followed by RS3 and the PB group had the lowest. The effects of RS treatment on fecal SCFAs concentrations follow the same pattern as its effects on bone. There was a significant dietary effect on total SCFA levels (Pdiet < 0.0001) with RS4 group having the highest total SCFAs followed by RS3 group. Similarly, there was a significant dietary effect on β-glucuronidase activity (Pdiet = 0.0031), specifically RS3 tending (P = 0.059) to have higher β-glucuronidase activity than the control group. Mice in the RS dietary groups, regardless of estrogen status, had an increase in microbiome Beta Diversity and Genus Abundance. Summary: Our findings demonstrate that RS supplementation made more notable improvements in bone densitometric parameters. uCT analysis will give us further insight on the effects of treatment on bone microarchitectural parameters. Moreover, data on gut bacterial diversity will provide more insight of its role on bone health. This study highlights the importance of fiber that are considered as prebiotic and its influence on the gut-bone axis and how this could be utilized to help alleviate conditions related to estrogen deficiency such as osteoporosis.