Bacillus velezensis S141, a soybean growth-promoting bacterium, hydrolyzes isoflavone glycosides into aglycones.

Bacillus velezensis S141, a plant growth-promoting rhizobacteria (PGPR), was isolated from a soybean field in Thailand. Previous studies demonstrated that S141 enhanced soybean growth, stimulating nodulation for symbiotic nitrogen fixation with soybean root nodule bacteria, including Bradyrhizobium diazoefficience USDA110. Isoflavone glycosides are produced in soybean roots and hydrolyzed into their aglycones, triggering nodulation. This study revealed that S141 efficiently hydrolyzed two isoflavone glycosides in soybean roots (daidzin and genistin) to their aglycones (daidzein and genistein, respectively). However, S141, Bacillus subtilis 168, NCIB3610, and B. velezensis FZB42 hydrolyzed isoflavone glucosides into aglycones. A BLASTp search suggested that S141 and the other three strains shared four genes encoding ß-glucosidases corresponding to bglA, bglC, bglH, and gmuD in B. subtilis 168. The gene inactivation analysis of B. subtilis 168 revealed that bglC encoded the major ß-glucosidase, contributing about half of the total activity to hydrolyze isoflavone glycosides and that bglA, bglH, and gmuD, all barely committed to the hydrolysis of isoflavone glycosides. Thus, an unknown ß-glucosidase exists, and our genetic knowledge of ß-glucosidases was insufficient to evaluate the ability to hydrolyze isoflavone glycosides. Nevertheless, S141 could predominate in the soybean rhizosphere, releasing isoflavone aglycones to enhance soybean nodulation.

Development of sandwich ELISAs for detecting glucagon-like peptide-1 secretion from intestinal L-cells and their application in STC-1 cells and mice.

Certain nutrients stimulate glucagon-like peptide-1 (GLP-1) secretion from the intestinal enteroendocrine L-cells, but due to rapid degradation by the DPP-4 enzyme, >90% is converted to inactive metabolite before reaching the target organs via circulation. Plants are a source of potent bioactive compounds that promote endogenous secretion of GLP-1 from L-cells. To search for the effective bioactive compound from a vast library of natural compounds, a reliable and low-cost assay is required considering the high cost of commercial assays. We developed a low-cost sandwich enzyme-linked immunosorbent assays (s-ELISAs) for detecting 'total', 'sensitive active', and 'wide-range active' GLP-1. The s-ELISAs exhibited high sensitivity with measurement ranges between 0.94~240, 0.98~62.5, and 4.8~4,480 pmol/L, respectively. High precision was observed; i.e., CVs within 5% and 20% for intra- and inter-assay variations, respectively, and excellent recovery of exogenous GLP-1 from assay buffer. The developed s-ELISAs had the same performance as the commercial kits and approximately 80% cheaper cost. For their application, cinnamtannin A2-induced GLP-1 secretion was confirmed in STC-1 cells consistent with our previous findings. The s-ELISAs were further validated by measuring plasma GLP-1 level in mice after oral administration of black soy bean seed coat extract containing cinnamtannin A2.

Black soybean seed coat polyphenol ameliorates the abnormal feeding pattern induced by high-fat diet consumption.

High-fat diet (HFD) consumption induces chronic inflammation and microglial accumulation in the mediobasal hypothalamus (MBH), the central regulator of feeding behavior and peripheral metabolism. As a result, the diurnal feeding rhythm is disrupted, leading to the development of obesity. Diet-induced obesity (DIO) can be prevented by restoring the normal feeding pattern. Therefore, functional foods and drugs that ameliorate hypothalamic inflammation and restore the normal feeding pattern may prevent or ameliorate DIO. Numerous functional foods and food-derived compounds with anti-obesity effects have been identified; however, few studies have been performed that assessed their potential to prevent the HFD-induced hypothalamic inflammation and disruption of feeding rhythm. In the present study, we found that polyphenols derived from black soybean seed coat (BE) significantly ameliorated the accumulation of activated microglia and pro-inflammatory cytokine expression in the arcuate nucleus of the hypothalamus of HFD-fed mice, and restored their feeding pattern to one comparable to that of standard diet-fed mice, thereby ameliorating DIO. Furthermore, cyanidin 3-O-glucoside-the principal anthocyanin in BE-was found to be a strong candidate mediator of these effects. This is the first study to show that BE has the potential to provide a variety of beneficial effects on health, which involve amelioration of the HFD-induced hypothalamic inflammation and abnormal feeding pattern. The results of this study provide new evidence for the anti-obesity effects of black soybean polyphenols.

Kaempferol Promotes Glucose Uptake in Myotubes through a JAK2-Dependent Pathway.

Kaempferol possesses various health-promoting functions including antihyperglycemic activity, but its underlying molecular mechanism is poorly understood. Glucose transporter 4 (GLUT4) plays an important role in the uptake of blood glucose into muscle cells after its translocation to the plasma membrane. In this study, we demonstrated that kaempferol at 1.0 nM or more significantly increased the uptake of 2-[3H]- deoxy-d-glucose by 1.3-1.4-fold in L6 myotubes. Kaempferol at 10 pM or more also significantly increased GLUT4 translocation by 1.3-1.6-fold. Kaempferol at 1.0 nM significantly increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) by 2.9-fold, liver kinase B1 and Janus kinase 2 (JAK2) by 1.9-fold, and signal transducer and activator of transcription 3 by 3.7-fold. In addition, kaempferol increased phosphorylation of phosphoinositide 3-kinase (PI3K) by 1.8-fold but not the insulin receptor. Small interfering RNA (siRNA) for AMPK, JAK2, or PI3K canceled kaempferol-induced glucose uptake and GLUT4 translocation. Furthermore, siRNA for JAK2 canceled kaempferol-induced phosphorylation of AMPK and PI3K. These results indicate that a JAK2-depdendent pathway regulates kaempferol-induced glucose uptake and GLUT4 translocation in L6 myotubes and that kaempferol may be an effective compound for the prevention of hyperglycemia.

Cacao polyphenols regulate the circadian clock gene expression and through glucagon-like peptide-1 secretion.

Energy metabolism and circadian rhythms are closely related together, i.e., the timing of nutrient intake affects metabolism under the regulation of circadian rhythms. Previously, we have reported that cacao liquor procyanidin (CLPr) promotes energy metabolism, resulting in preventing obesity and hyperglycemia. However, it is not unclear whether CLPr regulates clock gene expression. In this study, we investigated whether the administration timing of CLPr affected clock gene expression and found that CLPr regulated the circadian clock gene expression through the glucagon-like peptide-1 (GLP-1) signaling pathway. CLPr administration at Zeitgeber time 3 increased the expression level of Per family and Dbp in the liver. At the same administration timing, CLPr increased GLP-1 and insulin concentration in the plasma and phosphorylation of AMPK in the liver. It was noteworthy that an antagonist for GLP-1 receptor Exendin (9-39) canceled CLPr-increased expression of Per family and Dbp and phosphorylation of AMPK in the liver, in addition to insulin secretion. These results strongly suggest that CLPr-induced GLP-1 regulates the changes in clock gene expression in the liver through increased insulin. Thus, CLPr is a possible functional food material for prevention and/or amelioration of metabolic disorders through preventing circadian disruption through GLP-1 and AMPK pathways.

The cacao procyanidin extract-caused anti-hyperglycemic effect was changed by the administration timings.

Mammals have the biological clocks with approximately 24 h-rhythm. Energy metabolism including glucose metabolism is regulated by the biological clocks. Glucose metabolism is affected by not only meal volume and its energy but also meal timing. We have reported that cacao liquor procyanidin-rich extract (CLPr) ameliorated the postprandial hyperglycemia through AMP-activated protein kinase pathway. However, the effect of administration timing of CLPr on the postprandial hyperglycemia and its signaling pathway are still unclear. In the present study, we compared the effect of CLPr-administration at the rest-phase (light-period) and active-phase (dark-period) on glucose metabolism. Single oral administration of CLPr to ICR mice at the rest-phase, but not at the active-phase, promoted phosphorylation of AMP-activated protein kinase and its upstream liver kinase B1 and translocation of glucose transporter 4 to the plasma membrane in the skeletal muscle, resulting in reduced postprandial hyperglycemia. These results indicated that the intake of CLPr at the rest-phase more effectively suppressed postprandial hyperglycemia.

Prevention effect of quercetin and its glycosides on obesity and hyperglycemia through activating AMPKα in high-fat diet-fed ICR mice.

Quercetin and its glycosides possess various health beneficial functions, but comparative study of them on energy metabolism in different tissues are not well studied. In this study, we investigated AMP-activated protein kinase regulated glucose metabolism in the skeletal muscle and lipid metabolism in the white adipose tissue and liver to compare the effectiveness of quercetin and its glycosides, namely isoquercitrin, rutin, and enzymatically modified isoquercitrin, in male ICR mice. The mice were fed a standard or high-fat diet supplemented with 0.1% quercetin and its glycosides for 13 weeks. Quercetin glycosides, but not quercetin, decreased body weight gain and fat accumulation in the mesenteric adipose tissue in high-fat groups. All compounds decreased high-fat diet-increased plasma glucose and insulin levels. Moreover, all compounds significantly increased AMP-activated protein kinase phosphorylation in either standard or high-fat diet-fed mice in all tissues tested. As its downstream events, all compounds induced glucose transporter 4 translocation in the muscle. In the white adipose tissue and liver, all compounds increased lipogenesis while decreased lipolysis. Moreover, all compounds increased browning markers and decreased differentiation markers in adipose tissue. Therefore, quercetin and its glycosides are promising food components for prevention of adiposity and hyperglycemia through modulating AMP-activated protein kinase-driven pathways.