The seed color of BB is determined mainly by the presence of anthocyanins, and condensed tannins . As one of the main flavonoid groups found in BB, anthocyanins have been shown to determine the color of the seed coats, but also demonstrated to be biologically active and have potential health properties . BB have been suggested to contribute to the treatment of T2DM. Anthocyanins from BB have a strong antioxidant ability to arrest free radicals along with anti-inflammatory activity . We previously showed through an in silico perspective, that polyphenols found in BB, particularly anthocyanins, could modulate the activity of proteins involved in different mechanisms of T2DM pathways . Molecular docking results highlighted that cyanidin 3-glucoside, delphinidin 3-glucoside, and petunidin 3-glucoside had a stronger affinity for 11β- HS, GFAT, PPARG, PTP, RTKs, and PTP, proteins related with mechanisms that can regulate different biomarkers linked to inflammation, insulin resistance, oxidative stress, glucose and lipid metabolism, insulin secretion, and carbohydrate absorption . The mode of action underlying the anthocyanin biological effects has been attributed only to their direct antioxidant properties. However in recent years, it has been shown that these bio-active compounds exert more complex molecular mechanisms of action, plastic gardening pots including modulation of gene expression, cell signaling, or DNA methylation .
Most of the studies performed used targeted approaches so global mechanisms are still little known. Taken together, the aim of this study was to characterize anti-diabetic properties of an anthocyanin-rich extract from BB and decipher, using RNAseq approach, molecular mechanisms of action of these bio-active compounds on adipose tissue in a diabetic rat model.We previously reported the chemical composition of BB extract . Polyphenol extraction from BB was performed on recently harvested BB which were finely ground until obtaining a flour consistency. The flour was mixed with a solution of ethanol and hydrochloric acid . The mix was stirred for 4 h at room temperature and covered from light. The extracts were centrifuged for 20 min at 13,000 rpm, the supernatant collected and evaporated at 38◦C and 90 rpm until ethanol was completely removed. The extracts were stored at −20◦C overnight and lyophilized during three days at −50◦C and 250 mBar. The polyphenolic powders were conserved at 4◦C until their use .The animal experiments were approved by the Internal Committee of Care and Use of Laboratory Animals of the Center for Research and Assistance in Technology and Design of the State of Jalisco, A.C., according with the Official Mexican Standard NOM-062-ZOO- 1999, concerning the technical specifications for the production, care and use of laboratory animals and NOM-087-ECOLSSA1-2002, related to the Management of Infectious Biological Hazardous Waste -RBPI. In addition, the Standard Bioterial Operation Procedure and the Animal Experimentation Laboratory Regulations were applied.
A total of 24 male Wistar rats around 150 ± 20g were purchased from Envigo RMS S.A de C.V. and placed in a SPF barrier environment under standard environmental conditions under 12 h light/dark cycle, with free access to water and standard diet during the acclimation stage. After 2 weeks of acclimatization, all the rats were randomly divided into three groups , namely the HE , BB , and DB animals with induced T2DM without treatment . During the experiment the HE group was fed with standard diet, while BB and DB groups were fed with a high fat diet consisting of 42.7% carbohydrates, 42% lipids and 15.2% proteins. After the HFD feeding for 5 weeks, the rats were fasted for 12 h with unlimited access to water. Rats from HE group received an intraperitoneal injection of citrate buffer. Our T2DM induction is supported by the methodology reported by Skovsko, , where the prolonged administration of HFD combined with a single low dose of Streptozotocin produce a partial damage of pancreatic B-cells accompanied by lipotoxicity, glucolipotoxicity, insulin resistance, and hyperinsulinemia caused by HFD. Rats in BB and DB groups received an intraperitoneal injection of a single low dose of Streptozotocin, N–α- D-glucosamine solution dissolved in sodium citrate buffer . The successful diabetes induction was confirmed with blood glucose measurements from the tail vein with a glucometer , all the measurements were taken during fasting and postprandial periods during 3 days after injection. Values of fasting glucose higher than 200 mg/dl indicated a successful establishment of T2DM rat model. The BB extract was diluted in 1ml of water and administrated by oral gavage for 31 days.
The fasting glucose levels in blood were tested every 2 days per week from the tail vein. Water and food consumption were quantified every day and body weight was monitored once a week.With the objective to inquire in the cellular functions of significantly moderated protein coding genes, we first conducted gene ontology enrichment analysis using Metascape and Cytoscape tools. Gene ontology analysis by p-value indicated that black bean extract impacted numerous biological functional categories that include cell substrate junction assembly, phosphatidylinositol phosphate binding, fat pad development, regulation of cysteine-type endopeptidase activity, among others . Additionally, we performed a network analysis of over-represented gene ontologies where terms with a p-value <0.05, a minimum count of 3, and an enrichment factor >1.5 are collected and grouped into clusters based on their similarities . To obtain a more detailed understanding of the cellular functions that are regulated by protein coding genes significantly modulated by black bean extract, we then performed pathway enrichment analysis of up- and down-regulated differentially expressed genes using Metascape tool . The results showed that anthocyanin-rich BB extract changed the expression of genes up regulating important pathways in T2DM pathogenesis like insulin secretion, cell-substrate junction assembly, ER organization, phosphatidylserine binding, phosphatidylinositol 3-kinase binding, among others. On the other hand, BB extract also altered the expression of genes that downregulate signaling pathways involved with regulation of NIK/NF-kappaB, regulation of response to extracellular stimulus, positive regulation of cell junction assembly, negative regulation of cell population proliferation, cell adhesion molecules and negative regulation of actin filament polymerization.Our next step was to use STRING database to explore the potential protein–protein interactions of genes identified as differentially expressed by black bean extract intake. The analysis revealed a network of interactions between identified proteins as presented in Figure 5A, as well as genes that form nodes in the network. The next step was to select the genes with the highest number of interactions with other genes and which potentially play an important role in multi-genomic effects. The number of interactions reached 12 for UBB , or 11 for MST1R and RRAS2 , or proteins like INS1 or INPPL1 with 5 or more interactions . Interestingly, pathway enrichment analyses of hub proteins conducted in GeneTrail revealed that these genes are involved in insulin signaling, mature onset of diabetes, insulin resistance, inositol phosphate metabolism or AMPK signaling pathway .Our next objective was to identify transcriptional regulators involved in the observed changes of genes, that is, transcription factors which could have their activity altered by black bean extract and affect the expression of identified significantly modulated genes. To this end, we used the database TRANSFAC and JASPAR using the Enrichr platform. Among the top ten transcription factors identified are GATA2, POU2AF1, IRF3, GATA1, NR2F2 or PPARA . It could be suggested that circulating polyphenol metabolites generated after BB extract intake could interact with transcription factors and/or cell signaling proteins regulating their activity. With the aim to test this hypothesis, we searched the capacity of major metabolites of black bean to interact and bind to these proteins using a 3D docking online server. We assessed the binding capacity of 3 major metabolites, delphinidin 3-glucoside, petunidin 3-glucoside and malvidin 3-glucoside: delphinidin 3-glucoside to GATA2 ; delphinidin 3-glucoside to POU2AF1 ; petunidin 3-glucoside to GATA2 ; malvidin 3-glucoside to GATA2 and malvidin 3-glucoside to POU2AF1 . We observed that petunidin 3-glucoside showed potential binding capacity of -6.4 kcal/mol to POU2AF1, as well as petunidin 3-glucoside and delphinidin 3-glucoside with GATA2 , and POU2AF1 , respectively. These results shows that anthocyanins in BB can interact with cell signaling proteins and produce changes in their kinase activity, this modulates the activity of downstream cell signaling proteins and consequently transcription factors. Our gene expression analysis also allowed us to surmise that BB can also lead to alterations in the expression of not only protein coding RNAs but also non-coding RNAs,such as miRNAs.
We observed changes in expression of 33 miRNAs, including Mir615, Mir152, Mir219a1 or Mir384. Using existing database, blueberry pot size we searched for target genes of the identified miRNAs, and nearly 500 target genes were identified. These target genes and identified miRNAs form a network of interactions as presented in the Figure 7A. To identify potential cellular functions affected by these miRNAs, we cross-examined the Mienturnet database to reveal over-represented pathways from both KEGG and Reactome databases, that is pathways associated with each of the miRNA recognized as differentially expressed by black bean extract. Among the pathways identified are PI3K- signaling pathway, Ras signaling pathway, type 1 diabetes mellitus, Insulin receptor substrate 1 related pathway, and regulation of insulin-like growth factor.As we mentioned in the materials and methods section, we were not able to perform the RNA-lncRNAs interaction analysis in LncRRIresearch web server. However, in Supplementary Figure 1 we show the list of 39 lncRNAs with their fold changes that were modulated by the anthocyanin-rich BB extract.Together with identification of cellular mechanisms affected by different types of RNAs, we also aimed to identify diseases associated with identified differentially expressed genes. We used the Enrichr database OMIM disease tool that interconnects differentially expressed genes with diseases, revealing their possible role in prevention or development of these disorders. We observed that our genes differentially expressed between the BB and DB groups are significantly associated with metabolic disease, nutrition disorder, cardiovascular disease, and immune system disease .In this research we investigated the potential health benefits and the multigenomic mode of action of a rich-anthocyanin extract from BB on adipose tissue in the context of dietstreptozocin-induced type 2 diabetes mellitus. After 4 weeks dietary supplementation, we found that black bean extract improved the symptoms of T2DM and insulin resistance, controlled the levels of blood glucose, and pro-inflammatory cytokines. The use of RNAseq revealed a complex multigenomic mode action of these bio-active compounds in adipose tissue by modulating expression of protein coding, miRNA, transcription factors, and lncRNAs , regulating processes like inflammation, metabolism and cell signaling. In the last years, special interest was given to research on natural and non-toxic antidiabetic agents. Plants are important sources of bio-active compounds with numerous valuable health effects. Functional foods contain bio-active compounds that can exert health advantages beyond their natural properties when consumed in a regular and consistent manner through diet . Anthocyanins are an important class of polyphenols featured by their promising effects on T2DM acting on suppression of carbohydrate-metabolizing enzymes; decrease of glucose transporters expression or activity; inhibition of glycogenolysis and modifying the gut microbiota by anthocyanin breakdown products . In this research we found that an anthocyanin-rich BB extract improved glucose levels on diabetic rats. One of the effects of anthocyanins in T2DM is the suppression of postprandial glycaemia through the inhibition of α-amylase and α-glucosidase enzymes. In a study conducted by Törrönen et al., the authors evaluated the effect of berries, naturally rich in anthocyanins, on postprandial glucose levels in healthy volunteer adults. Another study showed that consumption of a rich-anthocyanin puree containing bilberries, blackcurrants, cranberries, strawberries, and 35 g of sucrose resulted, after 15 and 30 min, in lower levels of glucose when compared with the control group that only consumed sucrose . Using in silico and in vivo studies, pelargonidin-3-O-rutinose present in strawberries exhibited the potential to improve postprandial hyperglycemia by inhibiting α-glucosidase . Another mechanism of action of anthocyanins is their impact on glucose transporters. It was demonstrated that an anthocyaninrich berry-extract considerably decreased sodium-dependent and sodium-independent transporters in Caco-2 cells and also reduced the expression of genes encoding SGLT1 and GLUT2, suggesting that anthocyanins can regulate the rate of glucose absorption . The over-expression of glycogenolysis in the liver releases glucose into the bloodstream; glycogen synthase kinase is a key liver enzyme that inhibits glycogen synthase enzyme to convert glycogen to glucose. Herrera-Balandrano et al., investigated the hypoglycemic effects of malvidin from a blueberry anthocyanin extract and observed that BAE could improve insulin sensitivity by inhibiting GSK3β and glycogen synthase in the insulin independent pathway . Moreover, a recent systematic review describes that anthocyanins can also exert their health effects by modulating the gut microbiota composition, particularly by increasing Bacteroidetes and decreasing Firmicutes.