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. At the end of the treatment period, the rats were anesthetized via intraperitoneal with 3% pentobarbital sodium . Blood samples were collected by cardiac puncture, the blood was centrifuged for 15 min at 13,000 rpm and the plasma was recovered and stored in aliquots of 500 µl at −20◦C until use. Retroperitoneal adipose tissue was aliquoted, isolated, and washed with PBS and frozen immediately with liquid nitrogen. The frozen samples were stored at −80◦C. The stored serum samples were thawed at 4◦C. Insulin levels were quantified using the Rat Insulin ELISA kit RayBio R. Peachtree Corners, GA. The levels of total cholesterol, triglycerides, HDL-Cholesterol, and LDL-Cholesterol in the serum samples were measured with commercial kits for colorimetric assays. Tumor Necrosis Factor alpha was quantified with ELISA kit according to manufacturer protocols. RNA extraction from adipose tissue samples were carried out according to the protocol proposed in the RNeasy Lipid Tissue Mini Kit . The sequencing library was prepared by random fragmentation of the cDNA,raspbery container followed by the 5 ’and 3’ ligation adapters. Adapter-ligated fragments were amplified by PCR and visualized on agarose gels. Double stranded next generation sequencing RNAseq was commercially performed by MAcrogen on Illumina HiSeq4000 by triplicate in each group.

All RNAseq data are available at GEO database under accession number GSE215903. fter a low dose injection of STZ, rats in BB and DB groups showed significantly increased fasting blood glucose levels compared with animals from HE group, this tendence was observable during the 5 weeks treatment and at the end of the treatment as showed in Figure 1C. Furthermore, STZ administration resulted in decreased body weight in BB and DB groups , but no difference was observed on adipose tissue weight . These findings demonstrate the success of T2DM induction in Wistar rats.Two of the main characteristics of diabetic patients are the body weight decrease and the chronic increase of blood glucose levels. As exposed in Figure 1, the body weight of all the rats increased progressively, and their blood glucose levels were normal. However, the growth rate of the rats in the HE group was slower. Conversely, after STZ administration, the body weight of all rats started to decrease, and their blood glucose levels augmented significantly compared with rats in HE group. Moreover, rats in both the BB and DB groups tended to show a decrease in weight after STZ administration. The effect of BB on the fasting blood glucose level of the rats is shown in Figure 1A, from which it is obvious that the blood glucose levels of rats in the BB group decreased gradually after 1 week of treatment. At the end of treatment period, rats in BB group showed significant decreases in fasting blood glucose levels. The levels of insulin showed a down trend, even though the decreases were not significant between BB and DB groups . Further, TNF-α levels in the BB group exhibited a significant decrease when compared with DB group . With the aim to identify genomic impact of BB extract on genomic profile in adipose tissue, we performed global RNAseq. Following statistical analysis, we identified 566 significantly differentially expressed genes. A closer understanding into identified differentially expressed genes demonstrated that there are 406 among them that are protein coding genes , 33 are miRNA family genes, 39 are long non-coding genes, 3 are snRNAs and 85 are unidentified . The fold changes of these genes were observed to fluctuate from −1.15 to −13.45 for down regulated genes and from 1.14 to 22.91 for up-regulated genes.

These data suggest that the consumption of BB extract can considerably affect the expression of genes, not only protein coding but also protein non-coding genes in adipose tissue. The list of differentially expressed genes were then submitted to functional bio-informatic analyses.With the objective to inquire in the cellular functions of significantly moderated protein coding genes, we first conducted gene ontology enrichment analysis using Meta scape 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 down regulate 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,growing raspberries in container this modulates the activity of downstream cell signaling proteins and consequently transcription factors.The described changes could result in the observed gene expression modifications. 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, 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 LncRRI research 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. 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 insulinindependent 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. These changes will result in higher production of short chain fatty acids, lower intestinal permeability and pH, greater number of goblet cells and improvement of villi anatomy .