A study conducted on diabetic Zucker rats supplemented with an anthocyanin-rich extract from purple potato and bilberry, showed a reduction in dysbiosis of colonic microbiota and anthocyanins gut microbiota-derived metabolites, increased cecal sugar levels together with the increase in the abundance of Peptostreptococcaceae sp. and Parabacteroides spp. in colon. These results demonstrate that anthocyanins by modulating the gut microbiota can affect gut function and consequently prevent T2DM . Our RNAseq analysis showed for the first time that an anthocyanin-rich BB extract can affect the expression of large number of genes in adipose tissue, not only protein coding but also non-coding genes like miRNAs or lncRNAs. According to our knowledge, very few studies have reported the multigenomic effect of BB anthocyanins, as well as isolated anthocyanins, on adipose tissue. It has been observed that cyanidin 3-glucoside, anthocyanin from BB, can up-regulate the expression of GLUT4 gene in vitro in 3T3-L1 cells . The BB extract rich in flavonoids and saponins was also shown to exert the capacity to modulate the expression of key genes involved in lipogenesis, such as SREBP1c, ABCG5, CPT1, SREBP2, FAS, HMGCR, INSIG1 and INSIG2 . Supplementation of mice diet with BB has been reported to modulate the expression of the SCFAG-protein coupled receptors in colonic tissue as well as the expression of genes regulated the integrity of the epithelial barrier: occludin, JAM-A, E-cadherin and the junction/cytoskeletal connector ZO-1 . Recently it has been observed that black bean concentrate improves hepatic steatosis by reducing lipogenesis and increasing fatty acid oxidation in rats fed a high fat-sucrose diet . However, these studies present an important drawback,large plastic pots for plants which is use of targeted approach to assess the expression of few specific genes.
Rare recent studies have shown that anthocyanins can affect large number of genes presenting multi-modal action. For example, anthocyanins from blackberries affected the expression of over 600 genes, including miRNAs, in circulating immune cells in human volunteers . Another study has demonstrated the capacity of anthocyanin-rich bilberry extract to modulate the expression of over 1,600 genes in hippocampus of ApoE-/- mice . Enrichment pathway analysis in adipose tissue were performed using 406 differentially expressed genes following BB extract intake. This allowed us to show that the black bean extract can affect genes regulating cell signaling pathways in adipose tissue from diabetic rats, pathways related with phosphatase activity, phospholipid metabolism, phosphatidyl inositol 3-kinase binding, phosphatidylserine binding, and ER organization. On the other hand, we also found down-regulated pathways that participate in the modulation of NIK/NF-κB signaling, regulation of response to extracellular stimulus, positive regulation of cell junction assembly, negative regulation of cell population proliferation and negative regulation of cytokine-mediated signaling pathway. Remarkably, while regulation of NIK/NF-κB signaling is up-regulated in the DB group, we observed that the treatment with BB extract down-regulated this pathway in the BB group, suggesting the capacity of the extract to counteract the effect of T2D. The biological relevance of this pathway on adipose tissue is the critical function in reprogramming the fat cell transcriptome to inflammation in response to over nutrition and metabolic stress. The inhibition of NF-κB signaling has metabolic advantages for adipose tissue inflammation caused by obesity . On the other hand, NF-κB inducing kinase is a key controller of immunity and inflammation at local and systemic levels on metabolic processes.
In adipose tissue, NIK promotes adipogenesis by activating non-canonical NF-κB pathway, demonstrated by Pflug et al., when NIK deficient mice fed with a high fat diet showed decreased overall fat mass, increased insulin sensitivity, and energy expenditure . Moreover, anti-inflammatory effects have also been attributed to anthocyanins from Hibiscus syriacus L. by the capacity to inhibit TLR4 in LPS-induced cells, followed by the decrease of the phosphorylation of MyD88 and IRAK4, which resulted in NF-κB inactivation . Black soybeans also demonstrated to inhibit IκB phosphorylation that impede NF-κB translocation resulting in the inhibition of iNOS transcription and iNOS and COX-2 translation . Taken together, these results suggest that extract from BB can exert health properties by countering the genomic modifications induced by T2DM. Elevated levels of FFAs, inflammation, excess of nutrients, inadequately folded proteins, and local hypoxia, are characteristics of obesity and can lead to ER stress. This results in increased oxidative stress in adipose tissue of obese animals . ER plays an important role in adipose tissue inflammation due to their participation in the activation of NF-κB signaling by the phosphorylation of JNK, IKK, and JNK-mediated phosphorylation of IRS1/2 triggering the unfolded protein reaction and involving pathways like PERK , IRE1 , and ATF6 . Exposure of J774A.1 macrophages to a cyanidin-3-O-galactoside-rich aqueous extract of Sambucus ebulus L. resulted in suppression of LPS-induced transcription of pro-inflammatory genes like IL-1β, IL-6, TNF-α, Ccl2, Icam-1, Fabp4, COX2, iNOS, Noxo1, IL-1ra and Sirt1. SE also showed to produce the decrease in protein levels of iNOS, peIFα, ATFα and C/EBP Homologous Protein . Similarly, Punica granatum L. a high-content polyphenols Chinese plant, was administrated to diabetic rats for 4 weeks. It was observed that doses between 50 and 100 mg/kg can improve ER stress signals including IRE1, activation of XBP-1, as well as lower levels of IREα, XBPs, and CHOP . Therefore, anthocyanins present in our extract also have the capacity to modulate the expression of genes regulating ER stress, presenting another important molecular target underlying the anti-diabetic effects.
Our bio-informatic analysis also revealed that phosphatidylinositol 3-kinase binding signaling pathway was up-regulated in BB group. The importance of this pathway in adipose tissue is due to its role in adipose hypertrophy which rises tissue immune cell infiltration, fibrosis, and lipolysis. Decreases IRS-1 activation and AKT-induced glucose uptake, as well as exacerbates systemic insulin resistance and the development of T2DM. Different factors like adipocytokines and adipose hypertrophy produce insulin resistance by obstructing PI3K/AKT-mediated inhibition of lipolysis reducing the capacity of glucose utilization and diminishing the capacity of SREBP to stimulate lipid synthesis . Recent research concluded that sweet potato leaf polyphenols might up-regulate the important mediators of the insulin-mediated PI3K/AKT/GSK-3β signaling pathway in a dose-dependent way in diabetic mice by regulating the mRNA expression of genes IR, IRS-1, PI3K, AKT and GLUT-4 . Bioinformatic analysis of differentially expressed genes permitted to identify potential transcription factors whose activity can be affected by BB extract and result in the detected nutrigenomic modifications. Among these transcription factors are GATA2, POU2AF1, IRF3, GATA1, NR2F2 or PPARA. Interestingly some of these transcription factors have been identified as playing a role in adipose tissue development and/or diabetes. For example, it has been shown that GATA2 plays an important role in diabetes development and associated diseases . It has been described that hyperinsulinemia, observed during Type 2 diabetes, can activate NR2F2 which can induce development of different diseases . PPARs have central role in lipid and glucose homeostasis, pathogenesis of insulin resistance and metabolic syndromes and are active in adipose tissue. It has been shown that binding phenolic and other molecules to PPARs result in significant changes in their activities and present important mode of treatment type 2 diabetes mellitus. . Similarly, IRF3 has been described as a main transcriptional regulator of adipose tissue inflammation and is involved in preserving systemic glucose and energy homeostasis . One study also observed that IRF3 in adipose tissue promotes adipose inflammation and insulin resistance . These observations from our bioinformatic analysis reveal significant regulators of BB extract underlying their observed health properties. Moreover, our genomic analysis revealed that black bean extract rich in anthocyanins can also modulate the expression of microRNAs. MicroRNAs are short, non-coding RNAs which can bind to mRNAs,plant pots with drainage resulting in modification in translational repression. It is estimated that there are approximately 2200 miRNA genes in the human genome that can regulate the transcriptional levels of over 60% of genes. Consequently, they can regulate many major cellular functions such as development, differentiation, growth, or metabolism . Moreover, it has been shown that modifications in their expression produce an important role in progress of diseases. Among these, it has been demonstrated that miRNA might have an important role in development of diabetes and metabolic disorders . On the other hand, several previous studies have shown that anthocyanins can modulate the expression of these non-coding RNAs. For example, it was shown that a mixture of anthocyanins as well as their gut microbiome derived metabolites at physiologically relevant concentrations can affect the expression of miRNAs in isolated human primary endothelial cells . Also, a study showed that supplementation of mice diets with different polyphenols at nutritionally relevant doses can significantly affect global miRNA expression profile in liver . Among the miRNAs identified as differentially expressed by black bean extract are miR-152, miR-219a1, miR-384 or miR-615.
It has been observed that miR-152 is expressed in adipocytes and can stimulate lipid accumulation in preadipocytes accompanied by higher expression of some pro-adipogenic genes, adipogenesis and intramuscular fat formation . Its expression is also altered in patients with T2D with or without medication . miR-219a1 has been suggested to show a role in fat development. Similarly, it has been demonstrated that miR-615 is expressed in adipose tissue in humans and that its expression changes with the obesity . Moreover, the expression of this miRNA has also been identified in patients with diabetes . Together with miRNAs, our analysis also revealed changes in the expression of long non-coding RNAs. Biogenesis of lncRNAs is different than mRNAs and can interact with DNA, RNA, and proteins, modulating chromatin function, changing the stability and translation of cytoplasmic mRNAs and interfering with signaling pathways . Because of their large mode of action, they affect numerous cellular and consequently physio-pathological processes. It has been shown that they regulate adipogenesis and adipose tissue function and diabetes . Only few studies have suggested that polyphenols are potent modulators of the expression of noncoding RNAs. For example, epicatechin metabolites can regulate the expression of these miRNAs in human brain endothelial cells in physiologically relevant conditions . Therefore, our study provides novel and original data about the capacity of legumes and their bio-actives to affect the expression of this class of RNAs. However due to the lack of known biological functions of these lncRNAs as well as their targets, regulated cellular and molecular processes remain to be identified. Taken together, our results suggest that black bean extract rich in anthocyanins exert protective properties by modulating the expression of miRNAs and lncRNAs in adipose tissue in diabetic conditions.From a microbiological perspective, soil is largely unexplored even though we know it has a rich diversity of microbial life. Depending on its physical and chemical properties, soil can contain 109-1010 microbial cells per gram, including tens of thousands of different bacterial, archaeal, and fungal species, plus viruses and protists. Soil microbes carry out life-sustaining functions for our planet, including cycling of nutrients and promoting plant growth. Respiring soil microorganisms, producing metabolic byproducts such as carbon dioxide and methane, cycle enormous volumes of carbon-containing gases from the terrestrial ecosystem into the atmosphere. Perturbing this ecosystem—for example, when global warming raises temperatures—potentially alters the flux of these gases. Despite the immensity of the carbon reservoir in soil , its fate in the face of climate change is not known. The high diversity of soil microbial communities makes them difficult to study. Exacerbating this difficulty, few soil microorganisms are amenable to isolation and cultivation, steps that in conventional terms are crucial for elucidating microbial physiology and biochemistry. Although most isolated soil bacteria are “weeds” such as pseudomonads and actinobacteria, 16S rRNA gene surveys reveal that few species from the great bulk of soil bacteria have cultured representatives. For example, members of the Acidobacteria phylum are widespread in soil but are notoriously difficult to cultivate. To address these issues, microbial ecologists are now using a metagenomics approach, sequencing soil DNA to develop a better understanding of the microbial identities and their potential functions in soils. Initially, investigators who used metagenomics to study soil microbial communities cloned DNA extracted from soil into bacterial artificial chromosome vectors or fosmid vectors. One advantage of this cloning-based approach is that it permits investigators to screen for expression of particular phenotypes, including production of antibiotics and enzymes.