Identification and characterization of multidomain monothiol glutaredoxin 3 from diploblastic Hydra.

Intracellular antioxidant glutaredoxin controls cell proliferation and survival. Based on the active site, structure, and conserved domain motifs, it is classified into two classes. Class I contains dithiol Grxs with two cysteines in the consensus active site sequence CXXC, while class II has monothiol Grxs with one cysteine residue in the active site. Monothiol Grxs can also have an additional N-terminal thioredoxin (Trx)-like domain. Previously, we reported the characterization of Grx1 from Hydra vulgaris (HvGrx1), which is a dithiol isoform. Here, we report the molecular cloning, expression, analysis, and characterization of another isoform of Grx, which is the multidomain monothiol glutaredoxin-3 from Hydra vulgaris (HvGrx3). It encodes a protein with 303 amino acids and is significantly larger and more divergent than HvGrx1. In-silico analysis revealed that Grx1 and Grx3 have 22.5% and 9.9% identical nucleotide and amino acid sequences, respectively. HvGrx3 has two glutaredoxin domains and a thioredoxin-like domain at its amino terminus, unlike HvGrx1, which has a single glutaredoxin domain. Like other monothiol glutaredoxins, HvGrx3 failed to reduce glutathione-hydroxyethyl disulfide. In the whole Hydra, HvGrx3 was found to be expressed all over the body column, and treatment with H2O2 led to a significant upregulation of HvGrx3. When transfected in HCT116 (human colon cancer cells) cells, HvGrx3 enhanced cell proliferation and migration, indicating that this isoform could be involved in these cellular functions. These transfected cells also tolerate oxidative stress better.

Characterization of palmitic acid toxicity induced insulin resistance in HepG2 cells.

BACKGROUND: An etiology of palmitic acid (PA) induced insulin resistance (IR) is complex for which two mechanisms are proposed namely ROS induced JNK activation and lipid induced protein kinase-C (PKCε) activation. However, whether these mechanisms act alone or in consortium is not clear. METHODS AND RESULTS: In this study, we have characterized PA induced IR in liver cells. These cells were treated with different concentrations of PA for either 8 or 16 h. Insulin responsiveness of cells treated with PA for 8 h was found to be same as that of control. However, cells treated with PA for 16 h, showed increased glucose output both in the presence and in absence of insulin only at higher concentrations, indicating development of IR. In these, both JNK and PKCε were activated in response to increased ROS and lipid accumulation, respectively. Activated JNK and PKCε phosphorylated IRS1 at Ser-307 resulting in inhibition of AKT which in turn inactivated GSK3ß, leading to reduced glycogen synthase activity. Inhibition of AKT also reduced insulin suppression of hepatic gluconeogenesis by activating Forkhead box protein O1 (FOXO1) and increased expression of the gluconeogenic enzymes and their transcription factors. CONCLUSION: Thus, our data clearly demonstrate that both these mechanisms work simultaneously and more importantly, identified a threshold of HepG2 cells, which when crossed led to the pathological state of IR in response to PA.

Evaluation of HbA1c from CGM traces in an Indian population.

Introduction: The development of continuous glucose monitoring (CGM) over the last decade has provided access to many consecutive glucose concentration measurements from patients. A standard method for estimating glycated hemoglobin (HbA1c), already established in the literature, is based on its relationship with the average blood glucose concentration (aBG). We showed that the estimates obtained using the standard method were not sufficiently reliable for an Indian population and suggested two new methods for estimating HbA1c. Methods: Two datasets providing a total of 128 CGM and their corresponding HbA1c levels were received from two centers: Health Centre, Savitribai Phule Pune University, Pune and Joshi Hospital, Pune, from patients already diagnosed with diabetes, non-diabetes, and pre-diabetes. We filtered 112 data-sufficient CGM traces, of which 80 traces were used to construct two models using linear regression. The first model estimates HbA1c directly from the average interstitial fluid glucose concentration (aISF) of the CGM trace and the second model proceeds in two steps: first, aISF is scaled to aBG, and then aBG is converted to HbA1c via the Nathan model. Our models were tested on the remaining 32 data- sufficient traces. We also provided 95% confidence and prediction intervals for HbA1c estimates. Results: The direct model (first model) for estimating HbA1c was HbA1cmmol/mol = 0.319 × aISFmg/dL + 16.73 and the adapted Nathan model (second model) for estimating HbA1c is HbA1cmmol/dL = 0.38 × (1.17 × ISFmg/dL) - 5.60. Discussion: Our results show that the new equations are likely to provide better estimates of HbA1c levels than the standard model at the population level, which is especially suited for clinical epidemiology in Indian populations.

Effect of long-term oral glutathione supplementation on gut microbiome of type 2 diabetic individuals.

The aim of this study was to check the effect of long-term oral glutathione (GSH) supplementation on alteration in gut microbiome of Indian diabetic individuals. Early morning fresh stool sample of diabetic individuals recruited in a randomized clinical trial wherein they were given 500 mg GSH supplementation orally once a day for a period of 6 months was collected and gut microbiome was analysed using high throughput 16S rRNA metagenomic sequencing. Long-term GSH supplementation as reported in our earlier work showed significant increase in body stores of GSH and stabilized decreased glycated haemoglobin (HbA1c). Analysis of gut microbiome revealed that abundance of phylum Proteobacteria significantly decreased (P < 0.05) in individuals with GSH supplementation after 6 months compared to those without it. Beneficial dominant genera such as Megasphaera, Bacteroides, and Megamonas were found to be significantly enriched (P < 0.05), while pathogenic Escherichia/Shigella was found to be depleted (P < 0.05) after supplementation. Data clearly demonstrate that GSH supplementation along with antidiabetic treatment helps restore the gut microbiome by enriching beneficial bacteria of healthy gut and reducing significantly the load of pathogenic bacteria of diabetic gut.

Genetic polymorphisms in Nrf2 and FoxO1: implications for antioxidant enzyme activity in diabetes.

In diabetes, persistent hyperglycemia generates excess reactive oxygen species (ROS), leading to oxidative stress (OS). In response to OS, transcription factors (TFs) Nrf2 and FoxO1 get activated, which induce the expression of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). It is well documented that the antioxidant response in diabetic individuals is very low. Since Nrf2 and FoxO1 are the major TFs activating these genes, we were interested in determining if single nucleotide polymorphisms (SNPs) in genes for these TFs have any association with lowered antioxidant enzyme activity in diabetic individuals. The activity of CAT and SOD and total antioxidant capacity (TAC) were quantified from the serum samples of diabetic (n = 98) and non-diabetic (n = 90) individuals. Genomic DNA was isolated, and Nrf2 and FoxO1 were amplified and sequenced by Illumina NextSeq500. Data were screened for SNPs in amplified regions. An independent samples t-test to find an association between CAT, SOD, and TAC and allele frequency of SNP with the diabetic condition was carried out. We found decreased CAT and SOD activity and significantly low TAC in diabetic individuals. Thirty-two and thirty-four SNPs and Single-nucleotide variants (SNVs) were observed in Nrf2 and FoxO1, respectively. However, a statistically significant difference in the allele frequency distribution between study groups was observed only in two intronic SNPs, rs17524059:A > C and rs60373589:Indel(A) of Nrf2 and FoxO1, respectively. SNPs, rs17524059 in the Nrf2 and rs60373589 of FoxO1, were not associated with reduced CAT and SOD activity and level of TAC in Indian diabetic individuals.Communicated by Ramaswamy H. Sarma.

Glutaredoxin 1 from Evolutionary Ancient Hydra: Characteristics of the Enzyme and Its Possible Functions in Cell.

Glutaredoxin (Grx) is an antioxidant redox protein that uses glutathione (GSH) as an electron donor. Grx plays a crucial role in various cellular processes, such as antioxidant defense, control of cellular redox state, redox control of transcription, reversible S-glutathionylation of specific proteins, apoptosis, cell differentiation, etc. In the current study, we have isolated and characterized dithiol glutaredoxin from Hydra vulgaris Ind-Pune (HvGrx1). Sequence analysis showed that HvGrx1 belongs to the Grx family with the classical Grx motif (CPYC). Phylogenetic analysis and homology modeling revealed that HvGrx1 is closely related to Grx2 from zebrafish. HvGrx1 gene was cloned and expressed in Escherichia coli cells; the purified protein had a molecular weight of 11.82 kDa. HvGrx1 efficiently reduced ß-hydroxyethyl disulfide (HED) with the temperature optimum of 25°C and pH optimum 8.0. HvGrx1 was ubiquitously expressed in all body parts of Hydra. Expression of HvGrx1 mRNA and enzymatic activity of HvGrx1 were significantly upregulated post H2O2 treatment. When expressed in human cells, HvGrx1 protected the cells from oxidative stress and enhanced cell proliferation and migration. Although Hydra is a simple invertebrate, HvGrx1 is evolutionary closer to its homologs from higher vertebrates (similar to many other Hydra proteins).

Pune GSH supplementation study: Analyzing longitudinal changes in type 2 diabetic patients using linear mixed-effects models.

Oral GSH supplementation along with antidiabetic treatment was shown to restore the body stores of GSH significantly and reduce oxidative DNA damage (8-OHdG) in Indian Type 2 diabetic (T2D) patients over 6 months in our recent clinical study. Post hoc analysis of the data also suggested that elder patients benefit from improved HbA1c and fasting insulin. We modeled longitudinal changes in diabetic individuals using a linear mixed-effects (LME) framework and obtained i) the distribution of individual trajectories with and without GSH supplementation and ii) the overall rates of changes in the different study arms. Serial changes in elder and younger diabetic individuals were also modeled independently to examine differences in their progression. The average linear trajectories obtained from the model explain how biochemical parameters in T2D patients progress over 6 months on GSH supplementation. Model estimates show improvements in erythrocytic GSH of 108 µM per month and a reduction in 8-OHdG at a rate of 18.5 ng/µg DNA per month in T2D patients. GSH replenishes faster in younger people than in the elder. 8-OHdG reduced more rapidly in the elder (24 ng/µg DNA per month) than in younger (12 ng/µg DNA per month) individuals. Interestingly, elder individuals show a substantial reduction in HbA1c (0.1% per month) and increased fasting insulin (0.6 µU/mL per month). Changes in GSH correlate strongly with changes in HbA1c, 8-OHdG, and fasting insulin in the elder cohort. The model estimates strongly suggest it improves the rate of replenishment in erythrocytic GSH stores and reduces oxidative DNA damage. Elder and younger T2D patients respond differently to GSH supplementation: It improves the rate of reduction in HbA1c and increases fasting insulin in elder patients. These model forecasts have clinical implications that aid in personalizing treatment targets for using oral GSH as adjuvant therapy in diabetes.

Andrographolide and pterostilbene inhibit adipocyte differentiation by downregulating PPARγ through different regulators.

Adipogenesis involves commitment of stem cells and their differentiation into mature adipocytes. It is tightly regulated by hormones, nutrients and adipokines. Many natural compounds are being tested for their anti-adipogenic activity which can be attributed to apoptosis induction in adipocytes, blocking adipocyte differentiation, or inhibiting intracellular triglyceride synthesis and accumulation. In this study, we have determined molecular mechanism of two phytocompounds: andrographolide (AN) and pterostilbene (PT) during differentiation of the human MSCs into adipocyte. Interestingly, AN upregulates miR27a, whereas, PT upregulated SIRT1 which inhibits the expression of PPARγ. Thus, our results clearly demonstrate that both AN and PT inhibited adipogenesis by blocking a surge of reactive oxygen species (ROS) during differentiation and inhibiting expression of crucial transcription factors like SREBP1c and PPARγ.

A novel thioredoxin glutathione reductase from evolutionary ancient metazoan Hydra.

Thioredoxin (Trx) and glutathione disulfide (GSSG), are regenerated in reduced state by thioredoxin reductase (TrxR) and glutathione reductase (GR) respectively. A novel protein thioredoxin glutathione reductase (TGR) capable of reducing Trx as well as GSSG, linking two redox systems, has only been reported so far from parasitic flat worms and mammals. For the first time, we report a multifunctional antioxidant enzyme TGR from the nonparasitic, nonmammalian cnidarian Hydra vulgaris (HvTGR) which is a selenoprotein with unusual fusion of a TrxR domain with glutaredoxin (Grx) domain. We have cloned and sequenced HvTGR which encodes a polypeptide of 73 kDa. It contains conserved sequence CPYC of Grx domain, and CVNVGC and GCUG domains of thioredoxin reductase. Phylogenetic analysis revealed HvTGR to be closer to TGR from mammals rather than to TGR from parasitic helminths. We then subcloned HvTGR in plasmid pSelExpress-1 and expressed it in HEK293T cells to ensure selenocysteine incorporation. Purified HvTGR showed Grx, glutathione reductase and TrxR activities. Both thioredoxin and GSSG disulfide reductase activities were inhibited by 1-Chloro-2,4-dinitrobenzene (DNCB) supporting the existence of an essential selenocysteine residue. HvTGR expression was induced in response to H2O2 in Hydra. Interestingly, inhibition of HvTGR by DNCB, inhibited regeneration in Hydra indicating its involvement in other cellular processes.

Randomized Clinical Trial of How Long-Term Glutathione Supplementation Offers Protection from Oxidative Damage and Improves HbA1c in Elderly Type 2 Diabetic Patients.

Complications in type 2 diabetes (T2D) arise from hyperglycemia-induced oxidative stress. Here, we examined the effectiveness of supplementation with the endogenous antioxidant glutathione (GSH) during anti-diabetic treatment. A total of 104 non-diabetic and 250 diabetic individuals on anti-diabetic therapy, of either sex and aged between 30 and 78 years, were recruited. A total of 125 diabetic patients were additionally given 500 mg oral GSH supplementation daily for a period of six months. Fasting and PP glucose, insulin, HbA1c, GSH, oxidized glutathione (GSSG), and 8-hydroxy-2-deoxy guanosine (8-OHdG) were measured upon recruitment and after three and six months of supplementation. Statistical significance and effect size were assessed longitudinally across all arms. Blood GSH increased (Cohen's d = 1.01) and 8-OHdG decreased (Cohen's d = −1.07) significantly within three months (p < 0.001) in diabetic individuals. A post hoc sub-group analysis showed that HbA1c (Cohen's d = −0.41; p < 0.05) and fasting insulin levels (Cohen's d = 0.56; p < 0.05) changed significantly in diabetic individuals above 55 years. GSH supplementation caused a significant increase in blood GSH and helped maintain the baseline HbA1c overall. These results suggest GSH supplementation is of considerable benefit to patients above 55 years, not only supporting decreased glycated hemoglobin (HbA1c) and 8-OHdG but also increasing fasting insulin. The clinical implication of our study is that the oral administration of GSH potentially complements anti-diabetic therapy in achieving better glycemic targets, especially in the elderly population.

Polyalanine polymorphism in the signal peptide of Glutathione peroxidase 1 (GPX1) gene & its association with osteoporosis.

Background & objectives: Osteoporosis is a systemic skeletal disease, characterized by a low bone mass leading to increased bone fragility and hence, a greater susceptibility to the risk of fracture. Since age-related oxidative stress is one of the factors that has been implicated in developing low bone mineral density (BMD), leading to osteoporosis, this study wanted to explore the expression of antioxidant enzymes in individuals with osteoporosis. The present study focused on mapping polymorphism in an important antioxidant enzyme glutathione peroxidase 1 (GPx1) among osteoporosis and healthy Asian Indians. Methods: Dual-energy X-ray absorptiometry was used to assess BMD of individuals and was classified into normal (n=96) and osteoporotic (n=88) groups. Biochemical parameters such as vitamin D, total oxidant status (TOS), and GPx1 enzyme activity were estimated from plasma samples of recruited individuals. Quantitative real-time qRT-PCR was carried out using GAPDH as an endogenous control. Genomic DNA was isolated from whole blood, and polymorphisms were evaluated by sequencing. Results: The BMD was lower in osteoporotic individuals, and further analysis of biochemical parameters indicated significantly low 25-hydroxy vitamin D and GPx1 with higher TOS levels in osteoporotic as compared to healthy individuals. Furthermore, qRT-PCR revealed low expression of GPX1 in osteoporotic individuals. GPX1 sequence analysis of the promoter and two exons revealed the lower frequency of five alanine repeats in the osteoporotic individuals. Interpretation & conclusions: In this study, the in silico analysis revealed the lower frequency of five alanine repeats in exon 1 of GPX1 and high TOS to be associated with osteoporosis. However, no polymorphism was found in exon 2 of GPX1 among the two study groups.

Cloning and characterization of Thioredoxin 1 from the Cnidarian Hydra.

Thioredoxins, small disulphide-containing redox proteins, play an important role in the regulation of cellular thiol redox balance through their disulfide reductase activity. In this study, we have identified, cloned, purified and characterized thioredoxin 1 (HvTrx1) from the Cnidarian Hydra vulgaris Ind-Pune. Bioinformatics analysis revealed that HvTrx1 contains an evolutionarily conserved catalytic active site Cys-Gly-Pro-Cys and shows a closer phylogenetic relationship with vertebrate Trx1. Optimum pH and temperature for enzyme activity of purified HvTrx1 was found to be pH 7.0 and 25°C, respectively. Enzyme activity decreased significantly at acidic or alkaline pH as well as at higher temperatures. HvTrx1 was found to be expressed ubiquitously in whole mount in situ hybridization. Treatment of Hydra with hydrogen peroxide (H2O2), a highly reactive oxidizing agent, led to a significant increase in gene expression and enzyme activity of Trx1. Further experiments using PX12, an inhibitor of Trx1, indicated that Trx1 plays an important role in regeneration in Hydra. Finally, by using growth assay in Escherichia coli and wound healing assay in human colon cancer cells, we demonstrate that HvTrx1 is functionally active in both prokaryotic and eukaryotic heterologous systems.

Molecular mechanism of anti-adipogenic effect of vitexin in differentiating hMSCs.

In this study, we evaluated a detailed molecular mechanism of anti-adipogenic activity of vitexin, apigenin flavone glucoside, present in germinated fenugreek seeds, in differentiating human mesenchymal stem cells (hMSCs). The lipid content of differentiated adipocytes was estimated by ORO staining. Effect on mitotic clonal expansion was checked by cell cycle analysis. Expression of early and terminal adipocyte differentiation markers, anti- and pro-adipogenic transcription factors and signalling intermediates regulating them was evaluated at RNA and protein level. We found vitexin to be non-cytotoxic up to 20 µM at which intracellular lipid accumulation was significantly decreased. Cell cycle analysis suggested that vitexin does not affect mitotic clonal expansion. Expression of early and late differentiation markers, such as CEBPα, CEBPß, PPARγ, FABP4, perilipin, adiponectin and Glut4 was significantly reduced in the presence of vitexin. Expression of KLF4 and KLF15, positive regulators of PPARγ, was decreased, whereas that of negative regulators, namely KLF2, GATA2, miR20a, miR27a, miR27b, miR128, miR130a, miR130b, miR182 and miR548 increased with vitexin treatment. This effect was mediated by the activation of the AMP-activated protein kinase (AMPK) pathway via the activation of LepR and additionally by inhibiting ROS. Thus, our results showed that vitexin regulates the expression of PPARγ and inhibits adipogenesis of hMSCs at an early stage of differentiation.

Xeroderma pigmentosum A homolog from Hydra partially complements DNA repair defect in human XPA-deficient cells.

Nucleotide excision repair (NER) pathway is a DNA repair mechanism that rectifies a wide spectrum of DNA lesions. Xeroderma pigmentosum group of proteins (XPA through XPG) orchestrate the NER pathway in humans. We have earlier studied XPA homolog from Hydra (HyXPA) and found it to be similar to human XPA. Here, we examined if HyXPA can functionally complement human XPA-deficient cells and reduce their sensitivity to UV radiation. We found that HyXPA was able to partially rescue XPA-deficient human cells from UV by its binding to chromatin of UV-irradiated cells. However, HyXPA failed to bind replication protein A (RPA70), a key interacting partner of human XPA in NER pathway. This could be attributed to changes in certain amino acid residues that have occurred during evolution, leading to prevention of some interactions between Hydra and human proteins.

DNA Repair Repertoire of the Enigmatic Hydra.

Since its discovery by Abraham Trembley in 1744, hydra has been a popular research organism. Features like spectacular regeneration capacity, peculiar tissue dynamics, continuous pattern formation, unique evolutionary position, and an apparent lack of organismal senescence make hydra an intriguing animal to study. While a large body of work has taken place, particularly in the domain of evolutionary developmental biology of hydra, in recent years, the focus has shifted to molecular mechanisms underlying various phenomena. DNA repair is a fundamental cellular process that helps to maintain integrity of the genome through multiple repair pathways found across taxa, from archaea to higher animals. DNA repair capacity and senescence are known to be closely associated, with mutations in several repair pathways leading to premature ageing phenotypes. Analysis of DNA repair in an animal like hydra could offer clues into several aspects including hydra's purported lack of organismal ageing, evolution of DNA repair systems in metazoa, and alternative functions of repair proteins. We review here the different DNA repair mechanisms known so far in hydra. Hydra genes from various DNA repair pathways show very high similarity with their vertebrate orthologues, indicating conservation at the level of sequence, structure, and function. Notably, most hydra repair genes are more similar to deuterostome counterparts than to common model invertebrates, hinting at ancient evolutionary origins of repair pathways and further highlighting the relevance of organisms like hydra as model systems. It appears that hydra has the full repertoire of DNA repair pathways, which are employed in stress as well as normal physiological conditions and may have a link with its observed lack of senescence. The close correspondence of hydra repair genes with higher vertebrates further demonstrates the need for deeper studies of various repair components, their interconnections, and functions in this early metazoan.

miR-3666 inhibits development of hepatic steatosis by negatively regulating PPARγ.

AIMS: PPARγ is a crucial transcription factor involved in development of hepatic steatosis, an early stage of NAFLD. PPARγ is tightly regulated through various positive and negative regulators including miRNAs. In this study, we report for the first time miR-3666 as a negative regulator of PPARγ and its involvement in development of hepatic steatosis. METHODS: Binding of miR-3666 to regulate PPARγ was checked by luciferase assay and was confirmed by mutating PPARγ 3'UTR. Regulation of PPARγ was determined by overexpression of miR-3666 in HepG2 cells. Hepatic steatotic state in HepG2 cells was developed by exposure to excess palmitic acid and expression of PPARγ, miR-3666 and some PPARγ target and non-target genes was checked. Involvement of mir-3666 by regulating PPARγ in hepatic steatosis was also examined in liver of HFD fed mice. RESULTS: On overexpression of miR-3666, PPARγ expression decreased significantly in a dose-dependent manner in HepG2 cells. Binding of miR-3666 to PPARγ was confirmed as the luciferase activity using pMIR-REPORT with PPARγ 3'UTR decreased in PA treated HepG2 cells overexpressing miR-3666 and remained unchanged when PPARγ 3'UTR was mutated. In PA treated HepG2 cells during development of hepatic steatosis PPARγ was significantly up-regulated concomitant with down-regulation of miR-3666. Overexpression of miR-3666 in these cells decreased the extent of hepatic steatosis. Significant up-regulation of PPARγ and down-regulation of miR-3666 was also observed in liver of HFD fed mice indicating that miR-3666 regulates PPARγ in vivo. CONCLUSIONS: miR-3666 negatively regulates PPARγ by binding to its 3'UTR during development of hepatic steatosis.

Excess hydrogen peroxide inhibits head and foot regeneration in hydra by affecting DNA repair and expression of essential genes.

Reactive oxygen species (ROS) are necessary for various cellular processes. However, excess ROS cause damage to many biological molecules and therefore must be tightly regulated in time and space. Hydrogen peroxide (H2 O2 ) is the most commonly used ROS as second messenger in the cell. It is a relatively long-lived freely diffusible signaling molecule during early events of injury. In the Cnidarian hydra, injury-induced ROS production is essential for regeneration to proceed. In the present study, we have examined influence of varying exposure to H2 O2 on head and foot regeneration in the middlepieces of trisected hydra. We find that longer (4 hours) exposure to 1 mM H2 O2 inhibits both head and foot regeneration while shorter exposure (2 hours) does not. Longer exposure to H2 O2 resulted in extensive damage to DNA that could not be repaired, probably due to suboptimal induction of APE1, an enzyme necessary for base excision repair (BER). Concomitantly, genes involved in activation of Wnt pathway, necessary for head regeneration, were significantly downregulated. This appeared to be due to failure of both stabilization and transient nuclear localization of ß-catenin. Similarly, genes involved in foot regeneration were also downregulated on longer exposure to H2 O2 . Thus, exposure to excess ROS inhibits regenerative processes in hydra through reduced expression of genes involved in regeneration and diminished DNA repair.

The Gut Microbial Diversity of Newly Diagnosed Diabetics but Not of Prediabetics Is Significantly Different from That of Healthy Nondiabetics.

Type 2 diabetes (T2D) is a complex metabolic syndrome characterized by insulin dysfunction and abnormalities in glucose and lipid metabolism. The gut microbiome has been recently identified as an important factor for development of T2D. In this study, a total of 102 subjects were recruited, and we have looked at the gut microbiota of prediabetics (PreDMs) (n = 17), newly diagnosed diabetics (NewDMs) (n = 11), and diabetics on antidiabetic treatment (KnownDMs) (n = 39) and compared them with healthy nondiabetics (ND) (n = 35). Twenty-five different serum biomarkers were measured to assess the status of diabetes and their association with gut microbiota. Our analysis revealed nine different genera as differentially abundant in four study groups. Among them, Akkermansia, Blautia, and Ruminococcus were found to be significantly (P < 0.05) decreased, while Lactobacillus was increased in NewDMs compared to ND and recovered in KnownDMs. Akkermansia was inversely correlated with HbA1c and positively correlated with total antioxidants. Compared to ND, there was increased abundance of Megasphaera, Escherichia, and Acidaminococcus and decreased abundance of Sutterella in KnownDMs. Among many taxa known to act as community drivers during disease progression, we observed genus Sutterella as a common driver taxon among all diabetic groups. On the basis of the results of random forest analysis, we found that the genera Akkermansia and Sutterella and that the serum metabolites fasting glucose, HbA1c, methionine, and total antioxidants were highly discriminative factors among studied groups. Taken together, our data revealed that gut microbial diversity of NewDMs but not of PreDMs is significantly different from that of ND. Interestingly, after antidiabetic treatment, the microbial diversity of KnownDMs tends to recover toward that of ND.IMPORTANCE Gut microbiota is considered to play a role in disease progression, and previous studies have reported an association of microbiome dysbiosis with T2D. In this study, we have attempted to investigate gut microbiota of ND, PreDMs, NewDMs, and KnownDMs. We found that the genera Akkermansia and Blautia decreased significantly (P < 0.05) in treatment-naive diabetics and were restored in KnownDMs on antidiabetic treatment. To the best of our knowledge, comparative studies on shifts in the microbial community in individuals of different diabetic states are lacking. Understanding the transition of microbiota and its association with serum biomarkers in diabetics with different disease states may pave the way for new therapeutic approaches for T2D.

Vitexin alleviates non-alcoholic fatty liver disease by activating AMPK in high fat diet fed mice.

Non-alcoholic fatty liver disease (NAFLD) is a most common liver disorder characterized by accumulation of fat in the liver and currently there is no approved treatment for it. Obesity and diabetes being leading cause of NAFLD, compounds having anti-obesity activity and potential to reduce insulin resistance are considered suitable candidate for NAFLD treatment. In this study, we checked effect of vitexin, a naturally occurring flavonoid, on high fat diet (HFD) induced NAFLD in C57BL/6J mice. In presence of vitexin, significant reduction in body and liver weight, triglyceride and cholesterol content in serum and liver was observed. Serum Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) levels were reduced significantly by vitexin which were elevated in HFD group whereas serum lipase activity remained unchanged. Vitexin suppressed de novo lipogenesis by downregulating expression of Peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein-α (C/EBP-α), sterol regulatory element-binding protein-1c (SREBP-1c), Fatty acid synthase (FAS) and Acetyl-CoA Carboxylase (ACC). Additionally, it also enhanced fatty acid oxidation and lipolysis by upregulating Peroxisome proliferator-activated receptor α (PPAR-α), carnitine palmitoyltransferase-1a (CPT-1a) and Adipose triglyceride lipase (ATGL). Inhibition of lipogenesis and activation of lipolysis and fatty acid oxidation by vitexin was found to be mediated by activation of AMP-activated protein kinase (AMPK). Vitexin also improved insulin signalling by activating insulin receptor substrate-1 (IRS-1) and its downstream target AKT. AMPK activation of vitexin was possibly through binding of vitexin to leptin receptor (LepR) which was confirmed by molecular docking studies and by observed enhanced expression of LepR. Thus, we propose that vitexin alleviates NAFLD by activating AMPK possibly by binding to LepR.