A gut microbial peptide and molecular mimicry in the pathogenesis of type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of pancreatic ß-cells. One of the earliest aspects of this process is the development of autoantibodies and T cells directed at an epitope in the B-chain of insulin (insB:9-23). Analysis of microbial protein sequences with homology to the insB:9-23 sequence revealed 17 peptides showing >50% identity to insB:9-23. Of these 17 peptides, the hprt4-18 peptide, found in the normal human gut commensal Parabacteroides distasonis, activated both human T cell clones from T1D patients and T cell hybridomas from nonobese diabetic (NOD) mice specific to insB:9-23. Immunization of NOD mice with P. distasonis insB:9-23 peptide mimic or insB:9-23 peptide verified immune cross-reactivity. Colonization of female NOD mice with P. distasonis accelerated the development of T1D, increasing macrophages, dendritic cells, and destructive CD8+ T cells, while decreasing FoxP3+ regulatory T cells. Western blot analysis identified P. distasonis-reacting antibodies in sera of NOD mice colonized with P. distasonis and human T1D patients. Furthermore, adoptive transfer of splenocytes from P. distasonis-treated mice to NOD/SCID mice enhanced disease phenotype in the recipients. Finally, analysis of human children gut microbiome data from a longitudinal DIABIMMUNE study revealed that seroconversion rates (i.e., the proportion of individuals developing two or more autoantibodies) were consistently higher in children whose microbiome harbored sequences capable of producing the hprt4-18 peptide compared to individuals who did not harbor it. Taken together, these data demonstrate the potential role of a gut microbiota-derived insB:9-23-mimic peptide as a molecular trigger of T1D pathogenesis.

Design, synthesis, and biological evaluation of a small molecule oral agonist of the glucagon-like-peptide-1 receptor.

An absolute or relative deficiency of pancreatic ß-cells mass and functionality is a crucial pathological feature common to type 1 diabetes mellitus and type 2 diabetes mellitus. Glucagon-like-peptide-1 receptor (GLP1R) agonists have been the focus of considerable research attention for their ability to protect ß-cell mass and augment insulin secretion with no risk of hypoglycemia. Presently commercially available GLP1R agonists are peptides that limit their use due to cost, stability, and mode of administration. To address this drawback, strategically designed distinct sets of small molecules were docked on GLP1R ectodomain and compared with previously known small molecule GLP1R agonists. One of the small molecule PK2 (6-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6H-indolo[2,3-b]quinoxaline) displays stable binding with GLP1R ectodomain and induces GLP1R internalization and increasing cAMP levels. PK2 also increases insulin secretion in the INS-1 cells. The oral administration of PK2 protects against diabetes induced by multiple low-dose streptozotocin administration by lowering high blood glucose levels. Similar to GLP1R peptidic agonists, treatment of PK2 induces ß-cell replication and attenuate ß-cell apoptosis in STZ-treated mice. Mechanistically, this protection was associated with decreased thioredoxin-interacting protein expression, a potent inducer of diabetic ß-cell apoptosis and dysfunction. Together, this report describes a small molecule, PK2, as an orally active nonpeptidic GLP1R agonist that has efficacy to preserve or restore functional ß-cell mass.

Low-dose naltrexone rescues inflammation and insulin resistance associated with hyperinsulinemia.

The incidence of diabetes, obesity, and metabolic diseases has reached an epidemic status worldwide. Insulin resistance is a common link in the development of these conditions, and hyperinsulinemia is a central hallmark of peripheral insulin resistance. However, how hyperinsulinemia leads to systemic insulin resistance is less clear. We now provide evidence that hyperinsulinemia promotes the release of soluble pro-inflammatory mediators from macrophages that lead to systemic insulin resistance. Our observations suggest that hyperinsulinemia induces sirtuin1 (SIRT1) repression and stimulates NF-κB p65 nuclear translocation and transactivation of NF-κB to promote the extracellular release of pro-inflammatory mediators. We further showed that low-dose naltrexone (LDN) abrogates hyperinsulinemia-mediated SIRT1 repression and prevents NF-κB p65 nuclear translocation. This, in turn, attenuates the hyperinsulinemia-induced release of pro-inflammatory cytokines and reinstates insulin sensitivity both in in vitro and in vivo diet-induced hyperinsulinemic mouse model. Notably, our data indicate that Sirt1 knockdown or inhibition blunts the anti-inflammatory properties of LDN in vitro Using numerous complementary in silico and in vitro experimental approaches, we demonstrated that LDN can bind to SIRT1 and increase its deacetylase activity. Together, these data support a critical role of SIRT1 in inflammation and insulin resistance in hyperinsulinemia. LDN improves hyperinsulinemia-induced insulin resistance by reorienting macrophages toward anti-inflammation. Thus, LDN treatment may provide a novel therapeutic approach against hyperinsulinemia-associated insulin resistance.

Zinc oxide nanoparticles attenuate hepatic steatosis development in high-fat-diet fed mice through activated AMPK signaling axis.

Insulin resistance is thought to be a common link between obesity and Non-Alcoholic Fatty Liver Disease (NAFLD). NAFLD has now reached epidemic status worldwide and identification of molecules or pathways as newer therapeutic strategies either to prevent or overcome insulin resistance seems critical. Dysregulated hepatic lipogenesis (DNL) is a hallmark of NAFLD in humans and rodents. Therefore, reducing DNL accretion may be critical in the development of therapeutics of NAFLD. In our in vivo model (high-fat-diet fed [HFD] obese mice) we found Zinc oxide nanoparticles (ZnO NPs) significantly decreased HFD-induced hepatic steatosis and peripheral insulin resistance. This protective mechanism of ZnO NPs was signaled through hepatic SIRT1-LKB1-AMPK which restricted SREBP-1c within the cytosol limiting its transcriptional ability and thereby ameliorating HFD mediated DNL. These observations indicate that ZnO NP can serve as a therapeutic strategy to improve the physiological homeostasis during obesity and its associated metabolic abnormalities.

Sex-Specific Cytokine, Chemokine, and Growth Factor Signatures in T1D Patients and Progressors.

While studies have reported altered levels of cytokines in type 1 diabetes (T1D) patients, the results are inconsistent, likely because of variable factors. This study tests the hypothesis that there are sex-based differences in cytokine levels in T1D, prior to and after disease onset. We analyzed 48 blood cytokine, chemokine, and growth factor levels using a multiplex assay. We found only two cytokines, M-CSF and IL-6, with significant differences between T1D patients (n=25) versus controls overall (n=25). However, we identified notable alterations when comparing sex-age-matched controls and T1D samples. Inflammatory cytokines (TNF-α, IL-6, IL-1a), Th2 cytokines (IL-4, IL-13), and chemokines (MIP-1α, RANTES, MIP-3) were lower in female T1D patients compared to female controls, but not in males. IL-22 was lower in female T1D patients compared to female controls, while it was higher in male T1D patients compared to male controls. In contrast, growth factors (EGF, PDGF-AB/BB) were higher in male T1D patients compared to male controls. In T1D progressors (children who developed the disease years after the sample collection, n=16-21), GROa was lower compared to controls in both sexes. Our findings underscore the importance of understanding sex-specific differences in T1D pathogenesis and their implications for developing personalized treatments.

Dynamics of the gut microbiome, IgA response, and plasma metabolome in the development of pediatric celiac disease.

BACKGROUND: Celiac disease (CD) is an autoimmune disorder triggered by gluten consumption. Almost all CD patients possess human leukocyte antigen (HLA) DQ2/DQ8 haplotypes; however, only a small subset of individuals carrying these alleles develop CD, indicating the role of environmental factors in CD pathogenesis. The main objective of this study was to determine the contributory role of gut microbiota and microbial metabolites in CD onset. To this end, we obtained fecal samples from a prospective cohort study (ABIS) at ages 2.5 and 5 years. Samples were collected from children who developed CD after the final sample collection (CD progressors) and healthy children matched by age, HLA genotype, breastfeeding duration, and gluten-exposure time (n=15-16). We first used 16S sequencing and immunoglobulin-A sequencing (IgA-seq) using fecal samples obtained from the same children (i) 16 controls and 15 CD progressors at age 2.5 and (ii) 13 controls and 9 CD progressors at age 5. We completed the cytokine profiling, and plasma metabolomics using plasma samples obtained at age 5 (n=7-9). We also determined the effects of one microbiota-derived metabolite, taurodeoxycholic acid (TDCA), on the small intestines and immune cell composition in vivo. RESULTS: CD progressors have a distinct gut microbiota composition, an increased IgA response, and unique IgA targets compared to healthy subjects. Notably, 26 plasma metabolites, five cytokines, and one chemokine were significantly altered in CD progressors at age 5. Among 26 metabolites, we identified a 2-fold increase in TDCA. TDCA treatment alone caused villous atrophy, increased CD4+ T cells, Natural Killer cells, and two important immunoregulatory proteins, Qa-1 and NKG2D expression on T cells while decreasing T-regulatory cells in intraepithelial lymphocytes (IELs) in C57BL/6J mice. CONCLUSIONS: Pediatric CD progressors have a distinct gut microbiota composition, plasma metabolome, and cytokine profile before diagnosis. Furthermore, CD progressors have more IgA-coated bacteria and unique targets of IgA in their gut microbiota. TDCA feeding alone stimulates an inflammatory immune response in the small intestines of C57BJ/6 mice and causes villous atrophy, the hallmark of CD. Thus, a microbiota-derived metabolite, TDCA, enriched in CD progressors' plasma, has the potential to drive inflammation in the small intestines and enhance CD pathogenesis. Video Abstract.

Organosulfur/Selenium-Based Highly Fluorogenic Molecular Probes for Live-Cell Nucleolus Imaging.

We present fluorogenic cationic organo chalcogens that are highly selective to RNA. We have demonstrated that the conformational dynamics and subsequently the optical properties of these dyes can be controlled to facilitate efficient bioimaging. We report the application of organoselenium and organosulfur-based cell-permeable red-emissive probes bearing a favorable cyclic sidearm for selective and high contrast imaging of cell nucleoli. The probes exhibit high quantum yield upon interacting with RNA in an aqueous solution. An in-depth multiscale simulation study reveals that the prominent rotational freezing of the electron-donating sidearm of the probes in the microenvironment of RNA helps in attaining more planar conformation when compared to DNA. It exerts a greater extent of intramolecular charge transfer and hence leads to enhanced fluorescence emission. A systematic structure-interaction relationship study highlighted the impact of heavy-chalcogens toward the improved emissive properties of the probes.

Gut Microbiota Regulate Pancreatic Growth, Exocrine Function, and Gut Hormones.

Growing evidence indicates an important link between gut microbiota, obesity, and metabolic syndrome. Alterations in exocrine pancreatic function are also widely present in patients with diabetes and obesity. To examine this interaction, C57BL/6J mice were fed a chow diet, a high-fat diet (HFD), or an HFD plus oral vancomycin or metronidazole to modify the gut microbiome. HFD alone leads to a 40% increase in pancreas weight, decreased glucagon-like peptide 1 and peptide YY levels, and increased glucose-dependent insulinotropic peptide in the plasma. Quantitative proteomics identified 138 host proteins in fecal samples of these mice, of which 32 were significantly changed by the HFD. The most significant of these were the pancreatic enzymes. These changes in amylase and elastase were reversed by antibiotic treatment. These alterations could be reproduced by transferring gut microbiota from donor C57BL/6J mice to germ-free mice. By contrast, antibiotics had no effect on pancreatic size or exocrine function in C57BL/6J mice fed the chow diet. Further, 1 week vancomycin administration significantly increased amylase and elastase levels in obese men with prediabetes. Thus, the alterations in gut microbiota in obesity can alter pancreatic growth, exocrine function, and gut endocrine function and may contribute to the alterations observed in patients with obesity and diabetes.

Viruses and Metabolism: The Effects of Viral Infections and Viral Insulins on Host Metabolism.

Over the past decades, there have been tremendous efforts to understand the cross-talk between viruses and host metabolism. Several studies have elucidated the mechanisms through which viral infections manipulate metabolic pathways including glucose, fatty acid, protein, and nucleotide metabolism. These pathways are evolutionarily conserved across the tree of life and extremely important for the host's nutrient utilization and energy production. In this review, we focus on host glucose, glutamine, and fatty acid metabolism and highlight the pathways manipulated by the different classes of viruses to increase their replication. We also explore a new system of viral hormones in which viruses mimic host hormones to manipulate the host endocrine system. We discuss viral insulin/IGF-1-like peptides and their potential effects on host metabolism. Together, these pathogenesis mechanisms targeting cellular signaling pathways create a multidimensional network of interactions between host and viral proteins. Defining and better understanding these mechanisms will help us to develop new therapeutic tools to prevent and treat viral infections.

Novel insights into the dynamics behavior of glucagon-like peptide-1 receptor with its small molecule agonists.

The glucagon-like peptide-1 receptor (GLP-1R) is a well-known target of therapeutics industries for the treatment of various metabolic diseases like type 2 diabetes and obesity. The structural-functional relationships of small molecule agonists and GLP-1R are yet to be understood. Therefore, an attempt was made on structurally known GLP-1R agonists (Compound 1, Compound 2, Compound A, Compound B, and (S)-8) to study their interaction with the extracellular domain of GLP-1R. In this study, we explored the dynamics, intrinsic stability, and binding mechanisms of these molecules through computational modeling, docking, molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) binding free energy estimation. Molecular docking study depicted that hydrophobic interaction (pi-pi stacking) plays a crucial role in maintaining the stability of the complex, which was also supported by intermolecular analysis from MD simulation study. Principal component analysis suggested that the terminal ends along with the turns/loops connecting adjacent helix and strands exhibit a comparatively higher movement of main chain atoms in most of the complexes. MM/PBSA binding free energy study revealed that non-polar solvation (van der Waals and electrostatic) energy subsidizes significantly to the total binding energy, and the polar solvation energy opposes the binding agonists to GLP-1R. Overall, we provide structural features information about GLP-1R complexes that would be conducive for the discovery of new GLP-1R agonists in the future for the treatment of various metabolic diseases. Communicated by Ramaswamy H. Sarma.

Long Range Emissive Water-Soluble Fluorogenic Molecular Platform for Imaging Carbon Monoxide in Live Cells.

Despite its murderous act, carbon monoxide (CO) is found to be a very crucial small gaseous messenger molecule in dictating prime biological and physiological processes. Determination of endogenous or exhaled CO levels can throw significant light on smoking status and can be used as a breath biomarker of inflammatory diseases. Therefore, fluorescence imaging of CO in biofluids will empower one with the minute details of various disease states that involve CO. Unfortunately, such efficient fluorescent probes are less in number and also associated with tedious protocols. This enticed our attention and inspired us to look upon developing perceptive imaging agents for CO in a living system. In this report, a resorufin-based "turn-on" orange emissive molecular probe has been successfully utilized to detect CO in an aqueous system. The mono protection of a resorufin unit with an allyl chloroformate furnished a weakly fluorescent small molecular probe P1. Further, the P1+Pd2+ ensemble has been successfully developed in situ using PdCl2 (as Pd2+) and utilized as a light-up signaling mechanism tool for the sensing of CO at the nanomolar level (62 nM) through deprotection mechanism. The probe selectively detects CO without any interference from other anions, gasotransmitters and fatty acids. The present integrated probe P1+Pd2+ system has been found to be highly sensitive to detect CO in cellular systems as well.

Interventions to improve ß-cell mass and function.

Diabetes mellitus (T2DM) has become an epidemiologically important disease worldwide and is also becoming a great matter of concern due to the effects associated with it like: high morbidity, elevated health care cost and shortened life span. T2DM is a chronic metabolic disease characterized by insulin resistance as well as ß-cell dysfunction. It is widely accepted that in the face of insulin resistance, euglycemia can be maintained by increase in pancreatic ß-cell mass and insulin secretion. This compensation is largely due to enhanced secretion of insulin by the ß-cell mass, which is present initially, and thereby subsequent increases in ß-cell mass provide additional insulin secretion. However, the mechanism by which ß-cell anatomical plasticity and functional plasticity for insulin secretion is coordinated and executed in different physiological and pathophysiological states is complex and has been poorly understood. As the incidence of T2DM continues to increase at an alarming rate, it is becoming imperative to shift the research focus towards the ß-cell physiology where identification of novel pathways that influence the ß-cell proliferation and/or contribute to increase insulin secretion has the potential to lead to new therapies for preventing or delaying onset of disease.