Visible light-initiated interfacial thiol-norbornene photopolymerization for forming islet surface conformal coating

A cytocompatible visible light-mediated interfacial thiol-norbornene photopolymerization scheme was developed for creating hydrogel conformal coating on pancreatic islets. The step-growth thiol-norbornene reaction affords high consistency and tunability in gel coating thickness. Furthermore, isolated islets coated with thiol-norbornene gel maintained their viability and function in vitro

Eukaryotic translation initiation factor 5A inhibition alters physiopathology and immune responses in a “humanized” transgenic mouse model of type 1 diabetes

Therapeutic options for treatment of type 1 diabetes (T1D) are still missing. New avenues for immune modulation need to be developed. Here we attempted at altering the diabetes outcome of our humanized model of T1D by inhibiting translation-initiation factor eIF5A hypusination in vivo. Double-transgenic (DQ8-GAD65) mice were immunized with adenoviral vectors carrying GAD65 for diabetes induction. Animals were subsequently treated with deoxyhypusine synthase (DHS) inhibitor GC7 and monitored for diabetes development over time. On one hand, helper CD4+ T cells were clearly affected by the downregulation of the eIF5A not just at the pancreas level but overall. On the other hand, the T regulatory cell component of CD4 responded with activation and proliferation significantly higher than in the non-GC7-treated controls. Female mice seemed to be more susceptible to these effects. All together, our results show for the first time that downregulation of eIF5A through inhibition of DHS altered the physiopathology and observed immune outcome of diabetes in an animal model that closely resembles human T1D. Although the development of diabetes could not be abrogated by DHS inhibition, the immunomodulatory capacity of this approach may supplement other interventions directed at increasing regulation of autoreactive T cells in T1D

Profiling of RNAs from Human Islet-Derived Exosomes in a Model of Type 1 Diabetes

Type 1 diabetes (T1D) is characterized by the immune-mediated destruction of insulin-producing islet β cells. Biomarkers capable of identifying T1D risk and dissecting disease-related heterogeneity represent an unmet clinical need. Toward the goal of informing T1D biomarker strategies, we profiled coding and noncoding RNAs in human islet-derived exosomes and identified RNAs that were differentially expressed under proinflammatory cytokine stress conditions. Human pancreatic islets were obtained from cadaveric donors and treated with/without IL-1β and IFN-γ. Total RNA and small RNA sequencing were performed from islet-derived exosomes to identify mRNAs, long noncoding RNAs, and small noncoding RNAs. RNAs with a fold change ≥1.3 and a p-value <0.05 were considered as differentially expressed. mRNAs and miRNAs represented the most abundant long and small RNA species, respectively. Each of the RNA species showed altered expression patterns with cytokine treatment, and differentially expressed RNAs were predicted to be involved in insulin secretion, calcium signaling, necrosis, and apoptosis. Taken together, our data identify RNAs that are dysregulated under cytokine stress in human islet-derived exosomes, providing a comprehensive catalog of protein coding and noncoding RNAs that may serve as potential circulating biomarkers in T1D

Expression of a functional non-ribosomal peptide synthetase module in Escherichia coli by coexpression with a phosphopantetheinyl transferase

AbstractBackground: Non-ribosomal peptide synthetases (NRPSs) found in bacteria abd fungi are multifunctional enzymes that catalyze the synthesis of a variety of biologically important peptides. These enzymes are composed of modular units, each responsible for the activation of an amino acid to an aminoacyl adenylate and for the subsequent formation of an aminoacyl thioester with the sulfhydryl group of a 4′-phosphopantetheine moiety. Attempts to express these modules in Escherichia coli have resulted in recombinant proteins deficient in 4′-phosphopantetheine. The recent identification of a family of phosphopantetheinyl transferases (P-pant transferases) associated with NRPS have led us to investigate whether coexpression of NRPS modules with P-pant transferases in E. coli would lead to the incorporation of 4′-phosphopantetheine.Results: A truncated module of gramicidin S synthetase, PheAT(His6), was expressed as a His6 fusion protein in E. coli with and without Gsp, the P-pant transferase associated with gramicidin S synthetase. Although PheAT(His6) expressed alone in E. coli catalyzed Phe-AMP formation from Phe and ATP, < 1% was converted to the Phe thioester. In contrast, >80% of the PheAT(His6) that was coexpressed with Gsp could form the Phe thioester in the presence of Phe and ATP.Conclusions: Our finding indicates the presence of an almost equimolar amount of 4′-phosphopantetheine covalently bound to the NRPS module PheAT(His6), and that the functional expression of NRPS modules in E. coli is possible, provided that they are coexpressed with an appropriate P-pant transferase

Protective effects of polyamine depletion in mouse models of type 1 diabetes: implications for therapy

The underlying pathophysiology of type 1 diabetes involves autoimmune-mediated islet inflammation, leading to dysfunction and death of insulin-secreting islet β cells. Recent studies have shown that polyamines, which are essential for mRNA translation, cellular replication, and the formation of the hypusine modification of eIF5A may play an important role in the progression of cellular inflammation. To test a role for polyamines in type 1 diabetes pathogenesis, we administered the ornithine decarboxylase inhibitor difluoromethylornithine to two mouse models--the low-dose streptozotocin model and the NOD model--to deplete intracellular polyamines, and administered streptozotocin to a third model, which was haploinsufficient for the gene encoding the hypusination enzyme deoxyhypusine synthase. Subsequent development of diabetes and/or glucose intolerance was monitored. In the low-dose streptozotocin mouse model, continuous difluoromethylornithine administration dose-dependently reduced the incidence of hyperglycemia and led to the preservation of β cell area, whereas in the NOD mouse model of autoimmune diabetes difluoromethylornithine reduced diabetes incidence by 50%, preserved β cell area and insulin secretion, led to reductions in both islet inflammation and potentially diabetogenic Th17 cells in pancreatic lymph nodes. Difluoromethylornithine treatment reduced hypusinated eIF5A levels in both immune cells and islets. Animals haploinsufficient for the gene encoding deoxyhypusine synthase were partially protected from hyperglycemia induced by streptozotocin. Collectively, these studies suggest that interventions that interfere with polyamine biosynthesis and/or eIF5A hypusination may represent viable approaches in the treatment of diabetes

Polyamine biosynthesis is critical for growth and differentiation of the pancreas

The pancreas, in most studied vertebrates, is a compound organ with both exocrine and endocrine functions. The exocrine compartment makes and secretes digestive enzymes, while the endocrine compartment, organized into islets of Langerhans, produces hormones that regulate blood glucose. High concentrations of polyamines, which are aliphatic amines, are reported in exocrine and endocrine cells, with insulin-producing β cells showing the highest concentrations. We utilized zebrafish as a model organism, together with pharmacological inhibition or genetic manipulation, to determine how polyamine biosynthesis functions in pancreatic organogenesis. We identified that inhibition of polyamine biosynthesis reduces exocrine pancreas and β cell mass, and that these reductions are at the level of differentiation. Moreover, we demonstrate that inhibition of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, phenocopies inhibition or knockdown of the enzyme deoxyhypusine synthase (DHS). These data identify that the pancreatic requirement for polyamine biosynthesis is largely mediated through a requirement for spermidine for the downstream posttranslational modification of eIF5A by its enzymatic activator DHS, which in turn impacts mRNA translation. Altogether, we have uncovered a role for polyamine biosynthesis in pancreatic organogenesis and identified that it may be possible to exploit polyamine biosynthesis to manipulate pancreatic cell differentiation

Biomarkers of β-Cell Stress and Death in Type 1 Diabetes

The hallmark of type 1 diabetes (T1D) is a decline in functional β-cell mass arising as a result of autoimmunity. Immunomodulatory interventions at disease onset have resulted in partial stabilization of β-cell function, but full recovery of insulin secretion has remained elusive. Revised efforts have focused on disease prevention through interventions administered at earlier disease stages. To support this paradigm, there is a parallel effort ongoing to identify circulating biomarkers that have the potential to identify stress and death of the islet β-cells. Whereas no definitive biomarker(s) have been fully validated, several approaches hold promise that T1D can be reliably identified in the pre-symptomatic phase, such that either β-cell preservation or immunomodulatory agents might be employed in at-risk populations. This review summarizes the most promising protein- and nucleic acid-based biomarkers discovered to date and reviews the context in which they have been studied

Measurement of Differentially Methylated INS DNA Species in Human Serum Samples as a Biomarker of Islet β Cell Death

The death of islet β cells is thought to underlie the pathogenesis of virtually all forms of diabetes and to precede the development of frank hyperglycemia, especially in type 1 diabetes. The development of sensitive and reliable biomarkers of β cell death may allow for early therapeutic intervention to prevent or delay the development of diabetes. Recently, several groups including our own have reported that cell-free, differentially methylated DNA encoding preproinsulin (INS) in the circulation is correlated to β cell death in pre-type 1 diabetes and new-onset type 1 diabetes. Here, we present a step-by-step protocol using digital PCR for the measurement of cell-free INS DNA that is differentially methylated at cytosine at position -69 bp (relative to the transcriptional start site). We demonstrate that the assay can distinguish between methylated and unmethylated cytosine at position -69 bp, is linear across several orders of magnitude, provides absolute quantitation of DNA copy numbers, and can be applied to samples of human serum from individuals with new-onset type 1 diabetes and disease-free controls. The protocol described here can be adapted to any DNA species for which detection of differentially methylated cytosines is desired, whether from circulation or from isolated cells and tissues, and can provide absolute quantitation of DNA fragments

Hypusinated eIF5A is expressed in the pancreas and spleen of individuals with type 1 and type 2 diabetes

The gene encoding eukaryotic initiation factor 5A (EIF5A) is found in diabetes-susceptibility loci in mouse and human. eIF5A is the only protein known to contain hypusine (hydroxyputrescine lysine), a polyamine-derived amino acid formed post-translationally in a reaction catalyzed by deoxyhypusine synthase (DHPS). Previous studies showed pharmacologic blockade of DHPS in type 1 diabetic NOD mice and type 2 diabetic db/db mice improved glucose tolerance and preserved beta cell mass, which suggests that hypusinated eIF5A (eIF5AHyp) may play a role in diabetes pathogenesis by direct action on the beta cells and/or altering the adaptive or innate immune responses. To translate these findings to human, we examined tissue from individuals with and without type 1 and type 2 diabetes to determine the expression of eIF5AHyp. We detected eIF5AHyp in beta cells, exocrine cells and immune cells; however, there was also unexpected enrichment of eIF5AHyp in pancreatic polypeptide-expressing PP cells. Interestingly, the presence of eIF5AHyp co-expressing PP cells was not enhanced with disease. These data identify new aspects of eIF5A biology and highlight the need to examine human tissue to understand disease

Molecular Mechanisms of Nonalcoholic Fatty Liver Disease: Potential Role for 12-Lipoxygenase

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of pathologies associated with fat accumulation in the liver. NAFLD is the most common cause of liver disease in the United States, affecting up to a third of the general population. It is commonly associated with features of metabolic syndrome, particularly insulin resistance. NAFLD shares the basic pathogenic mechanisms with obesity and insulin resistance, such as mitochondrial, oxidative and endoplasmic reticulum stress. Lipoxygenases catalyze the conversion of poly-unsaturated fatty acids in the plasma membrane—mainly arachidonic acid and linoleic acid—to produce oxidized pro-inflammatory lipid intermediates. 12-Lipoxygenase (12-LOX) has been studied extensively in setting of inflammation and insulin resistance. As insulin resistance is closely associated with development of NAFLD, the role of 12-LOX in pathogenesis of NAFLD has received increasing attention in recent years. In this review we discuss the role of 12-LOX in NAFLD pathogenesis and its potential role in emerging new therapeutics