Role of the 12-lipoxygenase pathway in diabetes pathogenesis and complications.

12-lipoxygenase (12-LOX) is one of several enzyme isoforms responsible for the metabolism of arachidonic acid and other poly-unsaturated fatty acids to both pro- and anti-inflammatory lipid mediators. Mounting evidence has shown that 12-LOX plays a critical role in the modulation of inflammation at multiple checkpoints during diabetes development. Due to this, interventions to limit pro-inflammatory 12-LOX metabolites either by isoform-specific 12-LOX inhibition, or by providing specific fatty acid substrates via dietary intervention, has the potential to significantly and positively impact health outcomes of patients living with both type 1 and type 2 diabetes. To date, the development of truly specific and efficacious inhibitors has been hampered by homology of LOX family members; however, improvements in high throughput screening have improved the inhibitor landscape. Here, we describe the function and role of human 12-LOX, and mouse 12-LOX and 12/15-LOX, in the development of diabetes and diabetes-related complications, and describe promise in the development of strategies to limit pro-inflammatory metabolites, primarily via new small molecule 12-LOX inhibitors.

A system for detecting high impact-low frequency mutations in primary tumors and metastases.

Tumor complexity and intratumor heterogeneity contribute to subclonal diversity. Despite advances in next-generation sequencing (NGS) and bioinformatics, detecting rare mutations in primary tumors and metastases contributing to subclonal diversity is a challenge for precision genomics. Here, in order to identify rare mutations, we adapted a recently described epithelial reprograming assay for short-term propagation of epithelial cells from primary and metastatic tumors. Using this approach, we expanded minor clones and obtained epithelial cell-specific DNA/RNA for quantitative NGS analysis. Comparative Ampliseq Comprehensive Cancer Panel sequence analyses were performed on DNA from unprocessed breast tumor and tumor cells propagated from the same tumor. We identified previously uncharacterized mutations present only in the cultured tumor cells, a subset of which has been reported in brain metastatic but not primary breast tumors. In addition, whole-genome sequencing identified mutations enriched in liver metastases of various cancers, including Notch pathway mutations/chromosomal inversions in 5/5 liver metastases, irrespective of cancer types. Mutations/rearrangements in FHIT, involved in purine metabolism, were detected in 4/5 liver metastases, and the same four liver metastases shared mutations in 32 genes, including mutations of different HLA-DR family members affecting OX40 signaling pathway, which could impact the immune response to metastatic cells. Pathway analyses of all mutated genes in liver metastases showed aberrant tumor necrosis factor and transforming growth factor signaling in metastatic cells. Epigenetic regulators including KMT2C/MLL3 and ARID1B, which are mutated in >50% of hepatocellular carcinomas, were also mutated in liver metastases. Thus, irrespective of cancer types, organ-specific metastases may share common genomic aberrations. Since recent studies show independent evolution of primary tumors and metastases and in most cases mutation burden is higher in metastases than primary tumors, the method described here may allow early detection of subclonal somatic alterations associated with metastatic progression and potentially identify therapeutically actionable, metastasis-specific genomic aberrations.

Lost in translation: endoplasmic reticulum stress and the decline of ß-cell health in diabetes mellitus.

Emerging data illustrate a pivotal role for activation of ß-cell endoplasmic reticulum (ER) stress pathways in diabetes pathophysiology. The purpose of this review is to appraise the evidence for ß-cell ER stress in human type 1 and 2 diabetes, review the molecular signalling pathways involved in the unfolded protein response and ER stress signalling, and to provide data from polyribosome profiling to illustrate the impact of ER stress on the mRNA translation response. Finally, we will discuss existing and novel therapeutic strategies that target ß-cell ER stress and discuss their use in rodent and human type 1 and 2 diabetes.

Effects of combination therapy with dipeptidyl peptidase-IV and histone deacetylase inhibitors in the non-obese diabetic mouse model of type 1 diabetes.

Type 1 diabetes (T1D) results from T helper type 1 (Th1)-mediated autoimmune destruction of insulin-producing ß cells. Novel experimental therapies for T1D target immunomodulation, ß cell survival and inflammation. We examined combination therapy with the dipeptidyl peptidase-IV inhibitor MK-626 and the histone deacetylase inhibitor vorinostat in the non-obese diabetic (NOD) mouse model of T1D. We hypothesized that combination therapy would ameliorate T1D by providing protection from ß cell inflammatory destruction while simultaneously shifting the immune response towards immune-tolerizing regulatory T cells (T(regs)). Although neither mono- nor combination therapies with MK-626 and vorinostat caused disease remission in diabetic NOD mice, the combination of MK-626 and vorinostat increased ß cell area and reduced the mean insulitis score compared to diabetic control mice. In prediabetic NOD mice, MK-626 monotherapy resulted in improved glucose tolerance, a reduction in mean insulitis score and an increase in pancreatic lymph node T(reg) percentage, and combination therapy with MK-626 and vorinostat increased pancreatic lymph node T(reg) percentage. We conclude that neither single nor combination therapies using MK-626 and vorinostat induce diabetes remission in NOD mice, but combination therapy appears to have beneficial effects on ß cell area, insulitis and T(reg) populations. Combinations of vorinostat and MK-626 may serve as beneficial adjunctive therapy in clinical trials for T1D prevention or remission.

Targeting regulatory T cells in the treatment of type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is a T cell-mediated autoimmune disease resulting in islet ß cell destruction, hypoinsulinemia, and severely altered glucose homeostasis. T1DM has classically been attributed to the pathogenic actions of auto-reactive effector T cells(Teffs) on the ß cell. Recent literature now suggests that a failure of a second T cell subtype, known as regulatory T cells (Tregs), plays a critical role in the development of T1DM. During immune homeostasis, Tregs counterbalance the actions of autoreactive Teff cells, thereby participating in peripheral tolerance. An imbalance in the activity between Teff and Tregs may be crucial in the breakdown of peripheral tolerance, leading to the development of T1DM. In this review, we summarize our current understanding of Treg function in health and in T1DM, and examine the effect of experimental therapies for T1DM on Treg cell number and function in both mice and humans.

Differential expression of cell cycle proteins during ageing of pancreatic islet cells.

One of the major challenges for developmental biologists and investigators in the field of diabetes over the last few decades has been to dissect the origin of pancreatic endocrine cells and to accurately understand the mechanisms that regulate islet cell regeneration. While significant advances have been made recently, there continues to be a paucity of knowledge regarding the growth factor signalling pathways that directly regulate the proteins involved in islet cell cycle control. We will discuss recent work in these areas and provide insights from our studies into age-dependent alterations in the expression of growth factor signalling proteins and cell cycle proteins in islet cells.

Regulation of the pancreatic pro-endocrine gene neurogenin3.

Neurogenin3 (ngn3), a basic helix-loop-helix (bHLH) transcription factor, functions as a pro-endocrine factor in the developing pancreas: by itself, it is sufficient to force undifferentiated pancreatic epithelial cells to become islet cells. Because ngn3 expression determines which precursor cells will differentiate into islet cells, the signals that regulate ngn3 expression control islet cell formation. To investigate the factors that control ngn3 gene expression, we mapped the human and mouse ngn3 promoters and delineated transcriptionally active sequences within the human promoter. Surprisingly, the human ngn3 promoter drives transcription in all cell lines tested, including fibroblast cell lines. In contrast, in transgenic animals the promoter drives expression specifically in regions of ngn3 expression in the developing pancreas and gut; and the addition of distal sequences greatly enhances transgene expression. Within the distal enhancer, binding sites for several pancreatic transcription factors, including hepatocyte nuclear factor (HNF)-1 and HNF-3, form a tight cluster. HES1, an inhibitory bHLH factor activated by Notch signaling, binds to the proximal promoter and specifically blocks promoter activity. Together with previous genetic data, these results suggest a model in which the ngn3 gene is activated by the coordinated activities of several pancreatic transcription factors and inhibited by Notch signaling through HES1.

Intramolecular control of transcriptional activity by the NK2-specific domain in NK-2 homeodomain proteins.

The developmentally important homeodomain transcription factors of the NK-2 class contain a highly conserved region, the NK2-specific domain (NK2-SD). The function of this domain, however, remains unknown. The primary structure of the NK2-SD suggests that it might function as an accessory DNA-binding domain or as a protein-protein interaction interface. To assess the possibility that the NK2-SD may contribute to DNA-binding specificity, we used a PCR-based approach to identify a consensus DNA-binding sequences for Nkx2.2, an NK-2 family member involved in pancreas and central nervous system development. The consensus sequence (T(C)(T)AAGT(G)(A)(G)(C)TT) is similar to the known binding sequences for other NK-2 homeodomain proteins, but we show that the NK2-SD does not contribute significantly to specific DNA binding to this sequence. To determine whether the NK2-SD contributes to transactivation, we used GAL4-Nkx2. 2 fusion constructs to map a powerful transcriptional activation domain in the C-terminal region beyond the conserved NK2-SD. Interestingly, this C-terminal region functions as a transcriptional activator only in the absence of an intact NK2-SD. The NK2-SD also can mask transactivation from the paired homeodomain transcription factor Pax6, but it has no effect on transcription by itself. These results demonstrate that the NK2-SD functions as an intramolecular regulator of the C-terminal activation domain in Nkx2.2 and support a model in which interactions through the NK2-SD regulate the ability of NK-2-class proteins to activate specific genes during development.

Transcriptional and translational regulation of beta-cell differentiation factor Nkx6.1.

In the mature pancreas, the homeodomain transcription factor Nkx6.1 is uniquely restricted to beta-cells. Nkx6.1 also is expressed in developing beta-cells and plays an essential role in their differentiation. Among cell lines, both beta- and alpha-cell lines express nkx6.1 mRNA; but no protein can be detected in the alpha-cell lines, suggesting that post-transcriptional regulation contributes to the restriction of Nkx6.1 to beta-cells. To investigate the regulator of Nkx6.1 expression, we outlined the structure of the mouse nkx6.1 gene, and we identified regions that direct cell type-specific expression. The nkx6.1 gene has a long 5'-untranslated region (5'-UTR) downstream of a cluster of transcription start sites. nkx6.1 gene sequences from -5.6 to +1.0 kilobase pairs have specific promoter activity in beta-cell lines but not in NIH3T3 cells. This activity is dependent on sequences located at about -800 base pairs and on the 5'-UTR. Electrophoretic mobility shift assays demonstrate that homeodomain transcription factors PDX1 and Nkx2.2 can bind to the sequence element located at -800 base pairs. In addition, dicistronic assays establish that the 5'-UTR region functions as a potent internal ribosomal entry site, providing cell type-specific regulation of translation. These data demonstrate that complex regulation of both Nkx6.1 transcription and translation provides the specificity of expression required during pancreas development.

Beta-cell differentiation factor Nkx6.1 contains distinct DNA binding interference and transcriptional repression domains.

beta-Cell differentiation factor Nkx6.1 is a homeodomain protein expressed in developing and mature beta-cells in the pancreatic islets of Langerhans. To understand how it contributes to beta-cell development and function, we characterized its DNA binding and transactivation properties. A single copy of the homeodomain of Nkx6. 1 binds to a strictly conserved 8-base pair DNA consensus sequence, TTAATTAC; even minor variations to this consensus reduce DNA binding affinity significantly. Full-length Nkx6.1, however, has markedly reduced DNA binding affinity due to an acidic domain at the carboxyl end of the molecule that functions as a mobile binding interference domain capable of interrupting the interaction between DNA and DNA binding domains of the helix-turn-helix type. When expressed in fibroblast cell lines, Nkx6.1 represses transcription through isolated Nkx6.1 binding sites; in beta-cell lines, Nkx6.1 specifically represses the intact insulin promoter through TAAT-containing sequences. In Gal4 one-hybrid fusion studies, transcriptional repression maps to a discreet region within the amino terminus. Our findings suggest a model in which Nkx6.1, regulated by interactions through its carboxyl terminus, directs the repression of specific genes in developing and mature beta-cells.

The homeodomain of PDX-1 mediates multiple protein-protein interactions in the formation of a transcriptional activation complex on the insulin promoter.

Activation of insulin gene transcription specifically in the pancreatic beta cells depends on multiple nuclear proteins that interact with each other and with sequences on the insulin gene promoter to build a transcriptional activation complex. The homeodomain protein PDX-1 exemplifies such interactions by binding to the A3/4 region of the rat insulin I promoter and activating insulin gene transcription by cooperating with the basic-helix-loop-helix (bHLH) protein E47/Pan1, which binds to the adjacent E2 site. The present study provides evidence that the homeodomain of PDX-1 acts as a protein-protein interaction domain to recruit multiple proteins, including E47/Pan1, BETA2/NeuroD1, and high-mobility group protein I(Y), to an activation complex on the E2A3/4 minienhancer. The transcriptional activity of this complex results from the clustering of multiple activation domains capable of interacting with coactivators and the basal transcriptional machinery. These interactions are not common to all homeodomain proteins: the LIM homeodomain protein Lmx1.1 can also activate the E2A3/4 minienhancer in cooperation with E47/Pan1 but does so through different interactions. Cooperation between Lmx1.1 and E47/Pan1 results not only in the aggregation of multiple activation domains but also in the unmasking of a potent activation domain on E47/Pan1 that is normally silent in non-beta cells. While more than one activation complex may be capable of activating insulin gene transcription through the E2A3/4 minienhancer, each is dependent on multiple specific interactions among a unique set of nuclear proteins.

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

BACKGROUND: Non-ribosomal peptide synthetases (NRPSs) found in bacteria and 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.

Interactions of a hybrid insulin/insulin-like growth factor-I analog with chimeric insulin/type I insulin-like growth factor receptors.

We have examined, by use of a hybrid insulin/insulin-like growth factor-I analog and chimeric insulin/type I insulin-like growth factor receptors, the interplay between ligand and receptor structure in determining the affinity and specificity of hormone-receptor interactions in the insulin and insulin-like growth factor-I systems. Our findings, obtained through the study of radiolabeled peptide binding to detergent-solubilized full-length receptors and to soluble truncated receptors, show that (a) the two-chain hybrid analog exhibits significant cross-reactivity with both receptor systems, (b) the exchange of appropriate domains in chimeric receptors enhances the receptor binding affinity of the analog by 3.5-21-fold, and (c) the affinity of the hybrid analog for the chimeric receptors actually exceeds that of either natural insulin or natural insulin-like growth factor-I. We conclude that the specificity-conferring domains of the insulin and type I insulin-like growth factor receptors reside in different regions of a common binding site, and that the exchange of domains between pairs of related hormones and between pairs of related receptors can yield new ligand-receptor systems with significantly altered affinities and selectivities.

Disposition of the phenylalanine B25 side chain during insulin-receptor and insulin-insulin interactions.

By the semisynthesis of both full-length insulin analogues and their des-pentapeptide-(B26-B30)-alpha-carboxamide counterparts, we have examined the importance of the electronic character and bulk of the position B25 side chain both in directing insulin interaction with its receptor on isolated canine hepatocytes and in determining the ability of insulin to self-associate in solution. Analogues include those in which PheB25 was replaced by cyclohexyl-Ala; Tyr; p-nitro-, p-fluoro-, p-iodo-, or p-amino-Phe; or p-amino-Phe in which the aromatic amino function had been acylated by the acetyl, hexanoyl, decanoyl, or 1-adamantanoyl group. Our findings identify that (a) the beta-aromatic side chain at position B25 is indeed critical for high-affinity ligand-receptor interactions, (b) neither electron withdrawal from nor electron donation to the beta-aromatic ring perturbs ligand-receptor interactions in major ways, (c) considerable latitude is allowed the placement of linear or polycyclic apolar mass at the para position in p-amino-PheB25-substituted analogues with respect both to receptor binding affinity and to biological activity in vivo, and (d) para apolar mass at position B25 is readily accommodated during the self-association of insulin monomers, as assessed by analytical tyrosine radioiodination and spectroscopic analysis of analogue complexes with Co2+ and Co3+. These findings are discussed in terms of a model for insulin-receptor interactions at the cell membrane in which the position B25 side chain defines the edge of intermolecular contact.

Physicochemical characterization of bovine retinal arrestin.

The native conformation of bovine retinal arrestin has been characterized by a variety of spectroscopic methods. The purified protein gives rise to a near uv absorption band centered at 279 nm which results from the absorbance of its 14 tyrosine and one tryptophan residue. The extinction coefficient for this absorption band was determined to be 38.64 mM-1, cm-1 using the tyrosinate-tyrosine difference spectrum method; this extinction coefficient is ca. 17% lower than the previously reported value, and provides estimates of protein concentration which are in good agreement with estimates from the Bradford colorimetric assay. When native arrestin is purified to homogeneity, it displays a fluorescence spectrum which is dominated by tyrosine emission with no discernible contribution from tryptophan. Observation of the tyrosine-like fluorescence is dependent on the purity and structural integrity of the protein. Denaturation of arrestin by guanidine hydrochloride results in a diminution of tyrosine fluorescence and the concomitant appearance of a second fluorescence maximum at ca. 340 nm, presumably due to the single tryptophan residue. Thermal denaturation of arrestin leads to a conformation characterized by a broad fluorescence band centered at ca. 325 nm. Study of the arrestin fluorescence spectrum as a function of temperature indicates that the thermal denaturation is well modeled as a two-state transition with a transition midpoint of 60 degrees C. Temperature-dependent far uv circular dichroism studies indicate that changes in secondary structure occur coincident with the change in fluorescence. Studies of the temperature dependence of arrestin binding to light-adapted phosphorylated rhodopsin shows a strong correlation between the fluorescence spectral features of arrestin and its ability to bind rhodopsin. These data suggest that the relative intensities of tyrosine and tryptophan fluorescence are sensitive to the structural integrity of the native (i.e., rhodopsin binding) state of arrestin, and can thus serve as useful markers of conformational transitions of this protein. The lack of tryptophan fluorescence for native arrestin suggests an unusual environment for this residue. Possible mechanisms for this tryptophan fluorescence quenching are discussed.

Importance of the character and configuration of residues B24, B25, and B26 in insulin-receptor interactions.

By use of isolated canine hepatocytes and insulin analogs prepared by trypsin-catalyzed semisynthesis, we have investigated the importance of the aromatic triplet PheB24-PheB25-TyrB26 of the COOH-terminal B-chain domain of insulin in directing the affinity of insulin-receptor interactions. Analysis of the receptor binding potencies of analogs bearing transpositions or replacements (by Tyr, D-Tyr or their corresponding 3,5-diiodo derivatives) in this region demonstrates a wide divergence in the acceptance both of configurational change (with [D-TyrB24,PheB26]insulin and [D-TyrB25,PheB26]insulin exhibiting 160 and 0.1% of the receptor binding potency of insulin, respectively) and of detailed side chain structure (with [TyrB24,PheB26]insulin and [TyrB25,PheB26]insulin exhibiting 2 and 80% of the receptor binding potency of insulin, respectively). Additional experiments addressed the solvent accessibilities of the 4 tyrosine residues of insulin and the insulin analogs at selected peptide concentrations by use of analytical radioiodination. Whereas two analogs ([TyrB25,PheB26]insulin and [D-TyrB24,PheB26]insulin) were found to undergo self aggregation, no strict correlation was found between the ability of an analog to aggregate and its potency for interaction with the insulin receptor. Related findings are discussed in terms of the interplay between side chain and main chain structure in the COOH-terminal domain of the insulin B-chain and the structural attributes of insulin that determine the affinity of insulin-receptor interactions.

An insulin-like growth factor I/insulin hybrid exhibiting high potency for interaction with the type I insulin-like growth factor and insulin receptors of placental plasma membranes.

We have prepared by semisynthetic methods a two-chain insulin/insulin-like growth factor I hybrid that contains a synthetic peptide related to residues 22-41 of insulin-like growth factor I linked via peptide bond to ArgB22 of des-octapeptide-(B23-B30)-insulin and have applied the analog to the analysis of ligand interactions with the type I insulin-like growth factor and insulin receptors of placental plasma membranes. Relative potencies for the inhibition of 125I-labeled insulin-like growth factor I binding to type I insulin-like growth factor receptors were 1.0:0.20:0.003 for insulin-like growth factor I, the hybrid analog, and insulin, respectively. Corresponding relative potencies for the inhibition of 125I-labeled insulin binding to insulin receptors were 0.007:0.28:1 for the three respective peptides. Additional studies identified that the hybrid analog interacts with only one of two populations of insulin-like growth factor I binding sites on placental plasma membranes and permitted the analysis of insulin-like growth factor I interactions with the separate populations of binding sites. We conclude that (a) des-octapeptide-(B23-B30)-insulin can serve well as a scaffold to support structural elements of insulin-like growth factor I and insulin necessary for high affinity binding to their receptors, (b) major aspects of structure relevant to the conferral of receptor binding affinity lie in the COOH-terminal region of the insulin B chain and in the COOH-terminal region of the insulin-like growth factor I B domain and in its C domain, and (c) the evolution of ligand-receptor specificity in these systems has relied as much on restricting interactions (through the selective introduction of negative structural elements) as it has on enhancing interactions (through the introduction of affinity conferring elements of structure).

Role of the phenylalanine B24 side chain in directing insulin interaction with its receptor. Importance of main chain conformation.

We have investigated (by use of semisynthetic insulin analogs and isolated canine hepatocytes) the role of invariant residue PheB24 in determining the affinity of insulin-receptor interactions. Our results confirm that replacement of PheB24 by D-Phe is not detrimental to ligand binding to receptor, show that D-Ala is well tolerated at position B24 (whereas Ala is not), and demonstrate that [GlyB24]insulin retains as much as 78% of the receptor binding potency of native insulin. Additional findings show that replacement of PheB24 by D-Pro or by alpha-aminoisobutyric acid results in analogs with severely decreased binding potency, and that the COOH-terminal domain containing residues B26-B30 plays a positive role in determining receptor binding potency in GlyB24-substituted insulin (whereas it plays a negative role in determining the receptor binding potency of its GlyB25-substituted counterpart). We interpret our results as identifying (a) a critical role for the insulin main chain near residue B24 in determining the affinity of receptor for ligand, (b) the importance of main chain flexibility in achieving a high affinity state of receptor-bound hormone, and (c) a potential interaction of the PheB24 side chain with receptor which initiates main chain structural changes in the natural hormone, but which does not itself confer affinity to ligand-receptor interactions.