Toll-like receptor 4 in pancreatic damage and immune infiltration in acute pancreatitis.

Acute pancreatitis is a complex inflammatory disease resulting in extreme pain and can result in significant morbidity and mortality. It can be caused by several factors ranging from genetics, alcohol use, gall stones, and ductal obstruction caused by calcification or neutrophil extracellular traps. Acute pancreatitis is also characterized by immune cell infiltration of neutrophils and M1 macrophages. Toll-like receptor 4 (TLR4) is a pattern recognition receptor that has been noted to respond to endogenous ligands such as high mobility group box 1 (HMGB1) protein and or exogenous ligands such as lipopolysaccharide both of which can be present during the progression of acute pancreatitis. This receptor can be found on a variety of cell types from endothelial cells to resident and infiltrating immune cells leading to production of pro-inflammatory cytokines as well as immune cell activation and maturation resulting in the furthering of pancreatic damage during acute pancreatitis. In this review we will address the various mechanisms mediated by TLR4 in the advancement of acute pancreatitis and how targeting this receptor could lead to improved outcomes for patients suffering from this condition.

Inhibition of Toll-like Receptor 4 Using Small Molecule, TAK-242, Protects Islets from Innate Immune Responses.

Islet transplantation is a therapeutic option to replace ß-cell mass lost during type 1 or type 3c diabetes. Innate immune responses, particularly the instant blood-mediated inflammatory reaction and activation of monocytes, play a major role in the loss of transplanted islet tissue. In this study, we aimed to investigate the inhibition of toll-like receptor 4 (TLR4) on innate inflammatory responses. We first demonstrate a significant loss of graft function shortly after transplant through the assessment of miR-375 and miR-200c in plasma as biomarkers. Using in vitro models, we investigate how targeting TLR4 mitigates islet damage and immune cell activation during the peritransplant period. The results of this study support the application of TAK-242 as a therapeutic agent to reduce inflammatory and innate immune responses to islets immediately following transplantation into the hepatic portal vein. Therefore, TLR4 may serve as a target to improve islet transplant outcomes in the future.

Clinical and biological significance of circulating miRNAs in chronic pancreatitis patients undergoing total pancreatectomy with islet autotransplantation.

BACKGROUND: Specific microRNAs (miRNAs) were elevated in chronic pancreatitis (CP) patients during islet infusion after total pancreatectomy (TPIAT). We aimed to identify circulating miRNA signatures of pancreatic damage, predict miRNA-mRNA networks to identify potential links to CP pathogenesis and identify islet isolation and transplantation functional outcomes. METHODS: Small RNA sequencing was performed to identify distinct circulating miRNA signatures in CP. Plasma miRNAs were measured using miRCURY LNA SYBR green quantitative real-time polymerase chain reaction assays. Correlation analyses were performed using R software. The miRNA target and disease interactions were determined using miRNet and the miRNA enrichment and annotation tool. RESULTS: Alterations were found in circulating miRNAs in CP patients compared to healthy controls. Further studies were conducted on 12 circulating miRNAs enriched in the pancreas, other tissues and other diseases including cancer and fibrosis. Approximately 2888 mRNAs in the pancreas were their targets, demonstrating interactions with 76 small molecules. Three miRNAs exhibited interactions with morphine and five exhibited interactions with glucose. The miRNA panel targeted 22 genes associated with pancreatitis. The islet-specific, acinar cell-specific and liver-specific miRNAs were elevated at 6 h after islet infusion and returned to baseline levels 3 months after TPIAT. Circulating levels of miRNAs returned to pre-transplant levels 1-year post-transplant. Circulating miRNAs measured before and 6 h after islet infusion were directly or inversely associated with metabolic outcomes at 3 and 6 months post-transplant. CONCLUSIONS: miRNAs may contribute to CP pathogenesis, and elevated circulating levels may be specific to pancreatic inflammation and fibrosis, warranting further investigation.

Interaction of a viral insulin-like peptide with the IGF-1 receptor produces a natural antagonist.

Lymphocystis disease virus-1 (LCDV-1) and several other Iridoviridae encode viral insulin/IGF-1 like peptides (VILPs) with high homology to human insulin and IGFs. Here we show that while single-chain (sc) and double-chain (dc) LCDV1-VILPs have very low affinity for the insulin receptor, scLCDV1-VILP has high affinity for IGF1R where it can antagonize human IGF-1 signaling, without altering insulin signaling. Consequently, scLCDV1-VILP inhibits IGF-1 induced cell proliferation and growth hormone/IGF-1 induced growth of mice in vivo. Cryo-electron microscopy reveals that scLCDV1-VILP engages IGF1R in a unique manner, inducing changes in IGF1R conformation that led to separation, rather than juxtaposition, of the transmembrane segments and hence inactivation of the receptor. Thus, scLCDV1-VILP is a natural peptide with specific antagonist properties on IGF1R signaling and may provide a new tool to guide development of hormonal analogues to treat cancers or metabolic disorders sensitive to IGF-1 without affecting glucose metabolism.

Single-chain insulin analogs threaded by the insulin receptor αCT domain.

Insulin is a mainstay of therapy for diabetes mellitus, yet its thermal stability complicates global transportation and storage. Cold-chain transport, coupled with optimized formulation and materials, prevents to some degree nucleation of amyloid and hence inactivation of hormonal activity. These issues hence motivate the design of analogs with increased stability, with a promising approach being single-chain insulins (SCIs), whose C domains (foreshortened relative to proinsulin) resemble those of the single-chain growth factors (IGFs). We have previously demonstrated that optimized SCIs can exhibit native-like hormonal activity with enhanced thermal stability and marked resistance to fibrillation. Here, we describe the crystal structure of an ultrastable SCI (C-domain length 6; sequence EEGPRR) bound to modules of the insulin receptor (IR) ectodomain (N-terminal α-subunit domains L1-CR and C-terminal αCT peptide; "microreceptor" [µIR]). The structure of the SCI-µIR complex, stabilized by an Fv module, was determined using diffraction data to a resolution of 2.6 Å. Remarkably, the αCT peptide (IR-A isoform) "threads" through a gap between the flexible C domain and the insulin core. To explore such threading, we undertook molecular dynamics simulations to 1) compare threaded with unthreaded binding modes and 2) evaluate effects of C-domain length on these alternate modes. The simulations (employing both conventional and enhanced sampling simulations) provide evidence that very short linkers (C-domain length of -1) would limit gap opening in the SCI and so impair threading. We envisage that analogous threading occurs in the intact SCI-IR complex-rationalizing why minimal C-domain lengths block complete activity-and might be exploited to design novel receptor-isoform-specific analogs.

Activation of the human insulin receptor by non-insulin-related peptides.

The human insulin receptor signalling system plays a critical role in glucose homeostasis. Insulin binding brings about extensive conformational change in the receptor extracellular region that in turn effects trans-activation of the intracellular tyrosine kinase domains and downstream signalling. Of particular therapeutic interest is whether insulin receptor signalling can be replicated by molecules other than insulin. Here, we present single-particle cryoEM structures that show how a 33-mer polypeptide unrelated to insulin can cross-link two sites on the receptor surface and direct the receptor into a signalling-active conformation. The 33-mer polypeptide engages the receptor by two helical binding motifs that are each potentially mimicable by small molecules. The resultant conformation of the receptor is distinct from-but related to-those in extant three-dimensional structures of the insulin-complexed receptor. Our findings thus illuminate unexplored pathways for controlling the signalling of the insulin receptor as well as opportunities for development of insulin mimetics.

How insulin-like growth factor I binds to a hybrid insulin receptor type 1 insulin-like growth factor receptor.

Monomers of the insulin receptor and type 1 insulin-like growth factor receptor (IGF-1R) can combine stochastically to form heterodimeric hybrid receptors. These hybrid receptors display ligand binding and signaling properties that differ from those of the homodimeric receptors. Here, we describe the cryoelectron microscopy structure of such a hybrid receptor in complex with insulin-like growth factor I (IGF-I). The structure (ca. 3.7 Å resolution) displays a single IGF-I ligand, bound in a similar fashion to that seen for IGFs in complex with IGF-1R. The IGF-I ligand engages the first leucine-rich-repeat domain and cysteine-rich region of the IGF-1R monomer (rather than those of the insulin receptor monomer), consistent with the determinants for IGF binding residing in the IGF-1R cysteine-rich region. The structure broadens our understanding of this receptor family and assists in delineating the key structural motifs involved in binding their respective ligands.

Calcineurin/NFATc2 and PI3K/AKT signaling maintains ß-cell identity and function during metabolic and inflammatory stress.

Pancreatic islets respond to metabolic and inflammatory stress by producing hormones and other factors that induce adaptive cellular and systemic responses. Here we show that intracellular Ca2+ ([Ca2+]i) and ROS signals generated by high glucose and cytokine-induced ER stress activate calcineurin (CN)/NFATc2 and PI3K/AKT to maintain ß-cell identity and function. This was attributed in part by direct induction of the endocrine differentiation gene RFX6 and suppression of several ß-cell "disallowed" genes, including MCT1. CN/NFATc2 targeted p300 and HDAC1 to RFX6 and MCT1 promoters to induce and suppress gene transcription, respectively. In contrast, prolonged exposure to stress, hyperstimulated [Ca2+]i, or perturbation of CN/NFATc2 resulted in downregulation of RFX6 and induction of MCT1. These findings reveal that CN/NFATc2 and PI3K/AKT maintain ß-cell function during acute stress, but ß-cells dedifferentiate to a dysfunctional state upon loss or exhaustion of Ca2+/CN/NFATc2 signaling. They further demonstrate the utility of targeting CN/NFATc2 to restore ß-cell function.

Symmetric and asymmetric receptor conformation continuum induced by a new insulin.

Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes and biochemical properties. Here, we report an active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy (cryo-EM) and protein engineering to elucidate its interactions with the human insulin receptor (IR) ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of human insulin but also compromise therapeutic utility by delaying dissolution from the site of subcutaneous injection. This finding suggests approaches to developing improved therapeutic insulins. Curiously, the receptor displays a continuum of conformations from the symmetric state to a highly asymmetric low-abundance structure that displays coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.

Role of Exosomes in Islet Transplantation.

Exosomes are known for their ability to transport nucleic acid, lipid, and protein molecules, which allows for communication between cells and tissues. The cargo of the exosomes can have a variety of effects on a wide range of targets to mediate biological function. Pancreatic islet transplantation is a minimally invasive cell replacement therapy to prevent or reverse diabetes mellitus and is currently performed in patients with uncontrolled type 1 diabetes or chronic pancreatitis. Exosomes have become a focus in the field of islet transplantation for the study of diagnostic markers of islet cell viability and function. A growing list of miRNAs identified from exosomes collected during the process of isolating islets can be used as diagnostic biomarkers of islet stress and damage, leading to a better understanding of critical steps of the isolation procedure that can be improved to increase islet yield and quality. Exosomes have also been implicated as a possible contributor to islet graft rejection following transplantation, as they carry donor major histocompatibility complex molecules, which are then processed by recipient antigen-presenting cells and sensed by the recipient immune cells. Exosomes may find their way into the therapeutic realm of islet transplantation, as exosomes isolated from mesenchymal stem cells have shown promising results in early studies that have seen increased viability and functionality of isolated and grafted islets in vitro as well as in vivo. With the study of exosomes still in its infancy, continued research on the role of exosomes in islet transplantation will be paramount to understanding beta cell regeneration and improving long-term graft function.

Insertion of a synthetic switch into insulin provides metabolite-dependent regulation of hormone-receptor activation.

Insulin-signaling requires conformational change: whereas the free hormone and its receptor each adopt autoinhibited conformations, their binding leads to structural reorganization. To test the functional coupling between insulin's "hinge opening" and receptor activation, we inserted an artificial ligand-dependent switch into the insulin molecule. Ligand-binding disrupts an internal tether designed to stabilize the hormone's native closed and inactive conformation, thereby enabling productive receptor engagement. This scheme exploited a diol sensor (meta-fluoro-phenylboronic acid at GlyA1) and internal diol (3,4-dihydroxybenzoate at LysB28). The sensor recognizes monosaccharides (fructose > glucose). Studies of insulin-signaling in human hepatoma-derived cells (HepG2) demonstrated fructose-dependent receptor autophosphorylation leading to appropriate downstream signaling events, including a specific kinase cascade and metabolic gene regulation (gluconeogenesis and lipogenesis). Addition of glucose (an isomeric ligand with negligible sensor affinity) did not activate the hormone. Similarly, metabolite-regulated signaling was not observed in control studies of 1) an unmodified insulin analog or 2) an analog containing a diol sensor without internal tethering. Although secondary structure (as probed by circular dichroism) was unaffected by ligand-binding, heteronuclear NMR studies revealed subtle local and nonlocal monosaccharide-dependent changes in structure. Insertion of a synthetic switch into insulin has thus demonstrated coupling between hinge-opening and allosteric holoreceptor signaling. In addition to this foundational finding, our results provide proof of principle for design of a mechanism-based metabolite-responsive insulin. In particular, replacement of the present fructose sensor by an analogous glucose sensor may enable translational development of a "smart" insulin analog to mitigate hypoglycemic risk in diabetes therapy.

Design and Catalyzed Activation of Mycophenolic Acid Prodrugs.

Mycophenolic acid (MPA) and its morpholino ester prodrug mycophenolate mofetil (MMF) are widely used in solid organ transplantation. These drugs prevent rejection due to their potent inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH), an enzyme vital for lymphocyte proliferation. As a strategy to provide localized immunosuppression in cell transplantation, four mycophenolic acid prodrugs designed to release MPA by two distinct mechanisms were synthesized and characterized. A nitrobenzyl ether prodrug was effectively converted to MPA upon exposure to bacterial nitroreductase, while a propargyl ether was converted to the active drug by immobilized Pd0 nanoparticles. In vitro, both prodrugs were inactive against IMPDH and exhibited reduced toxicity relative to the active drug, suggesting their potential for providing localized immunosuppression.

Understanding insulin and its receptor from their three-dimensional structures.

BACKGROUND: Insulin's discovery 100 years ago and its ongoing use since that time to treat diabetes belies the molecular complexity of its structure and that of its receptor. Advances in single-particle cryo-electron microscopy have over the past three years revolutionized our understanding of the atomic detail of insulin-receptor interactions. SCOPE OF REVIEW: This review describes the three-dimensional structure of insulin and its receptor and details on how they interact. This review also highlights the current gaps in our structural understanding of the system. MAJOR CONCLUSIONS: A near-complete picture has been obtained of the hormone receptor interactions, providing new insights into the kinetics of the interactions and necessitating a revision of the extant two-site cross-linking model of hormone receptor engagement. How insulin initially engages the receptor and the receptor's traversed trajectory as it undergoes conformational changes associated with activation remain areas for future investigation.

Author Correction: A structurally minimized yet fully active insulin based on cone-snail venom insulin principles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A structurally minimized yet fully active insulin based on cone-snail venom insulin principles.

Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone-snail venom with moderate human insulin-like bioactivity. Here, with insights from structural biology studies, we report the development of mini-Ins-a human des-octapeptide insulin analog-as a structurally minimal, full-potency insulin. Mini-Ins is monomeric and, despite the lack of the canonical B-chain C-terminal octapeptide, has similar receptor binding affinity to human insulin. Four mutations compensate for the lack of contacts normally made by the octapeptide. Mini-Ins also has similar in vitro insulin signaling and in vivo bioactivities to human insulin. The full bioactivity of mini-Ins demonstrates the dispensability of the PheB24-PheB25-TyrB26 aromatic triplet and opens a new direction for therapeutic insulin development.

Grafting Islets to a Dissected Peritoneal Pouch to Improve Transplant Survival and Function.

BACKGROUND: Although the liver is the primary site for clinical islet transplantation, it poses several restrictions, especially limited tissue volume due to portal vein pressure. We evaluated the preperitoneal space as an extrahepatic islet transplant site to deliver high tissue volumes and sustain long-term graft function. METHODS: A peritoneal pouch was formed by dissecting the parietal peritoneum from the transversalis fascia of mice. Syngeneic C57BL/6 donor islets were transplanted into the peritoneal pouch of diabetic mouse recipients. Blood glucose was monitored for islet function, and miR-375 was analyzed for islet damage. Islet graft morphology and vascularization were evaluated by immunohistochemistry. [F] fluoro-D-glucose positron emission tomography/computed tomography was used to image islet grafts. RESULTS: Transplantation of 300 syngeneic islets into the peritoneal pouch of recipients reversed hyperglycemia for >60 days. Serum miR-375 was significantly lower in the peritoneal pouch group than in the peritoneal cavity group. Peritoneal pouch islet grafts showed high neovascularization and sustained insulin and glucagon expression up to 80 days posttransplantation. A peritoneal pouch graft with high tissue volume (1000 islets) could be visualized by positron emission tomography/computed tomography imaging. Human islets transplanted into the peritoneal pouch of diabetic nude mice also reversed hyperglycemia successfully. CONCLUSIONS: Islets transplanted into a dissected peritoneal pouch show high efficiency to reverse diabetes and sustain islet graft function. The preperitoneal site has the advantages of capacity for high tissue volume, enriched revascularization and minimal inflammatory damage. It can also serve as an extrahepatic site for transplanting large volume of islets necessitated in islet autotransplantation.

How IGF-II Binds to the Human Type 1 Insulin-like Growth Factor Receptor.

Human type 1 insulin-like growth factor receptor (IGF-1R) signals chiefly in response to the binding of insulin-like growth factor I. Relatively little is known about the role of insulin-like growth factor II signaling via IGF-1R, despite the affinity of insulin-like growth factor II for IGF-1R being within an order of magnitude of that of insulin-like growth factor I. Here, we describe the cryoelectron microscopy structure of insulin-like growth factor II bound to a leucine-zipper-stabilized IGF-1R ectodomain, determined in two conformations to a maximum average resolution of 3.2 Å. The two conformations differ in the relative separation of their respective points of membrane entry, and comparison with the structure of insulin-like growth factor I bound to IGF-1R reveals long-suspected differences in the way in which the critical C domain of the respective growth factors interact with IGF-1R.

MicroRNA Signatures as Future Biomarkers for Diagnosis of Diabetes States.

Diabetes results from the inability of pancreatic islets to maintain blood glucose concentrations within a normal physiological range. Clinical features are usually not observed until islets begin to fail and irreversible damage has occurred. Diabetes is generally diagnosed based on elevated glucose, which does not distinguish between type 1 and 2 diabetes. Thus, new diagnostic approaches are needed to detect different modes of diabetes before manifestation of disease. During prediabetes (pre-DM), islets undergo stress and release micro (mi) RNAs. Here, we review studies that have measured and tracked miRNAs in the blood for those with recent-onset or longstanding type 1 diabetes, obesity, pre-diabetes, type 2 diabetes, and gestational diabetes. We summarize the findings on miRNA signatures with the potential to stage progression of different modes of diabetes. Advances in identifying selective biomarker signatures may aid in early detection and classification of diabetic conditions and treatments to prevent and reverse diabetes.