Clara Westwell-Roper

IL-1 Blockade Attenuates Islet Amyloid Polypeptide-Induced Proinflammatory Cytokine Release and Pancreatic Islet Graft Dysfunction

Islets from patients with type 2 diabetes exhibit β cell dysfunction, amyloid deposition, macrophage infiltration, and increased expression of proinflammatory cytokines and chemokines. We sought to determine whether human islet amyloid polypeptide (hIAPP), the main component of islet amyloid, might contribute to islet inflammation by recruiting and activating macrophages. Early aggregates of hIAPP, but not nonamyloidogenic rodent islet amyloid polypeptide, caused release of CCL2 and CXCL1 by islets and induced secretion of TNF-α, IL-1α, IL-1β, CCL2, CCL3, CXCL1, CXCL2, and CXCL10 by C57BL/6 bone marrow-derived macrophages. hIAPP-induced TNF-α secretion was markedly diminished in MyD88-, but not TLR2- or TLR4-deficient macrophages, and in cells treated with the IL-1R antagonist (IL-1Ra) anakinra. To determine the significance of IL-1 signaling in hIAPP-induced pancreatic islet dysfunction, islets from wild-type or hIAPP-expressing transgenic mice were transplanted into diabetic NOD/SCID recipients implanted with mini-osmotic pumps containing IL-1Ra (50 mg/kg/d) or saline. IL-1Ra significantly improved the impairment in glucose tolerance observed in recipients of transgenic grafts 8 wk following transplantation. Islet grafts expressing hIAPP contained amyloid deposits in close association with F4/80-expressing macrophages. Transgenic grafts contained 50% more macrophages than wild-type grafts, an effect that was inhibited by IL-1Ra. Our results suggest that hIAPP-induced islet chemokine secretion promotes macrophage recruitment and that IL-1R/MyD88, but not TLR2 or TLR4 signaling is required for maximal macrophage responsiveness to prefibrillar hIAPP. These data raise the possibility that islet amyloid-induced inflammation contributes to β cell dysfunction in type 2 diabetes and islet transplantation.

Resident Macrophages Mediate Islet Amyloid Polypeptide–Induced Islet IL-1β Production and β-Cell Dysfunction

Islet amyloid polypeptide (IAPP) aggregates to form amyloid fibrils in patients with type 2 diabetes and acts as a potent stimulus for interleukin (IL)-1β secretion by bone marrow–derived macrophages. We sought to determine the contribution of resident islet macrophages to IAPP-induced inflammation and β-cell dysfunction. In cultured islets, macrophages (F4/80+CD11b+CD11c+ cells) were required for IAPP-induced mRNA expression of the proinflammatory cytokines IL-1β, tumor necrosis factor-α, and IL-6 and the anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist. Moreover, IAPP-induced IL-1β synthesis and caspase-1 activation were detected in macrophages but not other islet cell types. Transgenic mice with β-cell human IAPP (hIAPP) expression had impaired glucose tolerance, elevated islet Il1b mRNA, and decreased Il10 and Il1rn expression following high-fat feeding. Islet macrophages were the major source of these transcripts and expressed increased cell surface Ly6C and CD11c in hIAPP transgenic mice. Clodronate liposome–mediated depletion of islet macrophages improved glucose tolerance and blocked proinflammatory gene expression in hIAPP-expressing mice, despite increasing the amount of islet amyloid. These data provide the first evidence that IAPP aggregates skew resident islet macrophages toward a proinflammatory phenotype and suggest a mechanism by which anti-inflammatory therapies may protect β-cells from IAPP-induced islet dysfunction.

Loss of Both ABCA1 and ABCG1 Results in Increased Disturbances in Islet Sterol Homeostasis, Inflammation, and Impaired β-Cell Function

Cellular cholesterol homeostasis is important for normal β-cell function. Disruption of cholesterol transport by decreased function of the ATP-binding cassette (ABC) transporter ABCA1 results in impaired insulin secretion. Mice lacking β-cell ABCA1 have increased islet expression of ABCG1, another cholesterol transporter implicated in β-cell function. To determine whether ABCA1 and ABCG1 have complementary roles in β-cells, mice lacking ABCG1 and β-cell ABCA1 were generated and glucose tolerance, islet sterol levels, and β-cell function were assessed. Lack of both ABCG1 and β-cell ABCA1 resulted in increased fasting glucose levels and a greater impairment in glucose tolerance compared with either ABCG1 deletion or loss of ABCA1 in β-cells alone. In addition, glucose-stimulated insulin secretion was decreased and sterol accumulation increased in islets lacking both transporters compared with those isolated from knockout mice with each gene alone. Combined deficiency of ABCA1 and ABCG1 also resulted in significant islet inflammation as indicated by increased expression of interleukin-1β and macrophage infiltration. Thus, lack of both ABCA1 and ABCG1 induces greater defects in β-cell function than deficiency of either transporter individually. These data suggest that ABCA1 and ABCG1 each make complimentary and important contributions to β-cell function by maintaining islet cholesterol homeostasis in vivo.

Metabolic stress, IAPP and islet amyloid

Amyloid forms within pancreatic islets in type 2 diabetes from aggregates of the β‐cell peptide islet amyloid polypeptide (IAPP). These aggregates are toxic to β‐cells, inducing β‐cell death and dysfunction, as well as inciting islet inflammation. The β‐cell is subject to a number of other stressors, including insulin resistance and hyperglycaemia, that may contribute to amyloid formation by increasing IAPP production by the β‐cell. β‐Cell dysfunction, evident as impaired glucose‐stimulated insulin secretion and defective prohormone processing and exacerbated by metabolic stress, is also a likely prerequisite for islet amyloid formation to occur in type 2 diabetes. Islet transplants in patients with type 1 diabetes face similar stressors, and are subject to rapid amyloid formation and impaired proinsulin processing associated with progressive loss of β‐cell function and mass. Declining β‐cell mass is predicted to increase metabolic demand on remaining β‐cells, promoting a feed‐forward cycle of β‐cell decline.

Toll-like receptors and NLRP3 as central regulators of pancreatic islet inflammation in type 2 diabetes.

The global health and economic burden of type 2 diabetes (T2D) has reached staggering proportions. Current projections estimate that 592 million people will have diabetes by 2035. T2D—which comprises 90% of cases—is a complex disease, in most cases resulting from a combination of predisposing genes and an unhealthy environment. Clinical onset of the disease occurs when pancreatic β cells fail in the face of insulin resistance. It has long been appreciated that chronic activation of the innate immune system is associated with T2D, and many organs critical to the regulation of glucose homeostasis show signs of a chronic inflammatory process, including the pancreatic islets of Langerhans. Recent clinical trials using IL‐1‐targeting agents have confirmed that inflammation contributes to β‐cell failure in humans with T2D. However, little is known about the nature of the pro‐inflammatory response within the islet, and there is considerable debate about the triggers for islet inflammation, which may be systemically derived and/or tissue‐specific. In this review, we present evidence that Toll‐like receptors 2 and 4 and the NLRP3 (Nucleotide‐binding oligomerization domain, Leucine‐rich Repeat and Pyrin domain containing 3) inflammasome are triggers for islet inflammation in T2D and propose that the activation of macrophages by these triggers mediates islet endocrine cell dysfunction. Therapeutically targeting these receptors may improve hyperglycemia and protect the β cell in T2D.

TLR2/6 and TLR4-activated macrophages contribute to islet inflammation and impair beta cell insulin gene expression via IL-1 and IL-6.

Aims/hypothesisInflammation contributes to pancreatic beta cell dysfunction in type 2 diabetes. Toll-like receptor (TLR)-2 and -4 ligands are increased systemically in recently diagnosed type 2 diabetes patients, and TLR2- and TLR4-deficient mice are protected from the metabolic consequences of a high-fat diet. Here we investigated the role of macrophages in TLR2/6- and TLR4-mediated effects on islet inflammation and beta cell function.MethodsGenetic and pharmacological approaches were used to determine the effects of TLR2/6 and TLR4 ligands on mouse islets, human islets and purified rat beta cells. Islet macrophages were depleted and sorted by flow cytometry and the effects of TLR2/6- and TLR4-activated bone-marrow-derived macrophages (BMDMs) on beta cell function were assessed.ResultsMacrophages contributed to TLR2/6- and TLR4-induced islet Il1a/IL1A and Il1b/IL1B mRNA expression in mouse and human islets and IL-1β secretion from human islets. TLR2/6 and TLR4 ligands also reduced insulin gene expression; however, this occurred in a non-beta cell autonomous manner. TLR2/6- and TLR4-activated BMDMs reduced beta cell insulin secretion partly via reducing Ins1, Ins2, and Pdx1 mRNA expression. Antagonism of the IL-1 receptor and neutralisation of IL-6 completely reversed the effects of activated macrophages on beta cell gene expression.Conclusions/interpretationWe conclude that islet macrophages are major contributors to islet IL-1β secretion in response to TLR2/6 and TLR4 ligands. BMDMs stimulated with TLR2/6 and TLR4 ligands reduce insulin secretion from pancreatic beta cells, partly via IL-1β- and IL-6-mediated decreased insulin gene expression.

Death and Dysfunction of Transplanted β-Cells: Lessons Learned From Type 2 Diabetes?

β-Cell replacement by islet transplantation is a potential curative therapy for type 1 diabetes. Despite advancements in islet procurement and immune suppression that have increased islet transplant survival, graft function progressively declines, and many recipients return to insulin dependence within a few years posttransplant. The progressive loss of β-cell function in islet transplants seems unlikely to be explained by allo- and autoimmune-mediated mechanisms alone and in a number of ways resembles β-cell failure in type 2 diabetes. That is, both following transplantation and in type 2 diabetes, islets exhibit decreased first-phase glucose-stimulated insulin secretion, impaired proinsulin processing, inflammation, formation of islet amyloid, signs of oxidative and endoplasmic reticulum stress, and β-cell death. These similarities suggest common mechanisms may underlie loss of insulin production in both type 2 diabetes and islet transplantation and point to the potential for therapeutic approaches used in type 2 diabetes that target the β-cell, such as incretin-based therapies, as adjuncts for immunosuppression in islet transplantation.

Thioredoxin-interacting protein promotes islet amyloid polypeptide expression through miR-124a and FoxA2.

Background: Islet amyloid polypeptide (IAPP) plays an important role in beta-cell biology, but its regulation is not fully understood. Results: Thioredoxin-interacting protein (TXNIP) induces IAPP by inhibiting miR-124a and promoting FoxA2-mediated transcription. Conclusion: The critical beta-cell signaling pathways of TXNIP and IAPP are linked. Significance: Identification of this novel TXNIP/miR-124a/FoxA2/IAPP signaling pathway provides new insight into an important aspect of transcriptional regulation and beta-cell biology. Thioredoxin-interacting protein (TXNIP) is up-regulated by glucose and diabetes and plays a critical role in glucotoxicity, inflammation, and beta-cell apoptosis, whereas we have found that TXNIP deficiency protects against diabetes. Interestingly, human islet amyloid polypeptide (IAPP) is also induced by glucose, aggregates into insoluble amyloid fibrils found in islets of most individuals with type 2 diabetes and promotes inflammation and beta-cell cytotoxicity. However, so far no connection between TXNIP and IAPP signaling had been reported. Using TXNIP gain and loss of function experiments, INS-1 beta-cells and beta-cell-specific Txnip knock-out mice, we now found that TXNIP regulates IAPP expression. Promoter analyses and chromatin-immunoprecipitation assays further demonstrated that TXNIP increases IAPP expression at the transcriptional level, and we discovered that TXNIP-induced FoxA2 (forkhead box A2) transcription factor expression was conferring this effect by promoting FoxA2 enrichment at the proximal FoxA2 site in the IAPP promoter. Moreover, we found that TXNIP down-regulates miR-124a expression, a microRNA known to directly target FoxA2. Indeed, miR-124a overexpression led to decreased FoxA2 expression and IAPP promoter occupancy and to a significant reduction in IAPP mRNA and protein expression and also effectively inhibited TXNIP-induced IAPP expression. Thus, our studies have identified a novel TXNIP/miR-124a/FoxA2/IAPP signaling cascade linking the critical beta-cell signaling pathways of TXNIP and IAPP and thereby provide new mechanistic insight into an important aspect of transcriptional regulation and beta-cell biology.

Islet allograft rejection is independent of toll-like receptor signaling in mice.

Background. Islet transplantation is a promising therapy for type 1 diabetes; however, most islet grafts fail within 5 years. Innate immunity has been suggested to play a role in islet allograft rejection, potentially mediated by toll-like receptors (TLRs), a class of innate immune receptors. Lack of TLR4, in particular, has been reported to improve allograft survival. Therefore, we hypothesized that TLRs may be involved in islet allograft rejection, and that deletion of TLR4 may improve islet graft survival. Methods. Islets were isolated from C57BL/10ScNJ (Tlr4−/−) and C57BL/10 (wild-type [WT]) animals and transplanted into Balb/cJ recipients with streptozotocin-induced diabetes. Blood glucose levels were used to determine graft viability and immunostaining to assess graft morphology and immune cell infiltration. The roles of the TLR4 adaptor molecules MyD88 and TLR adaptor molecule 1 (Ticam-1) were assessed using islets isolated from mice lacking MyD88 (MyD88−/−), Ticam-1 (Ticam-1−/−), or the combined double knockout (MyD88−/−/Ticam-1−/−). Results. Contrary to our hypothesis, Tlr4−/− and WT islet allografts had similar failure rates; grafts failed at 23.2±1.2 and 24.5±1.5 days posttransplant, respectively (P=NS). Syngeneic grafts of Tlr4−/− and WT islets maintained normoglycemia for up to 10 weeks posttransplant, indicating that failure of Tlr4−/− islet allografts could not be attributed to an intrinsic defect in Tlr4−/− islets. Similarly, islet allotransplants from MyD88−/−, Ticam-1−/−, and MyD88−/−/Ticam-1−/− donors did not have improved allograft survival compared with WT controls. Conclusions. These findings indicate that islet allograft rejection in mice is independent of TLR4 and the TLR adaptor molecules MyD88 and Ticam-1, speaking against an essential role for TLR signaling in islet allograft rejection.

Npas4 Is a Novel Activity–Regulated Cytoprotective Factor in Pancreatic β-Cells

Cellular homeostasis requires intrinsic sensing mechanisms to temper function in the face of prolonged activity. In the pancreatic β-cell, glucose is likely a physiological trigger that activates an adaptive response to stimulation, thereby maintaining cellular homeostasis. Immediate early genes (IEGs) are activated as a first line of defense in cellular homeostasis and are largely responsible for transmitting an environmental cue to a cellular response. Here we examine the regulation and function of the novel β-cell IEG, neuronal PAS domain protein 4 (Npas4). Using MIN6 cells, mouse and human islets, as well as in vivo infusions, we demonstrate that Npas4 is expressed within pancreatic islets and is upregulated by β-cell depolarizing agents. Npas4 tempers β-cell function through a direct inhibitory interaction with the insulin promoter and by blocking the potentiating effects of GLP-1 without significantly reducing glucose-stimulated secretion. Finally, Npas4 expression is induced by classical endoplasmic reticulum (ER) stressors and can prevent thapsigargin- and palmitate-induced dysfunction and cell death. These results suggest that Npas4 is a key activity-dependent regulator that improves β-cell efficiency in the face of stress. We posit that Npas4 could be a novel therapeutic target in type 2 diabetes that could both reduce ER stress and cell death and maintain basal cell function.