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.
Inflammation is an established pathogenic player in insulin resistance, islet demise and atherosclerosis. The complex interactions between cytokines, immune cells and affected tissues result in ...sustained inflammation in diabetes and atherosclerosis. 12‐ and 15‐lipoxygenase (LO), such as 12/15‐LO, produces a variety of metabolites through peroxidation of fatty acids and potentially contributes to the complex molecular crosstalk at the site of inflammation. 12‐ and 15‐LO pathways are frequently activated in tissues affected by diabetes and atherosclerosis including adipose tissue (AT), islets and the vasculature. Moreover, mice with whole body and tissue‐specific knockout of 12/15‐LO are protected against insulin resistance, hyperglycaemia and atherosclerosis supporting functional contribution of 12‐ and 15‐LO pathways in diabetes and atherosclerosis. Recently, it has emerged that there is a temporal regulation of the particular isoforms of 12‐ and 15‐LO in human AT and islets during the development of type 1 and type 2 diabetes and obesity. Analyses of tissues affected by diabetes and atherosclerosis also implied the roles of interleukin (IL)‐12 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase‐1 (NOX‐1) in islets and IL‐17A in atherosclerosis. Future studies should aim to test the efficacy of inhibitions of these mediators for treatment of diabetes and atherosclerosis.
► Pro-inflammatory cytokines induce NADPH oxidase-1 expression in beta cells. ► Inhibitors of NADPH oxidase block NOX-1 expression in beta cells. ► Cellular reactive oxygen species levels modulate ...NOX-1 expression in beta cells. ► Src-kinase activation by cellular reactive oxygen species activates NOX-1 expression. ► Feedback regulation of NOX-1 in β-cells, an enticing therapeutic target in diabetes.
NADPH oxidase-1 (NOX-1) is upregulated in beta cells in response to pro-inflammatory cytokines. Inhibition of NADPH oxidase activity blocked stimulated NOX-1 expression (p<0.05). Regulation of NOX-1 expression in beta cells followed modulation of cellular reactive oxygen species (ROS); pro-oxidants increased NOX-1 (p<0.001) and anti-oxidants decreased NOX-1 (p<0.05). Activation of Src-kinase followed ROS elevation. Inhibition of Src-kinase decreased NOX-1 expression (p<0.01). Beta cell dysfunction, measured by elevated MCP-1 expression, loss of glucose-sensitive insulin secretion or cell death, was induced by pro-inflammatory cytokine stimulation. Importantly, inhibition of Src-kinase or NOX-1 preserved beta cell function and survival. Collectively, these data indicate that expression of NOX-1 in beta cells is regulated in a feed-forward loop mediated by ROS and Src-kinase. Uncoupling of this feed-forward activation could provide new approaches to preserve and protect beta cells in diabetes.
Context: Inflammation is increasingly recognized as an important contributing factor in diabetes mellitus. Lipoxygenases (LOs) produce active lipids that promote inflammatory damage by catalyzing the ...oxidation of linoleic and arachidonic acid, and LO is expressed in rodent and human islets. Little is known about the differential effect of the various hydroxyeicosatetraenoic acids (HETEs) that result from LO activity in human islets.
Objective: We compared the effects of 12-LO products on human islet viability and function.
Design: Human islets were treated with stable compounds derived from LOs: 12(S)-HETE, 15HETE, 12HPETE, and 12RHETE and then examined for insulin secretion and islet viability. The p38-MAPK (p38) and JNK stress-activated pathways were investigated as mechanisms of 12-LO-mediated islet inhibition in rodent and human islets.
Results: Insulin secretion was consistently reduced by 12(S)-HETE and 12HPETE. 12(S)-HETE at 1 nm reduced viability activity by 32% measured by MTT assay and increased cell death by 50% at 100 nm in human islets. These effects were partially reversed with lisofylline, a small-molecule antiinflammatory compound that protects mitochondrial function. 12(S)-HETE increased phosphorylated p38-MAPK (pp38) protein activity in human islets. Injecting 12-LO siRNA into C57BL/6 mice reduced 12-LO and pp38-MAPK protein levels in mouse islets. The addition of proinflammatory cytokines increased pp38 levels in normal mouse islets but not in siRNA-treated islets.
Conclusions: These data suggest that 12(S)-HETE reduces insulin secretion and increases cell death in human islets. The 12-LO pathway is present in human islets, and expression is up-regulated by inflammatory cytokines. Reduction of 12-LO activity could thus provide a new therapeutic approach to protect human β-cells from inflammatory injury.
12-Lipoxygenase is an important inflammatory pathway leading to damage of human islets.
Aims/hypothesis
IL-12 is an important cytokine in early inflammatory responses and is implicated in the immune-mediated pathogenesis of pancreatic islets in diabetes. However, little is known about ...the direct effects of IL-12 on islets and beta cells.
Methods
In this study, beta cell function, gene expression and protein production were assessed in primary human donor islets and murine beta cell lines in response to stimulation with IL-12 or a pro-inflammatory cytokine cocktail (TNF-α, IL-1β and IFN-γ).
Results
The pro-inflammatory cytokine cocktail induced islet dysfunction and potently increased the expression and production of IL-12 ligand and IL-12 receptor in human islets. In human islets, the receptor for IL-12 co-localised to the cell surface of insulin-producing cells. Both IL-12 ligand and IL-12 receptor are expressed in the homogeneous beta cell line INS-1. IL-12 induced changes in gene expression, including a dose-dependent upregulation of
IFNγ
(also known as
IFNG
), in INS-1 cells. A neutralising antibody to IL-12 directly inhibited
IFNγ
gene expression in human donor islets induced by either IL-12 or pro-inflammatory cytokine stimulation. Functionally, IL-12 impaired glucose-stimulated insulin secretion (GSIS) in INS-1 cells and human donor islets. A neutralising antibody to IL-12 reversed the beta cell dysfunction (uncoupling of GSIS or induction of caspase-3 activity) induced by pro-inflammatory cytokines.
Conclusions/interpretation
These data identify beta cells as a local source of IL-12 ligand and suggest a direct role of IL-12 in mediating beta cell pathology.
Current immunosuppressive therapies act on T lymphocytes by modulation of cytokine production, modulation of signaling pathways or by inhibition of the enzymes of nucleotide biosynthesis. We have ...identified a previously unknown series of immunomodulatory compounds that potently inhibit human and rat T lymphocyte proliferation in vitro and in vivo in immune-mediated animal models of disease, acting by a novel mechanism. Here we identify the target of these compounds, the monocarboxylate transporter MCT1 (SLC16A1), using a strategy of photoaffinity labeling and proteomic characterization. We show that inhibition of MCT1 during T lymphocyte activation results in selective and profound inhibition of the extremely rapid phase of T cell division essential for an effective immune response. MCT1 activity, however, is not required for many stages of lymphocyte activation, such as cytokine production, or for most normal physiological functions. By pursuing a chemistry-led target identification strategy, we have discovered that MCT1 is a previously unknown target for immunosuppressive therapy and have uncovered an unsuspected role for MCT1 in immune biology.
The 12/15-lipoxygenase enzymes react with fatty acids producing active lipid metabolites that are involved in a number of significant disease states. The latter include type 1 and type 2 diabetes ...(and associated complications), cardiovascular disease, hypertension, renal disease, and the neurological conditions Alzheimer’s disease and Parkinson’s disease. A number of elegant studies over the last thirty years have contributed to unraveling the role that lipoxygenases play in chronic inflammation. The development of animal models with targeted gene deletions has led to a better understanding of the role that lipoxygenases play in various conditions. Selective inhibitors of the different lipoxygenase isoforms are an active area of investigation, and will be both an important research tool and a promising therapeutic target for treating a wide spectrum of human diseases.
ABSTRACT
Context
Inflammation is increasingly recognized as an important contributing factor in diabetes mellitus. Lipoxygenases (LOs) produce active lipids that promote inflammatory damage by ...catalyzing the oxidation of linoleic and arachidonic acid, and LO is expressed in rodent and human islets. Little is known about the differential effect of the various hydroxyeicosatetraenoic acids (HETEs) that result from LO activity in human islets.
Design
We compared the effect of stable compounds derived from LOs: 12(S)-HETE, 15HETE, 12HPETE, and 12RHETE.
Interventions
At both 1 and 100 nm, insulin secretion was consistently reduced by 12(S)-HETE and 12HPETE. 12(S)-HETE also reduced viability activity by 32% at 1 nm. Insulin and reduced viability were partially reversed by treatment with lisofylline, a small-molecule antiinflammatory compound that protects mitochondrial function. 12(S)-HETE also increased cell death rate of human islets by 50% at 100 nm. To investigate mechanisms of 12-LO-mediated islet inhibition, we examined the p38-MAPK and JNK stress-activated pathways.
Results
Treatment of 12(S)-HETE significantly increased phosphorylated p38-MAPK (pp38) protein activity in human islets. We further explored the in vivo role of 12-LO in regulating p38-MAPK using mouse models. Knockdown of 12-LO by injecting 12-LO siRNA into C57BL/6 mice decreased pp38 protein levels in mouse islets. The addition of proinflammatory cytokines increased pp38 levels in normal mouse islets but not in siRNA-treated islets.
Conclusions
These data suggest that 12(S)-HETE reduces insulin secretion and increases cell death in human islets. The 12-LO pathway is present in human islets, and expression is up-regulated by inflammatory cytokines. Reduction of 12-LO activity could thus provide a new therapeutic approach to protect human β-cells from inflammatory injury.
Islet inflammation contributes to beta cell demise in both type 1 and type 2 diabetes. 12-Lipoxygenase (12-LO, gene expressed as
ALOX12
in humans and
12-Lo
in rodents in this manuscript) produces ...proinflammatory metabolites such as 12(
S
)-hydroxyeicosatetraenoic acids through dioxygenation of polyunsaturated fatty acids. 12-LO was first implicated in diabetes when the increase in
12-Lo
expression and 12(
S
)-hydroxyeicosatetraenoic acid was noted in rodent models of diabetes. Subsequently, germline
12-Lo
−/−
was shown to prevent the development of hyperglycemia in mouse models of type 1 diabetes and in high-fat fed mice. More recently, beta cell-specific
12-Lo
−/−
was shown to protect mice against hyperglycaemia after streptozotocin and a high-fat diet. In humans, 12-LO expression is increased in pancreatic islets of autoantibody-positive, type 1 diabetic and type 2 diabetic organ donors. Interestingly, the high expression of
ALOX12
is associated with the alteration in first-phase glucose-stimulated insulin secretion in human type 2 diabetic islets. To further clarify the role of islet 12-LO in diabetes and to validate 12-LO as a therapeutic target of diabetes, we have studied selective pharmacological inhibitors for 12-LO. The compounds we have identified show promise: they protect beta cell lines and human islets from apoptosis and preserve insulin secretion when challenged by proinflammatory cytokine mixture. Currently studies are underway to test the compounds in mouse models of diabetes. This review summarises a presentation given at the ‘Islet inflammation in type 2 diabetes’ symposium at the 2015 annual meeting of the EASD. It is accompanied two other mini-reviews on topics from this symposium (by Simone Baltrusch, DOI:
10.1007/s00125-016-3891-x
and Marc Donath, DOI:
10.1007/s00125-016-3873-z
) and a commentary by the Session Chair, Piero Marchetti (DOI:
10.1007/s00125-016-3875-x
).
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