Julie Webster

Inhibition of CCR6 Function Reduces the Severity of Experimental Autoimmune Encephalomyelitis via Effects on the Priming Phase of the Immune Response

Chemokines are essential for homeostasis and activation of the immune system. The chemokine ligand/receptor pairing CCL20/CCR6 is interesting because these molecules display characteristics of both homeostatic and activation functions. These dual characteristics suggest a role for CCR6 in the priming and effector phases of the immune response. However, while CCR6 has been implicated in the effector phase in several models, a role in the priming phase is less clear. Herein we analyze the role of CCR6 in these two important arms of the immune response during experimental autoimmune encephalomyelitis (EAE). Both CCR6 and its chemokine ligand CCL20 were up-regulated in the draining lymph nodes and spinal cord during EAE, and CCR6 was up-regulated on CD4+ T cells that had divided following induction of EAE. The functional role of this expression was demonstrated by impaired development of EAE in gene-targeted CCR6-deficient mice and in mice treated either with a neutralizing anti-CCR6 Ab or with a novel receptor antagonist. Inhibition of EAE was due to reduced priming of autoreactive CD4+ T cells probably as a result of impaired late-stage influx of dendritic cells into draining lymph nodes. This was accompanied by reduced egress of activated lymphocytes from the lymph nodes. These results demonstrate a novel role for CCR6 in the mechanism of autoreactive lymphocyte priming and emigration to the efferent lymphatics.

The carbonyl scavengers aminoguanidine and tenilsetam protect against the neurotoxic effects of methylglyoxal

Advanced glycation end products (AGEs) have been identified in age-related intracellular protein deposits of Alzheimer’s disease (amyloid plaques and neurofibrillary tangles) and Parkinson disease (Lewy bodies), suggesting that these protein deposits have been exposed to AGE precursors such as the reactive dicarbonyl compound methylglyoxal. In ageing tissue and under diabetic pseudohypoxia, intracellular methylglyoxal levels rise through an impairment of triosephosphate utilization. Furthermore, methylglyoxal detoxification is impaired when reduced glutathione levels are low, conditions, which have all been described in Alzheimer’s disease. However, there is less known about the toxicity of methylglyoxal, particularly about therapeutic strategies to scavenge such dicarbonyl compounds and attenuate their toxicity. In our study, extracellularly applied methylglyoxal was shown to be toxic to human neuroblastoma cells in a dose-dependent manner above concentrations of 150 µM with a LD50 of approximately 1.25 mM. Pre-incubation of methylglyoxal with a variety of carbonyl scavengers such as aminoguanidine or tenilsetam and the thiol antioxidant lipoic acid significantly reduced its toxicity. In summary, carbonyl scavengers might offer a promising therapeutic strategy to reduce the neurotoxicity of reactive carbonyl compounds, providing a potential benefit for patients with age-related neurodegenerative diseases.

ERp46 binds to AdipoR1, but not AdipoR2, and modulates adiponectin signalling

The pleiotropic effects of the insulin-sensitizing adipokine adiponectin are mediated, at least in part, by two seven-transmembrane domain receptors AdipoR1 and AdipoR2. Recent reports indicate a role for AdipoR-binding proteins, namely APPL1, RACK1 and CK2beta, in proximal signal transduction events. Here we demonstrate that endoplasmic reticulum protein 46 (ERp46) interacts specifically with AdipoR1 and provide evidence that ERp46 modulates adiponectin signalling. Co-immunoprecipitation followed by mass spectrometry identified ERp46 as an AdipoR1-, but not AdipoR2-, interacting protein. Analysis of truncated constructs and GST-fusion proteins revealed the interaction was mediated by the cytoplasmic, N-terminal residues (1-70) of AdipoR1. Indirect immunofluorescence microscopy and subcellular fractionation studies demonstrated that ERp46 was present in the ER and the plasma membrane (PM). Transient knockdown of ERp46 increased the levels of AdipoR1, and AdipoR2, at the PM and this correlated with increased adiponectin-stimulated phosphorylation of AMPK. In contrast, adiponectin-stimulated phosphorylation of p38MAPK was reduced following ERp46 knockdown. Collectively these results establish ERp46 as the first AdipoR1-specific interacting protein and suggest a role for ERp46 in adiponectin receptor biology and adiponectin signalling.

Sialic Acid Modification of Adiponectin Is Not Required for Multimerization or Secretion but Determines Half-Life in Circulation

Adiponectin is an adipocyte-secreted, insulin-sensitizing hormone the circulating levels of which are reduced in conditions of insulin resistance and diabetes. Previous work has demonstrated the importance of posttranslational modifications, such as proline hydroxylation and lysine hydroxylation/glycosylation, in adiponectin oligomerization, secretion, and function. Here we describe the first functional characterization of adiponectin sialylation. Using a variety of biochemical approaches we demonstrated that sialylation occurs on previously unidentified O-linked glycans on Thr residues of the variable domain in human adiponectin. Enzymatic removal of sialic acid or its underlying O-linked sugars did not affect adiponectin multimer composition. Expression of mutant forms of adiponectin (lacking the modified Thr residues) or of wild-type adiponectin in cells defective in sialylation did not compromise multimer formation or secretion, arguing against a structural role for this modification. Activity of desialylated adiponectin was comparable to control adiponectin in L6 myotubes and acute assays in adiponectin(-/-) mice. In contrast, plasma clearance of desialylated adiponectin was accelerated compared with that of control adiponectin, implicating a role for this modification in determining the half-life of circulating adiponectin. Uptake of desialylated adiponectin by isolated primary rat hepatocytes was also accelerated, suggesting a role for the hepatic asialoglycoprotein receptor. Finally, after chronic administration in adiponectin(-/-) mice steady-state levels of desialylated adiponectin were lower than control adiponectin and failed to recapitulate the improvements in glucose and insulin tolerance tests observed with control adiponectin. These data suggest an important role for sialic acid content in the regulation of circulating adiponectin levels and highlight the importance of understanding mechanisms regulating adiponectin sialylation/desialylation.

Identification of BMP and Activin Membrane-Bound Inhibitor (BAMBI) as a Potent Negative Regulator of Adipogenesis and Modulator of Autocrine/Paracrine Adipogenic Factors

Adipose tissue dysfunction underpins the association of obesity with type 2 diabetes. Adipogenesis is required for the maintenance of adipose tissue function. It involves the commitment and subsequent differentiation of preadipocytes and is coordinated by autocrine, paracrine, and endocrine factors. We previously reported that fibroblast growth factor-1 (FGF-1) primes primary human preadipocytes and Simpson Golabi Behmel syndrome (SGBS) preadipocytes and increases adipogenesis through a cascade involving extracellular signal–related kinase 1/2 (ERK1/2). Here, we aimed to use the FGF-1 system to identify novel adipogenic regulators. Expression profiling revealed bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) as a putative FGF-1 effector. BAMBI is a transmembrane protein and modulator of paracrine factors that regulate adipogenesis, including transforming growth factor (TGF) superfamily members (TGF-β and BMP) and Wnt. Functional investigations established BAMBI as a negative regulator of adipogenesis and modulator of the anti- and proadipogenic effects of Wnt3a, TGF-β1, and BMP-4. Further studies showed that BAMBI expression levels are decreased in a mouse model of diet-induced obesity. Collectively, these findings establish BAMBI as a novel, negative regulator of adipogenesis that can act as a nexus to integrate multiple paracrine signals to coordinate adipogenesis. Alterations in BAMBI may play a role in the (patho)physiology of obesity, and manipulation of BAMBI may present a novel therapeutic approach to improve adipose tissue function.

Different characteristics and nucleotide binding properties of Inosine Monophosphate Dehydrogenase (IMPDH) isoforms

We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP) would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i) the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii) communication occurs between the Bateman and catalytic domains and (iii) the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

An Inhibitor of Phospholipase A2 Group IIA Modulates Adipocyte Signaling and Protects Against Diet-Induced Metabolic Syndrome in Rats

Obesity, type 2 diabetes, and cardiovascular disease correlate with infiltration to adipose tissue of different immune cells, with uncertain influences on metabolism. Rats were fed a diet high in carbohydrates and saturated fats to develop diet-induced obesity over 16 weeks. This nutritional overload caused overexpression and secretion of phospholipase A2 group IIA (pla2g2a) from immune cells in adipose tissue rather than adipocytes, whereas expression of adipose-specific phospholipase A2 (pla2g16) was unchanged. These immune cells produce prostaglandin E2 (PGE2), which influences adipocyte signaling. We found that a selective inhibitor of human pla2g2a (5-(4-benzyloxyphenyl)-(4S)-(phenyl-heptanoylamino)-pentanoic acid [KH064]) attenuated secretion of PGE2 from human immune cells stimulated with the fatty acid, palmitic acid, or with lipopolysaccharide. Oral administration of KH064 (5 mg/kg/day) to rats fed the high-carbohydrate, high-fat diet prevented the overexpression of pla2g2a and the increased macrophage infiltration and elevated PGE2 concentrations in adipose tissue. The treatment also attenuated visceral adiposity and reversed most characteristics of metabolic syndrome, producing marked improvements in insulin sensitivity, glucose intolerance, and cardiovascular abnormalities. We suggest that pla2g2a may have a causal relationship with chronic adiposity and metabolic syndrome and that its inhibition in vivo may be a valuable new approach to treat obesity, type 2 diabetes, and metabolic dysfunction in humans.

Synergistic effects of ascorbic acid and thiazolidinedione on secretion of high molecular weight adiponectin from human adipocytes

Aim: To test the hypothesis that ascorbic acid (AA) and thiazolidinedione (TZD) would have additive effects on HMW adiponectin secretion by virtue of different modes of action.

Characterisation of the adiponectin receptors: The non-conserved N-terminal region of AdipoR2 prevents its expression at the cell-surface

Adiponectin is a beneficial adipokine with insulin-sensitizing, anti-inflammatory and anti-atherogenic effects. These effects are mediated by two poorly characterised, closely related, atypical seven-transmembrane receptors. In the current report we have used C-terminal, epitope-tagged AdipoR1 and AdipoR2 constructs to monitor cell-surface expression by indirect immunofluorescence microscopy and quantitative plate-based analysis. We demonstrate that only AdipoR1 is constitutively expressed on the cell-surface. Further investigations, involving characterisation of a number of chimeric and truncated constructs, show the non-conserved region of AdipoR2 (residues 1-81) restricts its cell-surface expression. Introduction or deletion of this region, into AdipoR1 or AdipoR2, resulted in inhibition or promotion of cell-surface expression, respectively. We also confirmed that AdipoR1 and AdipoR2 can form heterodimers when co-expressed and that co-expression leads to the cell-surface expression of AdipoR2. Collectively these studies demonstrate that the non-conserved region of AdipoR2 restricts its cell-surface expression and raise the possibility that the majority of cell-surface AdipoR2 may be present in the form of heterodimers.

Maillard reaction products in food as pro-inflammatory and pro-arteriosclerotic factors of degenerative diseases

ZusammenfassungMaillardprodukte (Maillard Reaction Products; MRPs) entstehen beim Erhitzen von Lebensmitteln durch Reaktion von reduzierenden Zuckern mit Proteinen oder Aminosäuren. Im menschlichen Körper werden über analoge Reaktionswege sog. „Advanced Glycation Endproducts“ (AGEs) gebildet, die sich im Alter in Geweben ablagern und degenerativ-entzündliche und proliferative Prozesse beschleunigen. Auch MRPs aus der Nahrung können—direkt im Darm—entzündliche Prozesse nach ähnlichen Mechanismen auslösen und—nach Resorption—im Körper zu diesen entzündlichen und degenerativen Prozessen beitragen oder sie verstärken. Gut aufgeklärt ist inzwischen der Beitrag von AGEs (und der zusätzliche durch MRPs) bei der Ausbildung von diabetischen Komplikationen wie Nephropathie, Neuropathie, Mikro- und Makroangiopathien. Es ist jedoch weitgehend unbekannt, welche der MRPs oder AGEs genau diese zellulären Prozesse auslösen. So könnte eine detaillierte Kenntnis über die chemischen Strukturen und Wirkungen der einzelnen MRPs helfen, die schädlichen unter ihnen in erhitzten Lebensmitteln zu minimieren. Da MRPs bei der erfolgreichen Vermarktung von Lebensmitteln aufgrund ihrer Eigenschaft als Aroma- und Geschmackskomponenten eine nicht unbedeutende Rolle spielen, ist es umso wichtiger, durch definierte Zubereitungsmethoden neutrale geschmackstragende MRPs zu maximieren und gleichzeitig signalaktive pro-inflammatorische MRPs zu minimieren. Zur quantitativen Bestimmung spezifischer MRPs/AGEs können preiswerte immunologische Messmethoden eingesetzt werden. Im medizinischem Bereich eröffnet die Kenntnis der signalaktiven MRP/AGE-Strukturen die Möglichkeit, deren Konzentrationen in Geweben und Körperflüssigkeiten gezielt zu messen und so ihren Einfluss auf entzündliche und altersbedingte degenerative Prozesse (z. B. diabetische Spätschäden, Arteriosklerose, Neurodegeneration) klinischdiagnostisch abzuklären. Aus pharmakotherapeutischer Sicht könnten dann—nach einer entsprechenden klinisch-chemischen Diagnostik—die sich momentan in der Entwicklung befindenden RAGE-Antagonisten als unterstützende Substanzen bei besonders betroffenen Patientengruppen, wie z. B. älteren Diabetikern und Dialysepatienten, in der Zukunft eine besonders wichtige Rolle spielen.SummaryHeating of food induces the formation of Maillard reaction products (MRPs) caused by the reaction of reducing sugars with proteins or amino acids. Analogous reactions occur in the human body, eventually forming “Advanced Glycation Endproducts” (AGEs). AGEs accumulate in aging tissues accelerating degenerative-inflammatory and proliferative processes. MRPs present in food can also directly cause inflammatory processes in the intestines and, once absorbed, would support and reinforce any inflammatory and degenerative process occurring in the body. The contribution of AGEs (and additional MRPs) in the development of diabetic complications as well as nephropathy, neuropathy, micro- and macroangiopathies is now well established. Which of the MRPs or AGEs in particular induce these cellular processes is currently unknown. Thus the exact knowledge of the chemical structures of the MRPs could help to minimize the formation of “harmful MRPs” that occur due to heating in food processing. Because MRPs play a decisive role in the successful marketing of edibles due to their characteristics as flavor components, it is important to increase the amount of innocuous and palatable MRPs, and minimize signal active pro-inflammatory MRPs by the use of defined preparation methods. It is practicable to use low-priced immunological methods for the quantitative determination of specific MRPs or AGEs. In the medical area, the knowledge of the signal active MRP/AGE structures provides the opportunity to measure their concentrations in body fluids and tissues and thus determine their influence on inflammatory and age-related degenerative processes (e. g., late diabetic complications, arteriosclerosis, degeneration of neurons). From a clinical perspective, the application of RAGE antagonists after an appropriate chemical diagnosis could be effective in supporting the treatment of affected patient groups, especially older diabetic and dialysis patients.