Identification of high-performing antibodies for the reliable detection of Tau proteoforms by Western blotting and immunohistochemistry.

Antibodies are essential research tools whose performance directly impacts research conclusions and reproducibility. Owing to its central role in Alzheimer's disease and other dementias, hundreds of distinct antibody clones have been developed against the microtubule-associated protein Tau and its multiple proteoforms. Despite this breadth of offer, limited understanding of their performance and poor antibody selectivity have hindered research progress.

Temporal regulation of interferon signalling in human EndoC-βH1 cells.

During the development of type 1 diabetes, interferons (IFN) are elaborated from islet-infiltrating immune cells and/or from virally infected β-cells. They act via specific receptors to increase, acutely, the phosphorylation of the transcription factors STAT1 and 2. However, the longer-term impacts of chronic IFN stimulation are poorly understood and were investigated in the current study.

Investigation of the utility of the 1.1B4 cell as a model human beta cell line for study of persistent enteroviral infection.

The generation of a human pancreatic beta cell line which reproduces the responses seen in primary beta cells, but is amenable to propagation in culture, has long been an important goal in diabetes research. This is particularly true for studies focussing on the role of enteroviral infection as a potential cause of beta-cell autoimmunity in type 1 diabetes. In the present work we made use of a clonal beta cell line (1.

Differential effects of saturated and unsaturated fatty acids on autophagy in pancreatic β-cells.

Long-chain saturated fatty acids are lipotoxic to pancreatic β-cells, whereas most unsaturates are better tolerated and some may even be cytoprotective. Fatty acids alter autophagy in β-cells and there is increasing evidence that such alterations can impact directly on the regulation of viability. Accordingly, we have compared the effects of palmitate (C16:0) and palmitoleate (C16:1) on autophagy in cultured β-cells and human islets.

Unexpected subcellular distribution of a specific isoform of the Coxsackie and adenovirus receptor, CAR-SIV, in human pancreatic beta cells.

Aims/hypothesis: The Coxsackie and adenovirus receptor (CAR) is a transmembrane cell-adhesion protein that serves as an entry receptor for enteroviruses and may be essential for their ability to infect cells. Since enteroviral infection of beta cells has been implicated as a factor that could contribute to the development of type 1 diabetes, it is often assumed that CAR is displayed on the surface of human beta cells. However, CAR exists as multiple isoforms and it is not known whether all isoforms subserve similar physiological functions.

Targeting surface voids to counter membrane disorders in lipointoxication-related diseases.

Saturated fatty acids (SFA), which are abundant in the so-called western diet, have been shown to efficiently incorporate within membrane phospholipids and therefore impact on organelle integrity and function in many cell types. In the present study, we have developed a yeast-based two-step assay and a virtual screening strategy to identify new drugs able to counter SFA-mediated lipointoxication. The compounds identified here were effective in relieving lipointoxication in mammalian β-cells, one of the main targets of SFA toxicity in humans.

GPR120 (FFAR4) is preferentially expressed in pancreatic delta cells and regulates somatostatin secretion from murine islets of Langerhans.

Aims/hypothesis: The NEFA-responsive G-protein coupled receptor 120 (GPR120) has been implicated in the regulation of inflammation, in the control of incretin secretion and as a predisposing factor influencing the development of type 2 diabetes by regulation of islet cell apoptosis. However, there is still considerable controversy about the tissue distribution of GPR120 and, in particular, it remains unclear which islet cell types express this molecule. In the present study, we have addressed this issue by constructing a Gpr120-knockout/β-galactosidase (LacZ) knock-in (KO/KI) mouse to examine the distribution and functional role of GPR120 in the endocrine pancreas.

Evaluation of the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, in human pancreas.

Aims/hypothesis: Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB).

Differential effects of interleukin-13 and interleukin-6 on Jak/STAT signaling and cell viability in pancreatic β-cells.

Pro-inflammatory cytokines are important mediators of β-cell demise in type 1 diabetes, and similar mechanisms are increasingly implicated in type 2 diabetes, where a state of chronic inflammation may persist. It is likely that the actions of anti-inflammatory cytokines are also altered in diabetes. Cytokines are released from immune cells, which may be recruited to the islets in diabetes, but they can also be produced by islet endocrine cells in response to environmental factors, including enteroviral infection.

The cytoprotective effects of oleoylethanolamide in insulin-secreting cells do not require activation of GPR119.

Background And Purpose: β-cells express a range of fatty acid-responsive G protein-coupled receptors, including GPR119, which regulates insulin secretion and is seen as a potential therapeutic target in type 2 diabetes. The long-chain unsaturated fatty acid derivative oleoylethanolamide (OEA) is an endogenous agonist of GPR119 and, under certain conditions, some long-chain unsaturated fatty acids can promote β-cell cytoprotection. It is not known, however, if OEA is cytoprotective in β-cells.

Structure-activity relationships influencing lipid-induced changes in eIF2α phosphorylation and cell viability in BRIN-BD11 cells.

Fatty acids influence the viability of eukaryotic cells differentially such that long chain saturated molecules are poorly tolerated, whereas unsaturated species are less detrimental and can be cytoprotective. The basis for these effects is unclear but studies in yeast imply that they reflect the spatial configuration of the molecules when incorporated into the ER membrane. Using BRIN-BD11 β-cells, we show that a wide range of unsaturated free fatty acids and their methyl-esters (having differing chain length and disposition of the double bonds) elicit cytoprotection and relief of protein kinase RNA-like endoplasmic reticulum kinase-dependent ER stress.

Dysregulation of Hnf1b gene expression in cultured beta-cells in response to cytotoxic fatty acid.

Context: Increased levels of circulating fatty acids deriving from over-nutrition are thought to contribute to the progressive beta-cell failure associated with type 2 diabetes. Pancreatic beta-cells in culture are sensitive to exposure to long-chain saturated fatty acids (e.g.

Pharmacological characterization of the cytoprotective effects of polyunsaturated fatty acids in insulin-secreting BRIN-BD11 cells.

Background And Purpose: Free fatty acids are important metabolic fuels for mammalian cells but, recently, it has become clear that they can also fulfil signalling functions, which are independent of their metabolic fate. We are investigating the ability of unsaturated free fatty acids to exert a cytoprotective response during exposure of insulin-secreting cells to toxic stimuli. The majority of earlier studies have focussed on monounsaturated fatty acids but this has now been extended to define the structural requirements of the cytoprotective effects of polyunsaturated species.

The significance of GPR119 agonists as a future treatment for type 2 diabetes.

GPR119 is a G protein-coupled receptor that is expressed on only a limited number of tissues, including pancreatic β-cells and enteroendocrine cells in the small intestine, and that appears to be involved in the regulation of metabolic homeostasis. The protein was originally defined as an orphan receptor, but it has subsequently been shown to bind a variety of lipid-derived ligands, as well as a range of small synthetic molecules. There is still debate as to the identity of its principal endogenous ligand, but certain lysophospholipids species, various fatty acyl-ethanolamides and N-oleoyldopamine have all been proposed as potential agonists.

Arachidonic acid actions on functional integrity and attenuation of the negative effects of palmitic acid in a clonal pancreatic β-cell line.

Chronic exposure of pancreatic β-cells to saturated non-esterified fatty acids can lead to inhibition of insulin secretion and apoptosis. Several previous studies have demonstrated that saturated fatty acids such as PA (palmitic acid) are detrimental to β-cell function compared with unsaturated fatty acids. In the present study, we describe the effect of the polyunsaturated AA (arachidonic acid) on the function of the clonal pancreatic β-cell line BRIN-BD11 and demonstrate AA-dependent attenuation of PA effects.

Unsaturated fatty acids as cytoprotective agents in the pancreatic beta-cell.

It is widely accepted that, in type 2 diabetes, elevated levels of free fatty acids and glucose contribute to a state of glucolipotoxicity in which beta-cell function declines and, ultimately, cell viability is compromised. This suggests that beta-cells do not readily tolerate chronic elevations in fatty acid levels. In vitro studies suggest, however, that beta-cells respond differentially to long chain fatty acids, such that saturated species are lipotoxic whereas long chain mono-unsaturated fatty acids can provide cytoprotection.

G-protein coupled receptors mediating long chain fatty acid signalling in the pancreatic beta-cell.

It is increasingly clear that some of the effects of both free and derivatised long chain fatty acids in pancreatic beta-cells are mediated by a group of G-protein coupled receptors. Some of these display close structural homology while others are more divergent. This Commentary reviews the expression and functional roles of three such molecules, GPR40, GPR119 and GPR120.

The cytoprotective actions of long-chain mono-unsaturated fatty acids in pancreatic beta-cells.

Chronic exposure of pancreatic beta-cells to long-chain fatty acids can cause loss of secretory function and enhanced apoptosis by a process of 'lipotoxicity', which may be a contributory factor to the rising incidence of Type 2 diabetes in humans. However, when incubated in vitro, beta-cells respond differentially to long-chain saturated and mono-unsaturated fatty acids, suggesting that these molecules may regulate cell functionality by different mechanisms. In particular, it is clear that, whereas saturated fatty acids [e.

Mechanisms involved in the cytotoxic and cytoprotective actions of saturated versus monounsaturated long-chain fatty acids in pancreatic beta-cells.

Long-chain saturated and monounsaturated fatty acids differ in their propensity to induce beta-cell death in vitro with palmitate (C16:0) being cytotoxic, whereas palmitoleate (C16:1n-7) is cytoprotective. We now show that this cytoprotective capacity extends to a poorly metabolised C16:1n-7 derivative, methyl-palmitoleate (0.25 mM palmitate alone: 92 +/- 4% death after 18 h; palmitate plus 0.