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. Here, we validate a large panel of Tau antibodies by Western blot (79 reagents) and immunohistochemistry (35 reagents). We address the reagents' ability to detect the target proteoform, selectivity, the impact of protein phosphorylation on antibody binding and performance in human brain samples. While most antibodies detected Tau at high levels, many failed to detect it at lower, endogenous levels. By WB, non-selective binding to other proteins affected over half of the antibodies tested, with several cross-reacting with the related MAP2 protein, whereas the "oligomeric Tau" T22 antibody reacted with monomeric Tau by WB, thus calling into question its specificity to Tau oligomers. Despite the presumption that "total" Tau antibodies are agnostic to post-translational modifications, we found that phosphorylation partially inhibits binding for many such antibodies, including the popular Tau-5 clone. We further combine high-sensitivity reagents, mass-spectrometry proteomics and cDNA sequencing to demonstrate that presumptive Tau "knockout" human cells continue to express residual protein arising through exon skipping, providing evidence of previously unappreciated gene plasticity. Finally, probing of human brain samples with a large panel of antibodies revealed the presence of C-term-truncated versions of all main Tau brain isoforms in both control and tauopathy donors. Ultimately, we identify a validated panel of Tau antibodies that can be employed in Western blotting and/or immunohistochemistry to reliably detect even low levels of Tau expression with high selectivity. This work represents an extensive resource that will enable the re-interpretation of published data, improve reproducibility in Tau research, and overall accelerate scientific progress.

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. In the present study, we have determined the profile of CAR isoforms present in human beta cells and monitored the subcellular localisation of the principal isoform within the cells. METHODS: Formalin-fixed, paraffin-embedded pancreatic sections from non-diabetic individuals and those with type 1 diabetes were studied. Immunohistochemistry, confocal immunofluorescence, electron microscopy and western blotting with isoform-specific antisera were employed to examine the expression and cellular localisation of the five known CAR isoforms. Isoform-specific qRT-PCR and RNA sequencing (RNAseq) were performed on RNA extracted from isolated human islets. RESULTS: An isoform of CAR with a terminal SIV motif and a unique PDZ-binding domain was expressed at high levels in human beta cells at the protein level. A second isoform, CAR-TVV, was also present. Both forms were readily detected by qRT-PCR and RNAseq analysis in isolated human islets. Immunocytochemical studies indicated that CAR-SIV was the principal isoform in islets and was localised mainly within the cytoplasm of beta cells, rather than at the plasma membrane. Within the cells it displayed a punctate pattern of immunolabelling, consistent with its retention within a specific membrane-bound compartment. Co-immunofluorescence analysis revealed significant co-localisation of CAR-SIV with zinc transporter protein 8 (ZnT8), prohormone convertase 1/3 (PC1/3) and insulin, but not proinsulin. This suggests that CAR-SIV may be resident mainly in the membranes of insulin secretory granules. Immunogold labelling and electron microscopic analysis confirmed that CAR-SIV was localised to dense-core (insulin) secretory granules in human islets, whereas no immunolabelling of the protein was detected on the secretory granules of adjacent exocrine cells. Importantly, CAR-SIV was also found to co-localise with protein interacting with C-kinase 1 (PICK1), a protein recently demonstrated to play a role in insulin granule maturation and trafficking. CONCLUSIONS/INTERPRETATION: The SIV isoform of CAR is abundant in human beta cells and is localised mainly to insulin secretory granules, implying that it may be involved in granule trafficking and maturation. We propose that this subcellular localisation of CAR-SIV contributes to the unique sensitivity of human beta cells to enteroviral infection.

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 ß-cells, one of the main targets of SFA toxicity in humans. In vitro reconstitutions and molecular dynamics simulations on bilayers revealed that these molecules, albeit according to different mechanisms, can generate voids at the membrane surface. The resulting surface defects correlate with the recruitment of loose lipid packing or void-sensing proteins required for vesicular budding, a central cellular process that is precluded under SFA accumulation. Taken together, the results presented here point at modulation of surface voids as a central parameter to consider in order to counter the impacts of SFA on cell function.

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/ß-galactosidase (LacZ) knock-in (KO/KI) mouse to examine the distribution and functional role of GPR120 in the endocrine pancreas. METHODS: A KO/KI mouse was generated in which exon 1 of the Gpr120 gene (also known as Ffar4) was replaced in frame by LacZ, thereby allowing for regulated expression of ß-galactosidase under the control of the endogenous GPR120 promoter. The distribution of GPR120 was inferred from expression studies detecting ß-galactosidase activity and protein production. Islet hormone secretion was measured from isolated mouse islets treated with selective GPR120 agonists. RESULTS: ß-galactosidase activity was detected as a surrogate for GPR120 expression exclusively in a small population of islet endocrine cells located peripherally within the islet mantle. Immunofluorescence analysis revealed co-localisation with somatostatin suggesting that GPR120 is preferentially produced in islet delta cells. In confirmation of this, glucose-induced somatostatin secretion was inhibited by a range of selective GPR120 agonists. This response was lost in GPR120-knockout mice. CONCLUSIONS/INTERPRETATION: The results imply that GPR120 is selectively present within the delta cells of murine islets and that it regulates somatostatin secretion.

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). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes. METHODS: Enteroviral VP1, CKB and ATP5B expression were analysed by western blotting, RT-PCR and immunocytochemistry in a range of cultured cell lines, isolated human islets and human tissue. RESULTS: Clone 5D8/1 labelled CKB, but not ATP5B, on western blots performed under denaturing conditions. In cultured human cell lines, isolated human islets and pancreas sections from patients with type 1 diabetes, the immunolabelling of ATP5B, CKB and VP1 by 5D8/1 was readily distinguishable. Moreover, in a human tissue microarray displaying more than 80 different cells and tissues, only two (stomach and colon; both of which are potential sites of enterovirus infection) were immunopositive when stained with clone 5D8/1. CONCLUSIONS/INTERPRETATION: When used under carefully optimised conditions, the immunolabelling pattern detected in sections of human pancreas with clone 5D8/1 did not reflect cross-reactivity with either ATP5B or CKB. Rather, 5D8/1 is likely to be representative of enteroviral antigen expression.

Temporal regulation of interferon signalling in human EndoC-ßH1 cells.

During the development of type 1 diabetes, interferons (IFN) are elaborated from islet-infiltrating immune cells and/or from virally infected ß-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. Human EndoC-ßH1 cells were treated with IFNα, IFNγ or IFNλ either acutely (<2 h) or chronically (≥24 h) and STAT phosphorylation, expression and activity were assessed by Western blotting and transcriptional reporter assays. Exposure of ß-cells to IFNα or IFNλ induced a swift increase in the phosphorylation of both STAT1 and STAT2, whereas IFNγ increased only pSTAT1. Over more extended periods (≥24 h), STAT phosphorylation declined but STAT1 and STAT2 expression were enhanced in a sustained manner. All IFNs stimulated ISRE transcriptional activity (but with different time courses), whereas GAS activity was responsive only to IFNγ. The re-addition of a second bolus of IFNα, 24 h after an initial dose, failed to cause renewed STAT1/2 phosphorylation. By contrast, when IFNγ was added 24 h after exposure to IFNα, rapid STAT1 phosphorylation was re-initiated. Exposure of ß-cells to IFNs leads to rapid, transient, STAT phosphorylation and to slower and more sustained increases in total STAT1/2 levels. The initial phosphorylation response is accompanied by marked desensitisation to the cognate agonist. Together, the results reveal that the response of ß-cells to IFNs is regulated both temporally and quantitatively to achieve effective signal integration.

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

Chronic exposure of pancreatic ß-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 ß-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 ß-cell line BRIN-BD11 and demonstrate AA-dependent attenuation of PA effects. When added to ß-cell incubations at 100 µM, AA can stimulate cell proliferation and chronic (24 h) basal insulin secretion. Microarray analysis and/or real-time PCR indicated significant AA-dependent up-regulation of genes involved in proliferation and fatty acid metabolism [e.g. Angptl (angiopoietin-like protein 4), Ech1 (peroxisomal Δ3,5,Δ2,4-dienoyl-CoA isomerase), Cox-1 (cyclo-oxygenase-1) and Cox-2, P<0.05]. Experiments using specific COX and LOX (lipoxygenase) inhibitors demonstrated the importance of COX-1 activity for acute (20 min) stimulation of insulin secretion, suggesting that AA metabolites may be responsible for the insulinotropic effects. Moreover, concomitant incubation of AA with PA dose-dependently attenuated the detrimental effects of the saturated fatty acid, so reducing apoptosis and decreasing parameters of oxidative stress [ROS (reactive oxygen species) and NO levels] while improving the GSH/GSSG ratio. AA decreased the protein expression of iNOS (inducible NO synthase), the p65 subunit of NF-κB (nuclear factor κB) and the p47 subunit of NADPH oxidase in PA-treated cells. These findings indicate that AA has an important regulatory and protective ß-cell action, which may be beneficial to function and survival in the 'lipotoxic' environment commonly associated with Type 2 diabetes mellitus.

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. palmitate) which cause cytotoxicity, whereas the monounsaturated equivalents (e.g. palmitoleate) are cytoprotective. OBJECTIVES: In this study we sought to determine whether of members of the hepatocyte nuclear factor (HNF) family of transcription factors, which are mutated in familial, young-onset, monogenic beta-cell diabetes, could play a role in fatty acid-mediated cytotoxicity in cultured beta-cells. DESIGN: We used real-time PCR to determine whether hepatocyte nuclear factor gene expression was altered in response to palmitate exposure in the BRIN-BD11 beta-cell line. RESULTS: We found that the Hnf isoforms expressed in BRIN-BD11 cells are dysregulated by palmitate exposure. The expression of Hnf1b is specifically reduced by exposure to palmitate, and this response is prevented by co-incubation with palmitoleate. CONCLUSIONS: Down-regulation of Hnf1b gene expression accompanies palmitate-mediated cytotoxicity in cultured beta-cells.

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 (1.1B4) available from the European Collection of Authenticated Cell Cultures, which had been generated by the fusion of primary human beta-cells with a pancreatic ductal carcinoma cell, PANC-1. Our goal was to study the factors allowing the development and persistence of a chronic enteroviral infection in human beta-cells. Since PANC-1 cells have been reported to support persistent enteroviral infection, the hybrid 1.1B4 cells appeared to offer an ideal vehicle for our studies. In support of this, infection of the cells with a Coxsackie virus isolated originally from the pancreas of a child with type 1 diabetes, CVB4.E2, at a low multiplicity of infection, resulted in the development of a state of persistent infection. Investigation of the molecular mechanisms suggested that this response was facilitated by a number of unexpected outcomes including an apparent failure of the cells to up-regulate certain anti-viral response gene products in response to interferons. However, more detailed exploration revealed that this lack of response was restricted to molecular targets that were either activated by, or detected with, human-selective reagents. By contrast, and to our surprise, the cells were much more responsive to rodent-selective reagents. Using multiple approaches, we then established that populations of 1.1B4 cells are not homogeneous but that they contain a mixture of rodent and human cells. This was true both of our own cell stocks and those held by the European Collection of Authenticated Cell Cultures. In view of this unexpected finding, we developed a strategy to harvest, isolate and expand single cell clones from the heterogeneous population, which allowed us to establish colonies of 1.1B4 cells that were uniquely human (h1.1.B4). However, extensive analysis of the gene expression profiles, immunoreactive insulin content, regulated secretory pathways and the electrophysiological properties of these cells demonstrated that they did not retain the principal characteristics expected of human beta cells. Our data suggest that stocks of 1.1B4 cells should be evaluated carefully prior to their use as a model human beta-cell since they may not retain the phenotype expected of human beta-cells.

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 ß-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. GPR119 is coupled to the signal transducer Gαs and activation of the receptor leads to increased adenylate cyclase activity via Gαs and a rise in intracellular cAMP. This then potentiates glucose-induced insulin secretion or promotes the release of intestinal incretin hormones, according to cell type. Both mechanisms ultimately lead to a rise in insulin secretion (either directly or indirectly) and improved glucose control. Thus, GPR119 may represent an important new therapeutic target for the design of insulin secretagogues able to promote improvements in blood glucose control in patients with type 2 diabetes. Accordingly, a range of lead compounds are in development as potential therapeutic agents.

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

Long-chain saturated fatty acids are lipotoxic to pancreatic ß-cells, whereas most unsaturates are better tolerated and some may even be cytoprotective. Fatty acids alter autophagy in ß-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 ß-cells and human islets. Treatment of BRIN-BD11 ß-cells with palmitate led to enhanced autophagic activity, as judged by cleavage of microtubule-associated protein 1 light chain 3-I (LC3-I) and this correlated with a marked loss of cell viability in the cells. In addition, transfection of these cells with an mCherry-YFP-LC3 reporter construct revealed the accumulation of autophagosomes in palmitate-treated cells, indicating an impairment of autophagosome-lysosome fusion. This was also seen upon addition of the vacuolar ATPase inhibitor, bafilomycin A1. Exposure of BRIN-BD11 cells to palmitoleate (C16:1) did not lead directly to changes in autophagic activity or flux, but it antagonised the actions of palmitate. In parallel, palmitoleate also improved the viability of palmitate-treated BRIN-BD11 cells. Equivalent responses were observed in INS-1E cells and in isolated human islets. Taken together, these data suggest that palmitate may cause an impairment of autophagosome-lysosome fusion. These effects were not reproduced by palmitoleate which, instead, antagonised the responses mediated by palmitate suggesting that attenuation of ß-cell stress may contribute to the improvement in cell viability caused by the mono-unsaturated fatty acid.

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 0.25 mM methyl-palmitoleate: 12 +/- 2%; P < 0.001). Palmitoleate and its methylated derivative also acted as mitogens in cultured beta-cells (5-bromo-2-deoxyuridine incorporation - control: 0.15 +/- 0.01 units; 0.25 mM palmitoleate: 0.22 +/- 0.01 units; P < 0.05). It has been proposed that alterations in neutral lipid synthesis (particularly triacylglycerol (TAG) formation) might mediate the differential responses to saturated and unsaturated fatty acids and we have examined this proposition. Palmitate and palmitoleate both promoted beta-cell phospholipid remodelling and increased TAG formation (control: 0.9 +/- 0.1 nmol TAG/10(6) cells; 0.25 mM palmitate: 1.55 +/- 0.07; 0.25 mM palmitoleate: 1.4 +/- 0.05; palmitate plus palmitoleate: 2.3 +/- 0.1). By contrast, methyl-palmitoleate failed to influence TAG levels (0.25 mM methyl-palmitoleate alone: 0.95 +/- 0.06 nmol TAG/10(6) cells; methyl-palmitoleate plus palmitate: 1.5 +/- 0.05) or its fatty acid composition in beta-cells exposed to palmitate. The results suggest that monounsaturated fatty acids can promote cell viability and mitogenesis by a mechanism that does not require their metabolism and is independent of alterations in TAG formation.

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

Pro-inflammatory cytokines are important mediators of ß-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. Since enteroviral infection of islet cells may influence the development of diabetes in humans, we examined the actions of two cytokines, IL-13 and IL-6, whose expression are reported to be altered in ß-cells during enteroviral infection. Human and rodent islet cells were shown to express receptors for both IL-13 and IL-6, and treatment with either cytokine resulted in the rapid phosphorylation of STAT3 and STAT6. However, while ß-cells were protected against a range of cytotoxic insults during exposure to IL-13, treatment with IL-6 enhanced cytotoxicity and western blotting revealed that IL-13 induced one specific isoform of phospho-STAT6 preferentially. Upon incubation with both cytokines together, the isoform of STAT6 that was upregulated by IL-13 alone was again induced, and the effects of IL-6 on ß-cell viability were attenuated. Overall, the results suggest that induction of specific isoforms of STAT family transcription factors may underlie the cytoprotective actions of IL-13, and they imply that selective targeting of specific STAT-mediated signaling components could provide a means to ameliorate the loss of ß-cell viability in diabetes.

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

BACKGROUND AND PURPOSE: ß-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 ß-cell cytoprotection. It is not known, however, if OEA is cytoprotective in ß-cells. The present study has examined this and determined whether GPR119 is involved. METHODS: Clonal rat insulin-secreting cell lines, BRIN-BD11 or INS-1E, were exposed to fatty acids complexed with BSA. cAMP levels, insulin release and cell viability were measured. Protein expression was studied by Western blotting and receptor expression by RT-PCR. KEY RESULTS: GPR119 was expressed in both BRIN-BD11 and INS-1E cells and OEA was cytoprotective in these cells. However, cytoprotection was not reproduced by any of a range of selective, synthetic ligands of GPR119. The cytoprotective response to OEA was lost during exposure to inhibitors of fatty acid amide hydrolase (FAAH) suggesting that OEA per se is not the cytoprotective species but that release of free oleate is required. Similar data were obtained with anandamide, which was cytoprotective only under conditions favouring release of free arachidonate. CONCLUSIONS AND IMPLICATIONS: Activation of GPR119 is not required to mediate the cytoprotective actions of OEA in BRIN-BD11 or INS-1E cells. Rather, OEA is internalised and subjected to hydrolysis by FAAH to release free oleate, which then mediates the cytoprotection.

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. EXPERIMENTAL APPROACH: Clonal rat insulin-secreting cell lines, BRIN-BD11 or INS-1, were exposed to fatty acids or their derivatives complexed with BSA and the viability of the cells was analysed by flow cytometry after staining with propidium iodide. KEY RESULTS: A variety of polyunsaturated fatty acids with chain lengths between C18-C22 attenuated the cytotoxic actions of the saturated fatty acid, palmitate (C16:0) in BRIN-BD11 and INS-1 cells. These effects were dose-dependent and displayed potencies that were much higher than those achieved with monounsaturated fatty acids. Methyl esters of the polyunsaturates were also effective. The cytoprotective responses were not altered by incubation of cells with inhibitors of cyclooxygenase or lipoxygenase enzymes although they were antagonized dose-dependently by arachidonyltrifluoromethylketone (AACOCF(3)). CONCLUSIONS AND IMPLICATIONS: The results are consistent with the involvement of a specific fatty acid binding site having loose, but defined, structural criteria, in mediating the cytoprotective effects of unsaturated fatty acids. AACOCF(3) may be of value in defining this site in molecular terms.

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 ß-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. Thus, both physical properties and specific signalling events may regulate fatty acid responses in ß-cells.

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. This difference does not appear to be mediated by a mutual metabolic antagonism between saturated and unsaturated species (although differential alterations in neutral lipid disposition may occur in response to these fatty acids) and the mechanisms remain unclear. This review summaries the current understanding of the actions of mono-unsaturated fatty acids in beta-cells and highlights areas of controversy as well as key unresolved issues which require to be addressed.

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. GPR40 is the best characterised of this group and appears to mediate the acute stimulatory effects of long chain fatty acids on insulin secretion. GPR40 has also been proposed as a potential mediator of fatty acid toxicity but this is more controversial. GPR119 is also involved in stimulation of insulin secretion and responds primarily to ethanolamide derivatives of long chain fatty acids and also to some lysophospholipids rather than to free fatty acids. It may represent a useful target for the development of new insulin secretagogues aimed to enhance insulin release in patients with type 2 diabetes. GPR120 is the most enigmatic of the lipid-responsive cell-surface receptors and its function remains to be established. It has been proposed to play a cytoprotective role in certain other cell types but it is unclear whether it fulfils a similar function in beta-cells.

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.g. palmitate (C16:0)] exert detrimental effects on beta-cells, the equivalent mono-unsaturated species [e.g. palmitoleate (C16:1)] are well tolerated. Indeed, mono-unsaturated species are potently cytoprotective. The present review explores the differential effects of these various fatty acids on beta-cell viability and considers the possible mechanisms involved in cytoprotection by mono-unsaturates.