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Eleftheria Diakogiannaki, Hannah J Welters, and Noel G Morgan

Exposure of pancreatic β-cells to long-chain fatty acids leads to the activation of some components of the endoplasmic reticulum (ER) stress pathway and this mechanism may underlie the ability of certain fatty acids to promote β-cell death. We have studied ER stress in BRIN-BD11 β-cells exposed to either the saturated fatty acid palmitate (C16:0) or the monounsaturated palmitoleate (C16:1). Palmitate (0.025–0.25 mM) induced the expression of various markers of the RNA-dependent protein kinase-like ER eukaryotic initiation factor 2α (eIF2α) kinase (PERK)-dependent pathway of ER stress (phospho-eIF2α; ATF4, activating transcription factor 4 and C/EBP homologous protein (CHOP-10)) although it failed to promote the expression of the ER chaperone GRP78. By contrast, palmitoleate did not induce any markers of the ER stress pathway even at concentrations as high as 1 mM. When palmitate and palmitoleate were added in combination, a marked attenuation of the ER stress response occurred. Under these conditions, the levels of phospho-eIF2α, ATF4 and CHOP-10 were reduced to less than those found in control cells. Palmitoleate also attenuated the ER stress response to the protein glycosylation inhibitor, tunicamycin, and improved the viability of the cells exposed to this agent. Exposure of the BRIN-BD11 cells to the protein phosphatase inhibitor, salubrinal, in the absence of fatty acids resulted in increased eIF2α phosphorylation but this was abolished by co-incubation with palmitoleate. We conclude that saturated fatty acids activate components of the PERK-dependent ER stress pathway in β-cells, ultimately leading to increased apoptosis. This effect is antagonised by monounsaturates that may exert their anti-apoptotic actions by regulating the activity of one or more kinase enzymes involved in mediating the phosphorylation of eIF2α.

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Hannah J Welters, Alina Oknianska, Kai S Erdmann, Gerhart U Ryffel, and Noel G Morgan

In pancreatic β-cells, increased expression of the MODY5 gene product, HNF1β, leads to enhanced rates of apoptosis and altered regulation of the cell cycle, suggesting that control of HNF1β expression may be important for the control of β-cell proliferation and viability. It is unclear how these effects of HNF1β are mediated, but previously we have identified a protein tyrosine phosphatase, (PTP)-BL, as an HNF1β-regulated protein in β-cells and have now studied the role of this protein in INS-1 β-cells. Stably transfected cells were generated, which express either wild-type (WT) or a phosphatase-deficient mutant (PTP-BL-CS) of PTP-BL conditionally under the control of a tetracycline-regulated promoter. Enhanced expression of WT PTP-BL inhibited INS-1 cell growth dose dependently, but this effect was not observed when PTP-BL-CS was expressed. Neither construct altered the rate of apoptosis. PTP-BL has been reported to interact with components of the Wnt signalling pathway, and we observed that addition of exogenous Wnt3a resulted in an increase in cell proliferation and a rise in β-catenin levels, consistent with the operation of this pathway in INS-1 cells. Up-regulation of WT PTP-BL antagonised these responses but PTP-BL-CS failed to inhibit Wnt3a-induced proliferation. The rise in β-catenin caused by Wnt3a was also suppressed by over-expression of HNF1β, suggesting that HNF1β may interact with the Wnt signalling pathway via an increase in PTP-BL levels. We conclude that PTP-BL plays an important role in the regulation of cell cycle progression in pancreatic β-cells, and that it interacts functionally with components of the Wnt signalling pathway.

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Eleftheria Diakogiannaki, Shalinee Dhayal, Caroline E Childs, Philip C Calder, Hannah J Welters, and Noel G Morgan

Long-chain saturated and monounsaturated fatty acids differ in their propensity to induce β-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 β-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 β-cell phospholipid remodelling and increased TAG formation (control: 0.9 ± 0.1 nmol TAG/106 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/106 cells; methyl-palmitoleate plus palmitate: 1.5 ± 0.05) or its fatty acid composition in β-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.

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Hannah J Welters, Sabine Senkel, Ludger Klein-Hitpass, Silke Erdmann, Heike Thomas, Lorna W Harries, Ewan R Pearson, Coralie Bingham, Andrew T Hattersley, Gerhart U Ryffel, and Noel G Morgan

Mutations in the gene encoding hepatocyte nuclear factor (HNF)1β result in maturity-onset diabetes of the young-(MODY)5, by impairing insulin secretory responses and, possibly, by reducing β-cell mass. The functional role of HNF1β in normal β-cells is poorly understood; therefore, in the present study, wild-type (WT) HNF1β, or one of two naturally occurring MODY5 mutations (an activating mutation, P328L329del, or a dominant-negative form, A263insGG) were conditionally expressed in the pancreatic β-cell line, insulin-1 (INS-1), and the functional consequences examined. Surprisingly, overexpression of the dominant-negative mutant did not modify any of the functional properties of the cells studied (including insulin secretion, cell growth and viability). By contrast, expression of WT HNF1β was associated with a time- and dose-dependent inhibition of INS-1 cell proliferation and a marked increase in apoptosis. Induction of WT HNF1β also inhibited the insulin secretory response to nutrient stimuli, membrane depolarisation or activation of protein kinases A and C and this correlated with a significant decrease in pancrease-duodenum homeobox-1 protein levels. The attenuation of insulin secretion was, however, dissociated from the inhibition of proliferation and loss of viability, since expression of the P328L329del mutant led to a reduced rate of cell proliferation, but failed to induce apoptosis or to alter insulin secretion. Taken together, the present results suggest that mature rodent β-cells are sensitive to increased expression of WT HNF1β and they imply that the levels of this protein are tightly regulated to maintain secretory competence and cell viability.