Differential routing and disposition of the long-chain saturated fatty acid palmitate in rodent vs human beta-cells.

BACKGROUND: Rodent and human ß-cells are differentially susceptible to the "lipotoxic" effects of long-chain saturated fatty acids (LC-SFA) but the factors accounting for this are unclear. Here, we have studied the intracellular disposition of the LC-SFA palmitate in human vs rodent ß-cells and present data that reveal new insights into the factors regulating ß-cell lipotoxicity. METHODS: The subcellular distribution of the LC-SFA palmitate was studied in rodent (INS-1E and INS-1 823/13 cells) and human (EndoC-ßH1) ß-cells using confocal fluorescence and electron microscopy (EM). Protein expression was assessed by Western blotting and cell viability, by vital dye staining. RESULTS: Exposure of INS-1 cells to palmitate for 24 h led to loss of viability, whereas EndoC-ßH1 cells remained viable even after 72 h of treatment with a high concentration (1 mM) of palmitate. Use of the fluorescent palmitate analogue BODIPY FL C16 revealed an early localisation of the LC-SFA to the Golgi apparatus in INS-1 cells and this correlated with distention of intracellular membranes, visualised under the EM. Despite this, the PERK-dependent ER stress pathway was not activated under these conditions. By contrast, BODIPY FL C16 did not accumulate in the Golgi apparatus in EndoC-ßH1 cells but, rather, co-localised with the lipid droplet-associated protein, PLIN2, suggesting preferential routing into lipid droplets. When INS-1 cells were treated with a combination of palmitate plus oleate, the toxic effects of palmitate were attenuated and BODIPY FL C16 localised primarily with PLIN2 but not with a Golgi marker. CONCLUSION: In rodent ß-cells, palmitate accumulates in the Golgi apparatus at early time points whereas, in EndoC- ßH1 cells, it is routed preferentially into lipid droplets. This may account for the differential sensitivity of rodent vs human ß-cells to "lipotoxicity" since manoeuvres leading to the incorporation of palmitate into lipid droplets is associated with the maintenance of cell viability in both cell types.

Long-chain saturated fatty acid species are not toxic to human pancreatic ß-cells and may offer protection against pro-inflammatory cytokine induced ß-cell death.

Obesity is a major risk factor for type 2 diabetes (T2D) although the causal links remain unclear. A feature shared by both conditions however is systemic inflammation and raised levels of circulating fatty acids (FFA). It is widely believed that in obese individuals genetically prone to T2D, elevated levels of plasma FFA may contribute towards the death and dysfunction of insulin-producing pancreatic ß-cells in a process of (gluco)lipotoxicity. In support of this, in vitro studies have shown consistently that long-chain saturated fatty acids (LC-SFA) are toxic to rodent ß-cells during chronic exposure (> 24 h). Conversely, shorter chain SFA and unsaturated species are well tolerated, suggesting that toxicity is dependent on carbon chain length and/or double bond configuration. Despite the wealth of evidence implicating lipotoxicity as a means of ß-cell death in rodents, the evidence that a similar process occurs in humans is much less substantial. Therefore, the present study has evaluated the effects of chronic exposure to fatty acids of varying chain length and degree of saturation, on the viability of human ß-cells in culture. We have also studied the effects of a combination of fatty acids and pro-inflammatory cytokines. Strikingly, we find that LC-FFA do not readily promote the demise of human ß-cells and that they may even offer a measure of protection against the toxic effects of pro-inflammatory cytokines. Therefore, these findings imply that a model in which elevated circulating LC-FFA play a direct role in mediating ß-cell dysfunction and death in humans, may be overly simplistic.

Molecular pathways underlying the pathogenesis of pancreatic alpha-cell dysfunction.

Glucagon plays a critical role in glucose homeostasis by counteracting insulin action, especially during hypoglycemia. Glucagon secretion from pancreatic alpha-cells is regulated by various mechanisms including glycemia, neural input, and secretion from neighboring beta-cells. However, glucagon secretion is dysregulated in diabetic states, causing exacerbation of glycemic disorders. Recently, new therapeutic approaches targeting excess glucagon secretion are being explored for use in diabetes treatment. Therefore, understanding the molecular mechanism of how glucagon secretion is regulated is critical for treating the alpha-cell dysfunction observed in diabetes.

Wnt signaling: relevance to beta-cell biology and diabetes.

Interest in the importance of Wnt signaling in diabetes has risen after identification of the transcription factor TCF7L2, a component of this pathway, as a strong risk factor for type 2 diabetes. Here, we review emerging new evidence that Wnt signaling influences endocrine pancreas development and modulates mature beta-cell functions including insulin secretion, survival and proliferation. Alterations in Wnt signaling might also impact other metabolic tissues involved in the pathogenesis of diabetes, with TCF7L2 proposed to modulate adipogenesis and regulate GLP-1 production. Together, these studies point towards a role for Wnt signaling in the pathogenesis of type 2 diabetes, highlighting the importance of further investigation of this pathway to develop new therapies for this disease.

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.

A Kir6.2 mutation causing neonatal diabetes impairs electrical activity and insulin secretion from INS-1 beta-cells.

ATP-sensitive K(+) channels (K(ATP) channels) couple beta-cell metabolism to electrical activity and thereby play an essential role in the control of insulin secretion. Gain-of-function mutations in Kir6.2 (KCNJ11), the pore-forming subunit of this channel, cause neonatal diabetes. We investigated the effect of the most common neonatal diabetes mutation (R201H) on beta-cell electrical activity and insulin secretion by stable transfection in the INS-1 cell line. Expression was regulated by placing the gene under the control of a tetracycline promoter. Transfection with wild-type Kir6.2 had no effect on the ATP sensitivity of the K(ATP) channel, whole-cell K(ATP) current magnitude, or insulin secretion. However, induction of Kir6.2-R201H expression strongly reduced K(ATP) channel ATP sensitivity (the half-maximal inhibitory concentration increased from approximately 20 mumol/l to approximately 2 mmol/l), and the metabolic substrate methyl succinate failed to close K(ATP) channels or stimulate electrical activity and insulin secretion. Thus, these results directly demonstrate that Kir6.2 mutations prevent electrical activity and insulin release from INS-1 cells by increasing the K(ATP) current and hyperpolarizing the beta-cell membrane. This is consistent with the ability of the R201H mutation to cause neonatal diabetes in patients. The relationship between K(ATP) current and the membrane potential reveals that very small changes in current amplitude are sufficient to prevent hormone secretion.

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.

Life and death decisions of the pancreatic beta-cell: the role of fatty acids.

Both stimulatory and detrimental effects of NEFAs (non-esterified fatty acids) on pancreatic beta-cells have been recognized. Acute exposure of the pancreatic beta-cell to high glucose concentrations and/or saturated NEFAs results in a substantial increase in insulin release, whereas chronic exposure results in desensitization and suppression of secretion, followed by induction of apoptosis. Some unsaturated NEFAs also promote insulin release acutely, but they are less toxic to beta-cells during chronic exposure and can even exert positive protective effects. Therefore changes in the levels of NEFAs are likely to be important for the regulation of beta-cell function and viability under physiological conditions. In addition, the switching between endogenous fatty acid synthesis or oxidation in the beta-cell, together with alterations in neutral lipid accumulation, may have critical implications for beta-cell function and integrity. Long-chain acyl-CoA (formed from either endogenously synthesized or exogenous fatty acids) controls several aspects of beta-cell function, including activation of specific isoenzymes of PKC (protein kinase C), modulation of ion channels, protein acylation, ceramide formation and/or NO-mediated apoptosis, and transcription factor activity. In this review, we describe the effects of exogenous and endogenous fatty acids on beta-cell metabolism and gene and protein expression, and have explored the outcomes with respect to insulin secretion and beta-cell integrity.

Conditional expression of hepatocyte nuclear factor-1beta, the maturity-onset diabetes of the young-5 gene product, influences the viability and functional competence of pancreatic beta-cells.

Mutations in the gene encoding hepatocyte nuclear factor (HNF)1beta result in maturity-onset diabetes of the young-(MODY)5, by impairing insulin secretory responses and, possibly, by reducing beta-cell mass. The functional role of HNF1beta in normal beta-cells is poorly understood; therefore, in the present study, wild-type (WT) HNF1beta, or one of two naturally occurring MODY5 mutations (an activating mutation, P328L329del, or a dominant-negative form, A263insGG) were conditionally expressed in the pancreatic beta-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 HNF1beta was associated with a time- and dose-dependent inhibition of INS-1 cell proliferation and a marked increase in apoptosis. Induction of WT HNF1beta 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 beta-cells are sensitive to increased expression of WT HNF1beta and they imply that the levels of this protein are tightly regulated to maintain secretory competence and cell viability.

Mono-unsaturated fatty acids protect against beta-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure.

Long-chain saturated fatty acids are cytotoxic to pancreatic beta-cells while shorter-chain saturated and long-chain unsaturated molecules are better tolerated. Mono-unsaturated fatty acids are not, however, inert since they inhibit the pro-apoptotic effects of saturated molecules. In the present work we show that the mono-unsaturates palmitoleate (C16:1) or oleate (C18:1) also cause marked inhibition of apoptosis induced by exposure of clonal BRIN-BD11 beta-cells to serum withdrawal or a combination of interleukin-1beta plus interferon-gamma. This response was dose-dependent and not accompanied by changes in NO formation. Taken together, the results suggest that mono-unsaturated fatty acids regulate a distal step common to several apoptotic pathways in pancreatic beta-cells.

Expression and functional activity of PPARgamma in pancreatic beta cells.

Rosiglitazone is an agonist of peroxisome proliferator activated receptor-gamma (PPARgamma) and ameliorates insulin resistance in type II diabetes. In addition, it may also promote increased pancreatic beta-cell viability, although it is not known whether this effect is mediated by a direct action on the beta cell. We have investigated this possibility. Semiquantitative real-time reverse transcription-polymerase chain reaction analysis (Taqman) revealed that freshly isolated rat islets and the clonal beta-cell line, BRIN-BD11, express PPARgamma, as well as PPARalpha and PPARdelta. The levels of expression of PPARgamma were estimated by reference to adipose tissue and were found to represent approximately 60% (islets) and 30% (BRIN-BD11) of that found in freshly isolated visceral adipose tissue. Western blotting confirmed the presence of immunoreactive PPARgamma in rat (and human) islets and in BRIN-BD11 cells. Transfection of BRIN-BD11 cells with a PPARgamma-sensitive luciferase reporter construct was used to evaluate the functional competence of the endogenous PPARgamma. Luciferase activity was modestly increased by the putative endogenous ligand, 15-deoxy-Delta12,14 prostaglandin J2 (15dPGJ2). Rosiglitazone also caused activation of the luciferase reporter construct but this effect required concentrations of the drug (50-100 microm) that are beyond the expected therapeutic range. This suggests that PPARgamma is relatively insensitive to activation by rosiglitazone in BRIN-BD11 cells. Exposure of BRIN-BD11 cells to the lipotoxic effector, palmitate, caused a marked loss of viability. This was attenuated by treatment of the cells with either actinomycin D or cycloheximide suggesting that a pathway of programmed cell death was involved. Rosiglitazone failed to protect BRIN-BD11 cells from the toxic actions of palmitate at concentrations up to 50 microm. Similar results were obtained with a range of other PPARgamma agonists. Taken together, the present data suggest that, at least under in vitro conditions, thiazolidinediones do not exert direct protective effects against fatty acid-mediated cytotoxicity in pancreatic beta cells.

A role for SPARC in the moderation of human insulin secretion.

AIMS/HYPOTHESIS: We have previously shown the implication of the multifunctional protein SPARC (Secreted protein acidic and rich in cysteine)/osteonectin in insulin resistance but potential effects on beta-cell function have not been assessed. We therefore aimed to characterise the effect of SPARC on beta-cell function and features of diabetes. METHODS: We measured SPARC expression by qRT-PCR in human primary pancreatic islets, adipose tissue, liver and muscle. We then examined the relation of SPARC with glucose stimulated insulin secretion (GSIS) in primary human islets and the effect of SPARC overexpression on GSIS in beta cell lines. RESULTS: SPARC was expressed at measurable levels in human islets, adipose tissue, liver and skeletal muscle, and demonstrated reduced expression in primary islets from subjects with diabetes compared with controls (p< = 0.05). SPARC levels were positively correlated with GSIS in islets from control donors (p< = 0.01). Overexpression of SPARC in cultured beta-cells resulted in a 2.4-fold increase in insulin secretion in high glucose conditions (p< = 0.01). CONCLUSIONS: Our data suggest that levels of SPARC are reduced in islets from donors with diabetes and that it has a role in insulin secretion, an effect which appears independent of SPARC's modulation of obesity-induced insulin resistance in adipose tissue.

Rosiglitazone promotes PPARγ-dependent and -independent alterations in gene expression in mouse islets.

The glitazone class of insulin-sensitizing agents act, in part, by the activation of peroxisome proliferator-activated receptor (PPAR)-γ in adipocytes. However, it is unclear whether the expression of PPARγ in the islets is essential for their potential ß-cell-sparing properties. To investigate the in vivo effects of rosiglitazone on ß-cell biology, we used an inducible, pancreatic and duodenal homeobox-1 enhancer element-driven, Cre recombinase to knockout PPARγ expression specifically in adult ß-cells (PPARgKO). Subjecting the PPARgKO mice to a chow diet led to virtually undetectable changes in glucose or insulin sensitivity, which was paralleled by minimal changes in islet gene expression. Similarly, challenging the mutant mice with a high-fat diet and treatment with rosiglitazone did not alter insulin sensitivity, glucose-stimulated insulin secretion, islet size, or proliferation in the knockout mice despite PPARγ-dependent and -independent changes in islet gene expression. These data suggest that PPARγ expression in the ß-cells is unlikely to be directly essential for normal ß-cell function or the insulin-sensitizing actions of rosiglitazone.

Human and rodent pancreatic beta-cells express IL-4 receptors and IL-4 protects against beta-cell apoptosis by activation of the PI3K and JAK/STAT pathways.

Secretion of pro-inflammatory cytokines is associated with loss of pancreatic beta-cell viability and cell death. IL-4 (interleukin-4) has been reported to mediate a protective effect against the loss of pancreatic beta-cells, and IL-4 receptors have been found in rat pancreatic beta-cells at both the RNA and the protein level. The aim of the present study was to investigate IL-4 receptor expression in human islet cells and to examine the signalling pathways by which IL-4 exerts its effects using the rat beta-cell lines, BRIN-BD11 and INS-1E. By means of immunohistochemistry, it was demonstrated that IL-4 receptors are present on human islet cells. Using a flow cytometric method for evaluating cell death, it was confirmed that incubating beta-cells with IL-4 attenuated cell death induced by IL-1beta and interferon-gamma by approx. 65%. This effect was abrogated by the presence of the PI3K (phosphoinositide 3-kinase) inhibitor, wortmannin, suggesting that activation of the PI3K pathway is involved. In support of this, Western blotting revealed that incubation of cells with IL-4 resulted in increased phosphorylation of Akt (also called protein kinase B), a downstream target of PI3K. Increased tyrosine phosphorylation of STAT6 (signal transducer and activator of transcription 6) also occurred in response to IL-4 and a selective JAK3 (Janus kinase 3) inhibitor reduced the cytoprotective response. Both effects were prevented by overexpression of the tyrosine phosphatase, PTP-BL (protein tyrosine phosphatase-BL). We conclude that IL-4 receptors are functionally competent in pancreatic beta-cells and that they signal via PI3K and JAK/STAT pathways. These findings may have implications for future therapeutic strategies for the management of diabetes.

Differential regulation of the endoplasmic reticulum stress response in pancreatic beta-cells exposed to long-chain saturated and monounsaturated fatty acids.

Exposure of pancreatic beta-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 beta-cell death. We have studied ER stress in BRIN-BD11 beta-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 2 alpha (eIF2 alpha) kinase (PERK)-dependent pathway of ER stress (phospho-eIF2 alpha; 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-eIF2 alpha, 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 eIF2 alpha 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 beta-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 eIF2 alpha.

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.

The protein tyrosine phosphatase-BL, modulates pancreatic beta-cell proliferation by interaction with the Wnt signalling pathway.

In pancreatic beta-cells, increased expression of the MODY5 gene product, HNF1 beta, leads to enhanced rates of apoptosis and altered regulation of the cell cycle, suggesting that control of HNF1 beta expression may be important for the control of beta-cell proliferation and viability. It is unclear how these effects of HNF1 beta are mediated, but previously we have identified a protein tyrosine phosphatase, (PTP)-BL, as an HNF1 beta-regulated protein in beta-cells and have now studied the role of this protein in INS-1 beta-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 beta-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 beta-catenin caused by Wnt3a was also suppressed by over-expression of HNF1 beta, suggesting that HNF1 beta 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 beta-cells, and that it interacts functionally with components of the Wnt signalling pathway.

Differential protective effects of palmitoleic acid and cAMP on caspase activation and cell viability in pancreatic beta-cells exposed to palmitate.

Saturated and mono-unsaturated fatty acids exert differential effects on pancreatic beta-cell viability during chronic exposure. Long chain saturated molecules (e.g. palmitate) are cytotoxic to beta-cells and this is associated with caspase activation and induction of apoptosis. By contrast, mono-unsaturated fatty acids (e.g. palmitoleate) are not toxic and can protect against the detrimental effects of palmitate. In the present study, we show that the protective actions of palmitoleate in BRIN-BD11 beta-cells result in attenuated caspase activation following exposure to palmitate and that a similar response occurs in cells having elevated levels of cAMP. However, unlike palmitoleate, elevation of cAMP was unable to prevent the cytotoxic actions of palmitate since it caused a diversion of the pathway of cell death from apoptosis to necrosis. Palmitoleate did not alter cAMP levels in BRIN-BD11 cells and the results suggest that a change in cAMP is not involved in mediating the protective effects of this fatty acid. Moreover, they reveal that attenuated caspase activation does not always correlate with altered cell viability in cultured beta-cells and suggest that mono-unsaturated fatty acids control cell viability by regulating a different step in the apoptotic pathway from that influenced by cAMP.