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

in Journal of Endocrinology
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Eleftheria Diakogiannaki
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Shalinee Dhayal
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Caroline E Childs
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Philip C Calder
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Hannah J Welters
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Noel G Morgan
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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.

Abstract

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.

Introduction

The prevalence of type 2 diabetes is rising rapidly in many regions of the world, driven, in part, by changing lifestyles and dietary habits. In particular, it appears to be associated with increases in obesity, leading to alterations in the circulating lipid profile of affected individuals ( Rhodes 2005, Kahn et al. 2006). This is significant since the disease is characterised by impaired insulin secretion and loss of insulin sensitivity followed by a progressive decline in β-cell mass, all of which may be influenced by alterations in lipid availability ( Shimabukuro et al. 1998b, Ahren 2005, Marchetti et al. 2006, Newsholme et al. 2007).

Acute exposure of β-cells to free fatty acids (FFA) in vitro leads to enhanced rates of insulin secretion ( Sako & Grill 1990, Newsholme et al. 2007), whereas more prolonged treatment can be associated with toxicity ( Lupi et al. 2002, El-Assaad et al. 2003, Haber et al. 2003, 2006, Rhodes 2005, Azevedo-Martins et al. 2006). However, the latter occurs more dramatically during exposure to long-chain saturated FFA (e.g. palmitate (C16:0) or stearate (C18:0)) than in response to either shorter chain saturated (C14 or less) or longer chain unsaturated FFA (such as palmitoleate (C16:1n-7) or oleate (C18:1n-9)). Indeed, there is increasing evidence that when used within the physiological range of free concentrations, the latter species are directly cytoprotective to β-cells ( Eitel et al. 2002, Maedler et al. 2003, Welters et al. 2004, 2006) although, when present in sufficiently high free concentrations, all fatty acids are likely to exert detrimental effects ( Azevedo-Martins et al. 2006).

Since long-chain saturated and long-chain monounsaturated FFA can exert differential effects on β-cell viability, this implies that they must also elicit different intracellular responses in order to mediate these effects. Such differences have proved elusive to define but changes in neutral lipid accumulation could be involved since β-cells can store excess FFA in the form of triacylglycerol (TAG; Briaud et al. 2001, Unger & Zhou 2001, Unger & Orci 2002, Moffitt et al. 2005). However, the mechanistic consequences of this response have become the source of considerable debate with some investigators, proposing that diversion of FFA into intracellular TAG serves a cytoprotective function ( Cnop et al. 2001, Listenberger et al. 2003), while others (e.g. Briaud et al. 2001, Moffitt et al. 2005) have seen this as a potentially toxic response.

In the present project, we have studied the responses of pancreatic β-cells to incubation with extracellular FFA in vitro in more detail. We have employed a range of modified FFA species, both saturated and unsaturated, and have investigated whether the propensity of these to regulate cell viability correlates with their potential for intracellular metabolism to TAG.

Materials and Methods

Cell culture

The rat pancreatic β-cell line BRIN-BD11 was used in all experiments. The cells were cultured in RPMI-1640 medium (Invitrogen) containing 11 mM glucose supplemented with 10% fetal calf serum (PAA Laboratories, Yeovil, Somerset, UK), 2 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). The cells were grown in 75 cm2 flasks at 37 °C and 5% CO2.

Treatment of cells with fatty acids

Stock solutions of 50 mM palmitate (Sigma) or 2-bromopalmitate (Avocado research chemicals, Morecambe, Lancs, UK) were prepared in 50% ethanol by heating to 70 °C. Palmitoleate (MP Biomedicals, Cambridge, UK) and methyl-palmitoleate (Sigma) were prepared by mixing with 90% ethanol at room temperature to produce stock solutions of 90 mM. The fatty acid preparations were then bound to 10% fatty acid-free BSA (MP Biomedicals) by incubation for 1 h at 37 °C. The mixture was added to RPMI-1640 medium (containing 11 mM glucose) deprived of fetal calf serum. The final concentrations present in the cell environment were 1% for BSA and 0.5% for ethanol. Cells were seeded into six-well plates at densities of 1 × 105 cells/well and incubated for 24 h in complete RPMI-1640 medium. The medium was then removed and replaced with relevant fatty acid/BSA complexes (in RPMI-1640 devoid of foetal calf serum) for a further 18 h. Controls received BSA and vehicle only.

Vital dye staining for estimation of viability

Following incubation, floating and attached cells were collected, centrifuged at 200 g for 5 min and resuspended in 250 μl complete RPMI-1640 plus 250 μl Trypan blue (0.4% in PBS). Viable and dead cells were counted using a haemocytometer and the number of dead cells expressed as a percentage of the total for each condition.

Fluorescence microscopic assays of cell viability

For the assessment of viable, necrotic and apoptotic cells, BRIN-BD11 cells previously treated with fatty acids were incubated for 5 min with a mixture of 50 μg/ml Hoechst 33342 (Sigma) and 5 μg/ml propidium iodide (Sigma) dissolved in PBS. Hoechst 33342 penetrates the plasma membrane of all cells, staining the DNA blue. Viable cells appeared pale blue, while apoptotic cells showed strong dark blue fluorescence due to DNA condensation. Propidium iodide can penetrate the cells only when their plasma membrane is damaged, leading to fluorescent pink staining of the nucleus. Consequently, necrotic cells were identified by their fluorescent pink colour. The stained cells were examined by fluorescence microscopy ( Jankowska et al. 1997). The percentages of viable, necrotic and apoptotic cells were calculated after counting at least 100 cells for each experimental condition.

5-Bromo-2-deoxyuridine (BrdU) cell proliferation assay

Incorporation of BrdU, as an indication of cell proliferation, was measured with a commercial kit (Amersham). BRIN-BD11 cells were initially treated with fatty acids and then seeded into 96-well plates at a density of 5000 cells/well for 24 h. BrdU label was added to the culture medium during either 4 or 20 h of incubation, after which the cells were fixed and BrdU incorporation was assessed according to the manufacturer’s instructions.

Analysis of phospholipid fatty acid composition and TAG formation

Gas chromatography was used for assessment of the fatty acid profile of β-cell phospholipids. BRIN-BD11 cells were seeded into 25 cm2 flasks at a density of 1 × 106 cells/flask and were then exposed to fatty acids. The cells were trypsinised and centrifuged (200 g; 5 min). Lipids were extracted by using a mixture of chloroform:methanol (2:1, v/v) containing butylated hydroxy-toluene (50 mg/l) as an antioxidant ( Folch et al. 1957). Phospholipids were separated from other lipid classes by solid phase extraction using methods described elsewhere ( Burdge et al. 2000) and were then saponified and methylated in methanol containing 2% (v/v) H2SO4 at 50 °C for 2 h. Fatty acid methyl esters (FAME) were recovered by extraction with hexane. FAME separation and analysis were carried out using a Hewlett Parckard 6890 gas chromatograph (Agilent Technologies, Wokingham, Berks, UK) with a BPX-70 capillary column (30 m length, 220 μm internal diameter, 0.25 μm film thickness, SGE Europe Limited, Milton Keynes, Bucks, UK). The gas chromatograph oven was programmed to hold for 2 min at the starting temperature of +115 °C then increased at +10 °C/min to 200 °C and hold there for 10 min and finally raised at +60 °C/ min to 245 °C and hold for 4 min at the temperature of 245 °C. FAME were identified by comparison of peak retention times to those of known standards. Chemstation software was used for peak area quantification. The quantityof fatty acids was calculated using appropriate internal standards.

Triglyceride (TG) levels of BRIN-BD11 cells were measured with a commercial kit designed for analysis of cellular triglyceride content (Zen-bio Triglyceride Assay Kit # TG-1-NC). Cells were seeded into 24-well plates at densities of 1 × 105cells/well and treated with the appropriate test reagents for 18 h. They were then lysed and processed according to the manufacturer’s instructions in parallel with standard glycerol solutions provided in the kit. TG levels were estimated from the release of glycerol in experimental samples and were measured spectrophotometrically at 540 nm.

Insulin secretion

BRIN-BD11 cells were seeded into 24-well plates at a density of 5 × 104 cells/well and incubated for 18 h in the absence or presence of fatty acids, as appropriate. The medium was then removed and the cells washed and incubated in a bicarbonate-buffered physiological saline solution ( Gey & Gey 1936) containing 4 mM glucose and 1 mg/ml BSA for 1 h. This buffer was then replaced with fresh incubation buffer containing test reagents and insulin secretion measured by RIA after incubation at 37 °C for 1 h.

Data analysis

All experiments were performed on at least three separate occasions and triplicates of each condition were used in each experiment. The results are expressed as mean ± s.e.m. and the level of significance was calculated by using Student’s t-test or ANOVA and was regarded as significant when P < 0.05.

Results

Effects of fatty acids on β-cell viability

In confirmation of previous observations ( Welters et al. 2004, 2006), incubation of BRIN-BD11 cells with palmitate dose-dependently reduced their viability as judged by either trypan blue exclusion or staining with propidium iodide/Hoechst 33342. This effect was primarily due to increased apoptosis as judged by fluorescence microscopy after staining with propidium iodide and Hoechst 33342 (Fig. 1 ). By contrast, palmitoleate was not toxic to the cells at concentrations up to 0.25 mM. Cells exposed to both palmitate and palmitoleate simultaneously also retained viability (0.25 mM palmitate alone: 89 ± 8% non-viable cells; 0.25 mM palmitoleate alone: 9 ± 3% non-viable cells; palmitate plus palmitoleate: 19 ± 3% non-viable cells (P < 0.001 versus palmitate alone)), suggesting that palmitoleate was actively protective against palmitate-induced cytotoxicity.

Examination of the cytoprotective effect of palmitoleate at the level of β-cell growth

It was important to establish whether the protective actions of palmitoleate result in true preservation of β-cell viability or whether this fatty acid simply delays the onset of apoptosis beyond the time course of the present experiments. A variety of methods were employed to determine this and, in particular, studies were undertaken to examine β-cell growth and proliferation following exposure to different fatty acids. Initially, BRIN-BD11 cells were treated with 0.25 mM palmitate alone, 0.25 mM palmitoleate alone or a combination of the two fatty acids for 18 h, after which time the fatty acids were removed and the culture medium replaced. Cell growth was then monitored over the ensuing period (Fig. 2 ). The results revealed that 18-h exposure to palmitate led to a marked loss of viability (up to 80%) and, as expected, these cells failed to proliferate during subsequent incubation. By contrast, those cells that had been treated with palmitoleate alone or with palmitoleate in the presence of palmitate exhibited an increased rate of proliferation over the period following initial fatty acid treatment. Thus, the protection afforded by palmitoleate extended not only to maintaining cell viability but also to facilitating cell proliferation. Indeed, the rate of proliferation seen in cells exposed to either palmitoleate alone or palmitoleate plus palmitate was significantly higher than that measured in cells treated with vehicle alone (Fig. 2 ).

Further confirmation that palmitoleate treatment results in enhanced cell growth was achieved by measuring β-cell DNA synthesis more directly following fatty acid exposure (Fig. 3 ). To achieve this, BRIN-BD11 cells were exposed to BrdU following the relevant culture period with fatty acids. Cells treated with palmitate alone showed reduced proliferation when compared with control (as evidenced by attenuated incorporation of BrdU), whereas those treated with palmitoleate (either alone or in the combined presence of palmitate) demonstrated the highest levels of DNA synthesis (Fig. 3 ). Indeed, the rate of DNA synthesis measured under these conditions surpassed that of cells grown in complete medium throughout (complete medium: 0.18 ± 0.01 A450 units; palmitoleate: 0.22 ± 0.01 units; P < 0.01).

The cytoprotection afforded by palmitoleate over an 18-h incubation period also extended to the preservation of the insulin secretory function of the cells (Fig. 4 ). Insulin secretion from cells exposed to palmitate alone could not be tested due to the extensive loss of viability under these conditions. However, cells exposed to palmitate in the presence of palmitoleate (where viability was maintained) responded to a nutrient stimulus, α-ketoisocaproate, with an equivalent response to that seen in untreated controls (Fig. 4 ) or those exposed to palmitoleate alone. Thus, at least during 18 h of fatty acid exposure, insulin secretion was not compromised when the monounsaturate was present.

Measurement of the lipid content of β-cells cultured in the presence of fatty acids

It has been proposed that the behaviour of β-cells following fatty acid exposure is determined by alterations in lipid disposition, but this area remains controversial since both protective and detrimental affects have been attributed to altered lipid handling. Therefore, to evaluate the effects of fatty acids on lipid formation in cultured β-cells, lipids were extracted from BRIN-BD11 cells and their fatty acid composition analysed by gas chromatography. Initial experiments were undertaken to establish the total fatty acid composition of the neutral and phospholipid fractions within BRIN-BD11 cells incubated under control conditions. The results demonstrated that the most abundant fatty acid species were palmitate (C16:0), oleate (C18:1n-9), stearate (C18:0) and linoleate (C18:2n-6) with lower, but measurable amounts of palmitoleate (C16:1n-7) and arachidonate (C20:4n-6; Fig. 5 ).

Treatment of the cells with exogenous fatty acids resulted in an alteration in their phospholipid fatty acid profile, without any change in the total phospholipid content. The total content of fatty acids present in phospholipids was ~120 μg/106 cells, suggesting that this pool of fatty acid (FA) is large with respect to the total that is esterified within TAG (see below). As expected, the alterations in the phospholipids FA profile mirrored the relative availability of each fatty acid in the culture medium (Fig. 6 ). Thus, palmitate-treated cells displayed an elevation of palmitate in the phospholipid fraction, consistent with increased incorporation of this fatty acid under these conditions. This occurred primarily at the expense of oleate and stearate. By contrast, palmitoleate-treated cells displayed an elevation of this fatty acid in their phospholipids with a concomitant reduction in palmitate, stearate and oleate. Cells exposed to both palmitate and palmitoleate showed an intermediate increase in the incorporation of each of these species within their phospholipids.

In addition to altering the phospholipid fatty acid composition of BRIN-BD11 cells, exposure to elevated concentrations of exogenous fatty acids also affected their TAG content (Fig. 7a ). The total amount of FA contained within the TAG fraction amounted to ~25 μg/106 cells and, as such, this represents a smaller pool than that held in phospholipids. Nevertheless, addition of either 0.25 mM palmitate or 0.25 mM palmitoleate to the culture medium significantly raised the total TAG content of BRIN-BD11 cells by comparison with control cells. Culture of cells in the simultaneous presence of palmitate and palmitoleate for 18 h resulted in a further rise in TAG, such that the net effect was additive (Fig. 7a ).

To investigate these effects further and to discover whether altered TAG levels show any correlation with the changes in viability observed in response to different fatty acids, the effects of a range of additional conditions were also examined. Initially, 2-bromopalmitate (2-BP), an analogue of palmitate that can be thioesterified to Coenzyme-A but cannot then be further metabolised, was studied. The 2-BP is expected to reduce the total Coenzyme-A pool by sequestration ( Chase & Tubbs 1972, Oakes et al. 1999) and thereby to cause a reduction in TAG synthesis from exogenous palmitate. In support of this, TAG formation was inhibited in cells exposed to palmitate plus 2-BP when compared with cells exposed to palmitate alone (Fig. 7a ). This result confirms that β-cell TAG formation is influenced by the availability of FFA for esterification.

Next, methyl-palmitoleate, an analogue of palmitoleate that cannot be esterified to Co-enzyme A, was employed. As expected, exposure of cells to methyl-palmitoleate alone, failed to influence their TAG content (Fig. 7a ). More significantly, the co-presence of this fatty acid failed to alter the TAG content of palmitate-treated cells, suggesting that it did not alter the metabolic routing of palmitate into TAG (Fig. 7a ). To confirm this result, additional studies were conducted in which the fatty acid composition of BRIN-BD11 cell TAG was analysed after incubation of cells with palmitate and methyl-palmitoleate. As predicted, the increased incorporation of palmitate into TAG seen under these conditions was not attenuated by the presence of methyl-palmitoleate (control cells: 1.77 ± 0.04 μg palmitate in TAG/106 cells; 0.25 mM palmitate alone: 2.57 ± 0.28 μg (P < 0.01); 0.25 mM methyl-palmitoleate alone: 1.02 ± 0.14 μg; palmitate plus methyl-palmitoleate: 2.39 ± 0.11 μg (not significantly different from palmitate alone)). Despite this, methyl-palmitoleate abolished the cytotoxicity induced by palmitate as revealed by cell viability assays (Fig. 7b ). Indeed, its protective actions were equipotent with palmitoleate (Fig. 8 ) and, like palmitoleate, it also promoted β-cell growth following co-incubation with palmitate (not presented). Thus, the cytoprotective and growth-promoting properties of methyl-palmitoleate and palmitoleate were equivalent even though these fatty acids exerted differential effects on the TAG content of palmitate-treated cells.

Etomoxir, an inhibitor of carnityl palmitoyltransferase-1 (CPT-1), attenuates the uptake of fatty acids into mitochondria thereby reducing the rate of β-oxidation ( Chen et al. 1994) and promoting fatty acid availability for TAG synthesis. Accordingly, treatment of BRIN-BD11 cells with etomoxir in the presence of palmitate resulted in enhanced TAG formation by comparison with cells exposed to palmitate alone (Fig. 7a ). However, viability assays revealed that the extent of cell death under these conditions was lower than that seen with palmitate alone (Fig. 7b ).

Discussion

It has become increasingly clear that long-chain fatty acids exert complex, time-dependent effects on pancreatic β-cells, with short-term exposure leading to enhancement of insulin secretion, while chronic treatment is associated with attenuated insulin secretion and reduced viability ( Zhou & Grill 1994, Milburn et al. 1995, Unger 1995, Bollheimer et al. 1998, Shimabukuro et al. 1998b, McGarry & Dobbins 1999). The latter has sometimes been called ‘lipotoxicity’ and could be taken as a relatively non-specific process arising from a generalised elevation in circulating fatty acid concentrations in vivo. Lipotoxicity can also be observed in vitro ( Shimabukuro et al. 1998b, Cnop et al. 2001, Eitel et al. 2002, Lupi et al. 2002, Piro et al. 2002, Maestre et al. 2003, Welters et al. 2004, Moffitt et al. 2005, Azevedo-Martins et al. 2006) when pancreatic β-cells are incubated in the presence of exogenous fatty acids although it is clear that under these conditions, loss of β-cell viability occurs over a much shorter timescale than that seen in vivo. Indeed, since the combinations of fatty acids used in vitro do not fully recapitulate the situation in vivo, the data must always be interpreted with caution. Nevertheless, it is accepted that in vitro models of lipotoxicity can provide important information about the underlying mechanisms involved ( Newsholme et al. 2007).

In the present work, we have used an in vitro system to demonstrate that saturated and unsaturated fatty acids differ markedly in their ability to regulate β-cell viability. In particular, the results reveal that the long-chain saturated fatty acid palmitate readily promotes β-cell apoptosis, whereas the monounsaturated palmitoleate is potently cytoprotective. Thus, at least under in vitro conditions, the ability to induce β-cell lipotoxicity appears to be a feature that is characteristic mainly of long-chain saturated fatty acids ( Eitel et al. 2002, El-Assaad et al. 2003, Maedler et al. 2003, Welters et al. 2004, 2006).

One area of specific controversy concerns the role played by intracellular TAG in mediating the cytotoxic actions of long-chain fatty acids. Some workers have considered intracellular TAG stores as potential mediators of lipotoxicity ( Shimabukuro et al. 1998a, b) by virtue of their capacity to mediate the physical disruption of cells ( Moffitt et al. 2005). Conversely, others have suggested that TAG represents a reservoir of intracellular lipid into which exogenous FFA can be sequestered as a means to minimise any cytotoxic potential ( Cnop et al. 2001, Listenberger et al. 2003). This issue remains unresolved and, in the present studies, we have attempted to clarify the situation by monitoring β-cell TAG formation in parallel with alterations in viability when cells are exposed to saturated and unsaturated fatty acids either alone or in combination.

As expected, fatty acid treatment caused a net increase in total TAG formation in BRIN-BD11 cells. The magnitude of this increase was similar when cells were exposed to either palmitate or palmitoleate alone (0.25 mM) and, when the two fatty acids were combined, an additive increase in TAG formation was seen. These results confirm that the intracellular TAG pool plays an active role in sequestration of exogenous fatty acids during incubation of BRIN-BD11 β-cells in vitro ( Dixon et al. 2004).

Moffitt et al.(2005) have argued that the species of TAG formed during fatty acid exposure of β-cells may be critical in determining the final outcome in terms of overall cell viability. In particular, they suggest that TAG species that contain predominantly palmitate may be particularly cytotoxic and that the apparent protective actions of monounsaturates could be due to their ability to alter the TAG composition. In order to address this issue, we employed a derivative of palmitoleate which is methylated on the carboxyl group and, as a consequence, cannot be incorporated into TAG. In support of this, incubation of BRIN-BD11 cells with 0.25 mM methyl-palmitoleate failed to generate an increase in TAG and it did not modify the extent of TAG formation seen in palmitate-treated cells. Moreover, this FA failed to attenuate the increase in palmitate incorporation into TAG when the two FAs were added in combination, suggesting that under these conditions, the TAG was still predominantly composed of tripalmitin.

Despite its failure to modify TAG levels in palmitate-treated cells, methyl-palmitoleate completely recapitulated the cytoprotective actions of its parent compound, palmitoleate, and it was equipotent with palmitoleate in this respect. These results suggest very strongly that the ability of the monounsaturates to promote β-cell viability in cells exposed to palmitate does not occur as a direct consequence of altered TAG formation or altered TAG composition. They also imply that the TAG formed in palmitate-treated cells is not directly cytotoxic since methyl-palmitoleate failed to modify TAG but maintained cell viability.

Additional support for these conclusions comes from the results of experiments performed with an inhibitor of CPT-1, etomoxir. CPT-1 catalyses the transit of fatty acids from the cytosol into the mitochondria where they are then available for metabolism by β-oxidation. Inhibition of CPT-1 therefore increases the availability of fatty acids in the cytosol, leading to their diversion away from oxidative metabolism and towards TAG formation. Accordingly, cells exposed to palmitate plus etomoxir contained more TAG than cells exposed to either compound alone. Moreover, since this increase in TAG occurred in response to the addition of exogenous palmitate, it is reasonable to deduce that it was relatively enriched in palmitate (i.e. that it was mainly tripalmitin). Despite this, the overall extent of cell viability was not further decreased under these conditions but, rather, etomoxir attenuated the loss of viability caused by palmitate.

Taken together, the data obtained with palmitoleate, methyl-palmitoleate and etomoxir reveal that changes in the formation of TAG from exogenous palmitate do not correlate with altered viability and they imply that TAG formation from palmitate is not directly cytotoxic under these conditions. Indeed, as proposed by Cnop et al.(2001), the results are not inconsistent with the possibility that TAG formation from palmitate can, under certain circumstances, be directly cytoprotective to β-cells (although this is unlikely to account completely for the protection afforded by monounsaturated fatty acids reported here).

In addition to causing alterations in TAG formation, exposure of β-cells to fatty acids also changed the relative composition of the cellular phospholipid pool, and this occurred in concert with the prevailing fatty acid availability. These observations imply that aspects of cell function that are determined largely by the physical properties of cellular membranes could vary in response to different exogenous fatty acids. Indeed, such effects may well contribute to the differential toxicity of saturated versus unsaturated molecules, perhaps by altering the activity of lipid-dependent enzymes such as phospholipases.

The results obtained in the current study also provide a number of additional insights into the actions of fatty acids in β-cells. Most importantly, they reveal that the effects of monounsaturates are not limited to the blockade of apoptosis but they show that as reported for breast cancer cells ( Hardy et al. 2005), these molecules can also directly promote the growth and proliferation of β-cells. The cytoprotection achieved by palmitoleate during an 18-h incubation period also preserved the insulin secretory responses of the cells.

The finding that the cytoprotective and mitogenic actions of palmitoleate were preserved when the carboxyl group of the fatty acid was methylated implies that these actions were not secondary to increased metabolism of palmitoleate. The present work has not disclosed the molecular mechanisms involved, but it is noteworthy that some actions of fatty acids in β-cells are now attributed to the activation of a subgroup of G-protein-coupled receptors (GPRs; Itoh et al. 2003, Kotarsky et al. 2003, Fujiwara et al. 2005, Hardy et al. 2005, Salehi et al. 2005, Steneberg et al. 2005, Gromada 2006, Schnell et al. 2007). One such molecule, GPR120, has recently been reported to mediate the anti-apoptotic effects of fatty acids in intestinal L-cells ( Katsuma et al. 2005), but it remains to be determined whether GPR120 may be responsible for mediating the actions of monounsaturates observed in the present study.

Figure 1
Figure 1

Mode of β-cell death following exposure to palmitate. BRIN-BD11 cells were treated for 18 h in the absence (control) or presence of 0.1 mM palmitate. The numbers of live (white bars), apoptotic (solid grey bars) and necrotic (cross-hatched bars) cells were estimated by fluorescence microscopy after staining with propidium iodide and Hoechst 33342. *Significantly reduced (P < 0.001) when compared with vehicle-treated cells; **significantly increased (P < 0.001) when compared with vehicle-treated cells.

Citation: Journal of Endocrinology 194, 2; 10.1677/JOE-07-0082

Figure 2
Figure 2

Effects of fatty acids on β-cell proliferation. BRIN-BD11 cells were initially incubated either in the absence of fatty acids (squares) or in the presence of 0.25 mM palmitate alone (triangles), 0.25 mM palmitoleate alone (circles) or the combination of 0.25 mM palmitate plus 0.25 mM palmitoleate (inverted triangles) for 18 h. After this time, the fatty acids were removed, the culture medium replaced and cell growth monitored over the subsequent 72 h. *P < 0.05 when compared with untreated cells; **P < 0.01 when compared with untreated cells.

Citation: Journal of Endocrinology 194, 2; 10.1677/JOE-07-0082

Figure 3
Figure 3

Measurement of DNA synthesis in cells exposed to fatty acids. BRIN-BD11 cells were cultured with vehicle (white bars) 0.25 mM palmitate (solid grey bars), 0.25 mM palmitoleate (hatched bars) or 0.25 mM palmitate plus 0.25 mM palmitoleate (cross-hatched bars) for 18 h. The level of cell proliferation was estimated via the incorporation of BrdU during the incubation period. *P < 0.01 when compared with untreated cells.

Citation: Journal of Endocrinology 194, 2; 10.1677/JOE-07-0082

Figure 4
Figure 4

Insulin secretory responses of BRIN-BD11 cells following exposure to fatty acids. Cells were cultured for 18 h either without fatty acids (UT) or in the presence of 0.25 mM palmitoleate alone (PO) or 0.25 mM palmitoleate plus 0.25 mM palmitate (P+PO). They were then washed and incubated in buffer containing 4 mM glucose alone (control; white bars) or 4 mM glucose plus 10 mM α-ketoisocaproate (KIC; grey bars) for 1 h. After this time, the medium was sampled and insulin was measured by RIA. *P < 0.01 relative to the absence of KIC.

Citation: Journal of Endocrinology 194, 2; 10.1677/JOE-07-0082

Figure 5
Figure 5

Fatty acid species present in untreated BRIN-BD11 cells. BRIN-BD11 cells were grown in complete medium and their total fatty acid composition analysed by gas chromatography after lipid extraction.

Citation: Journal of Endocrinology 194, 2; 10.1677/JOE-07-0082

Figure 6
Figure 6

Fatty acid composition of the phosphatidylcholine fraction of BRIN-BD11 cells following incubation with exogenous fatty acids. BRIN-BD11 cells were treated with vehicle (C) 0.25 mM palmitate (P) 0.25 mM palmitoleate (PO) or 0.25 mM palmitate plus 0.25 mM palmitoleate (P+PO) for 18 h. Phosphatidylcholine (PC) was extracted as a representative phospholipid and quantified by gas chromatography. Results are presented as the percentage of each fatty acid measured within the PC fraction under the relevant experimental conditions.

Citation: Journal of Endocrinology 194, 2; 10.1677/JOE-07-0082

Figure 7
Figure 7

Triglyceride (TG) content (a) and cell viability (b) of fatty acid treated β-cells. BRIN-BD11 cells were treated for 18 h with vehicle (white bars) 0.25 mM palmitoleate (solid grey bars), 0.25 mM 2-bromopalmitate (right-hatched bars) 0.25 mM methyl-palmitoleate (left-hatched bars) or 200 mg/ml etomoxir (cross-hatched bars) in the absence (control) or presence of 0.25 mM palmitate. The cells were then either lysed for analysis of intracellular triglyceride (a) or stained with trypan blue to assess their viability (b). (a)*P < 0.01 when compared with vehicle-treated cells. **P < 0.01 when compared with cells exposed to palmitate alone. ***P < 0.05 when compared with cells exposed to palmitate alone. (b) *P < 0.01 when compared with cells exposed to palmitate alone.

Citation: Journal of Endocrinology 194, 2; 10.1677/JOE-07-0082

Figure 8
Figure 8

Dose-dependent inhibition of palmitate-induced β-cell death by palmitoleate and methyl-palmitoleate. BRIN-BD11 cells were incubated with increasing concentrations of palmitoleate (squares) or methyl-palmitoleate (circles) in the presence of 0.25 mM palmitate for 18 h. The extent of cell death was estimated after staining with trypan blue. Significant inhibition (P < 0.05) of palmitate-induced cytotoxicity was achieved with concentrations of 0.025 mM or greater for both palmitoleate and methyl-palmitoleate.

Citation: Journal of Endocrinology 194, 2; 10.1677/JOE-07-0082

Work in the authors’ laboratories is supported by Diabetes UK, the Wellcome Trust, European Foundation for Study of Diabetes and the GB Sasakawa Foundation, to whom grateful thanks are expressed. Thanks are also due to Dr Peter Eickelmann at Boehringer-Ingelheim Pharma for providing a CASE-PhD studentship to support Eleftheria Diakogiannaki. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

  • Ahren B 2005 Type 2 diabetes, insulin secretion and β cell mass. Current Molecular Medicine 5 275–286.

  • Azevedo-Martins AK, Monteiro AP, Lima CL, Lenzen S & Curi R 2006 Fatty acid-induced toxicity and neutral lipid accumulation in insulin-producing RINm5F cells. Toxicology In Vitro 20 1106–1113.

    • Search Google Scholar
    • Export Citation
  • Bollheimer LC, Skelly RH, Chester MW, McGarry JD & Rhodes CJ 1998 Chronic exposure to free fatty acid reduces pancreatic β cell insulin content by increasing basal insulin secretion that is not compensated for by a corresponding increase in proinsulin biosynthesis translation. Journal of Clinical Investigation 101 1094–1101.

    • Search Google Scholar
    • Export Citation
  • Briaud I, Harmon JS, Kelpe CL, Segu VB & Poitout V 2001 Lipotoxicity of the pancreatic β-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids. Diabetes 50 315–321.

    • Search Google Scholar
    • Export Citation
  • Burdge GC, Wright P, Jones AE & Wootton SA 2000 A method for separation of phosphatidylcholine, triacylglycerol, non-esterified fatty acids and cholesterol esters from plasma by solid-phase extraction. British Journal of Nutrition 84 781–787.

    • Search Google Scholar
    • Export Citation
  • Chase JF & Tubbs PK 1972 Specific inhibition of mitochondrial fatty acid oxidation by 2-bromopalmitate and its coenzyme A and carnitine esters. Biochemical Journal 129 55–65.

    • Search Google Scholar
    • Export Citation
  • Chen S, Ogawa A, Ohneda M, Unger RH, Foster DW & McGarry JD 1994 More direct evidence for a malonyl-CoA-carnitine palmitoyltransferase I interaction as a key event in pancreatic β-cell signaling. Diabetes 43 878–883.

    • Search Google Scholar
    • Export Citation
  • Cnop M, Hannaert JC, Hoorens A, Eizirik DL & Pipeleers DG 2001 Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation. Diabetes 50 1771–1777.

    • Search Google Scholar
    • Export Citation
  • Dixon G, Nolan J, McClenaghan NH, Flatt PR & Newsholme P 2004 Arachidonic acid, palmitic acid and glucose are important for the modulation of clonal pancreatic β-cell insulin secretion, growth and functional integrity. Clinical Science 106 191–199.

    • Search Google Scholar
    • Export Citation
  • Eitel K, Staiger H, Brendel MD, Brandhorst D, Bretzel RG, Haring HU & Kellerer M 2002 Different role of saturated and unsaturated fatty acids in β-cell apoptosis. Biochemical and Biophysical Research Communications 299 853–856.

    • Search Google Scholar
    • Export Citation
  • El-Assaad W, Buteau J, Peyot ML, Nolan C, Roduit R, Hardy S, Joly E, Dbaibo G, Rosenberg L & Prentki M 2003 Saturated fatty acids synergize with elevated glucose to cause pancreatic β-cell death. Endocrinology 144 4154–4163.

    • Search Google Scholar
    • Export Citation
  • Folch J, Lees M & Sloane Stanley GH 1957 A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226 497–509.

    • Search Google Scholar
    • Export Citation
  • Fujiwara K, Maekawa F & Yada T 2005 Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet β-cells: mediation by PLC and L-type Ca2+ channel and link to insulin release. American Journal of Physiology 289 E670–E677.

    • Search Google Scholar
    • Export Citation
  • Gey GO & Gey MK 1936 Maintenance of human normal cells in continuous culture; preliminary report; cultivation of mesoblastic tumors and normal cells and notes on methods of cultivation. American Journal of Cancer 27 45–76.

    • Search Google Scholar
    • Export Citation
  • Gromada J 2006 The free fatty acid receptor GPR40 generates excitement in pancreatic β-cells. Endocrinology 147 672–673.

  • Haber EP, Ximenes HM, Procopio J, Carvalho CR, Curi R & Carpinelli AR 2003 Pleiotropic effects of fatty acids on pancreatic β-cells. Journal of Cellular Physiology 194 1–12.

    • Search Google Scholar
    • Export Citation
  • Haber EP, Procopio J, Carvalho CR, Carpinelli AR, Newsholme P & Curi R 2006 New insights into fatty acid modulation of pancreatic β-cell function. International Review of Cytology 248 1–41.

    • Search Google Scholar
    • Export Citation
  • Hardy S, St-Onge GG, Joly E, Langelier Y & Prentki M 2005 Oleate promotes the proliferation of breast cancer cells via the G-protein-coupled receptor GPR40. Journal of Biological Chemistry 280 13285–13291.

    • Search Google Scholar
    • Export Citation
  • Itoh Y, Kawamata Y, Harada M, Kobayashi M, Fujii R, Fukusumi S, Ogi K, Hosoya M, Tanaka Y, Uejima H et al.2003 Free fatty acids regulate insulin secretion from pancreatic β cells through GPR40. Nature 422 173–176.

    • Search Google Scholar
    • Export Citation
  • Jankowska A, Skonieczna D, Rommerts FF & Warchol JB 1997 Investigation on apoptosis in Leydig cells cultured in vitro. Folia Histochemica et Cytobiologica 35 97–98.

    • Search Google Scholar
    • Export Citation
  • Kahn SE, Hull RL & Utzschneider KM 2006 Mechanisms linking obesity to insulin resistance in type 2 diabetes. Nature 444 840–846.

  • Katsuma S, Hatae N, Yano T, Ruike Y, Kimura M, Hirasawa A & Tsujimoto G 2005 Free fatty acids inhibit serum deprivation-induced apoptosis through GPR120 in a murine enteroendocrine cell line STC-1. Journal of Biological Chemistry 280 19507–19515.

    • Search Google Scholar
    • Export Citation
  • Kotarsky K, Nilsson NE, Olde B & Owman C 2003 Progress in methodology. Improved reporter gene assays used to identify ligands acting on orphan seven-transmembrane receptors. Pharmacology and Toxicology 93 249–258.

    • Search Google Scholar
    • Export Citation
  • Listenberger LL, Han X, Lewis SE, Cases S, Farese RV Jr, Ory DS & Schaffer JE 2003 Triglyceride accumulation protects against fatty acid-induced lipotoxicity. PNAS 100 3077–3082.

    • Search Google Scholar
    • Export Citation
  • Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M, Santangelo C, Patane G, Boggi U, Piro S, Anello M et al.2002 Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that β-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated. Diabetes 51 1437–1442.

    • Search Google Scholar
    • Export Citation
  • Maedler K, Oberholzer J, Bucher P, Spinas GA & Donath MY 2003 Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic β-cell turnover and function. Diabetes 52 726–733.

    • Search Google Scholar
    • Export Citation
  • Maestre I, Jordan J, Calvo S, Reig JA, Cena V, Soria B, Prentki M & Roche E 2003 Mitochondrial dysfunction is involved in apoptosis induced by serum withdrawal and fattyacids in the β-cell line INS-1. Endocrinology 144 335–345.

    • Search Google Scholar
    • Export Citation
  • Marchetti P, Del Prato S, Lupi R & Del Guerra S 2006 The pancreatic β-cell in human Type 2 diabetes. Nutrition, Metabolism, and Cardiovascular Diseases 16 (Suppl 1) S3–S6.

    • Search Google Scholar
    • Export Citation
  • McGarry JD & Dobbins RL 1999 Fatty acids, lipotoxicity and insulin secretion. Diabetologia 42 128–138.

  • Milburn JL Jr, Hirose H, Lee YH, Nagasawa Y, Ogawa A, Ohneda M, BeltrandelRio H, Newgard CB, Johnson JH & Unger RH 1995 Pancreatic β-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids. Journal of Biological Chemistry 270 1295–1299.

    • Search Google Scholar
    • Export Citation
  • Moffitt JH, Fielding BA, Evershed R, Berstan R, Currie JM & Clark A 2005 Adverse physicochemical properties of tripalmitin in β cells lead to morphological changes and lipotoxicity in vitro. Diabetologia 48 1819–1829.

    • Search Google Scholar
    • Export Citation
  • Newsholme P, Keane D, Welters HJ & Morgan NG 2007 Life and death decisions for the pancreatic β-cell – the role of fatty acids. Clinical Science 112 27–42.

    • Search Google Scholar
    • Export Citation
  • Oakes ND, Kjellstedt A, Forsberg GB, Clementz T, Camejo G, Furler SM, Kraegen EW, Olwegard-Halvarsson M, Jenkins AB & Ljung B 1999 Development and initial evaluation of a novel method for assessing tissue-specific plasma free fatty acid utilization in vivo using (R)-2-bromopalmitate tracer. Journal of Lipid Research 40 1155–1169.

    • Search Google Scholar
    • Export Citation
  • Piro S, Anello M, Di Pietro C, Lizzio MN, Patane G, Rabuazzo AM, Vigneri R, Purrello M & Purrello F 2002 Chronic exposure to free fatty acids or high glucose induces apoptosis in rat pancreatic islets: possible role of oxidative stress. Metabolism 51 1340–1347.

    • Search Google Scholar
    • Export Citation
  • Rhodes CJ 2005 Type 2 diabetes – a matter of β-cell life and death? Science 307 380–384.

  • Sako Y & Grill VE 1990 A 48-h lipid infusion in the rat time-dependently inhibits glucose-induced insulin secretion and B cell oxidation through a process likely coupled to fatty acid oxidation. Endocrinology 127 1580–1589.

    • Search Google Scholar
    • Export Citation
  • Salehi A, Flodgren E, Nilsson NE, Jimenez-Feltstrom J, Miyazaki J, Owman C & Olde B 2005 Free fatty acid receptor 1 (FFA(1)R/GPR40) and its involvement in fatty-acid-stimulated insulin secretion. Cell and Tissue Research 322 207–215.

    • Search Google Scholar
    • Export Citation
  • Schnell S, Schaefer M & Schofl C 2007 Free fatty acids increase cytosolic free calcium and stimulate insulin secretion from β-cells through activation of GPR40. Molecular and Cellular Endocrinology 263 173–180.

    • Search Google Scholar
    • Export Citation
  • Shimabukuro M, Wang MY, Zhou YT, Newgard CB & Unger RH 1998a Protection against lipoapoptosis of β cells through leptin-dependent maintenance of Bcl-2 expression. PNAS 95 9558–9561.

    • Search Google Scholar
    • Export Citation
  • Shimabukuro M, Zhou YT, Levi M & Unger RH 1998b Fatty acid-induced β cell apoptosis: a link between obesity and diabetes. PNAS 95 2498–2502.

    • Search Google Scholar
    • Export Citation
  • Steneberg P, Rubins N, Bartoov-Shifman R, Walker MD & Edlund H 2005 The FFA receptor GPR40 links hyperinsulinemia, hepatic steatosis, and impaired glucose homeostasis in mouse. Cell Metabolism 1 245–258.

    • Search Google Scholar
    • Export Citation
  • Unger RH 1995 Lipotoxicity in the pathogenesis of obesity-dependent NIDDM. Genetic and clinical implications. Diabetes 44 863–870.

  • Unger RH & Orci L 2002 Lipoapoptosis: its mechanism and its diseases. Biochimica et Biophysica Acta 1585 202–212.

  • Unger RH & Zhou YT 2001 Lipotoxicity of β-cells in obesity and in other causes of fatty acid spillover. Diabetes 50 (Suppl 1) S118–S121.

    • Search Google Scholar
    • Export Citation
  • Welters HJ, Tadayyon M, Scarpello JH, Smith SA & Morgan NG 2004 Monounsaturated fatty acids protect against β-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure. FEBS Letters 560 103–108.

    • Search Google Scholar
    • Export Citation
  • Welters HJ, Diakogiannaki E, Mordue JM, Tadayyon M, Smith SA & Morgan NG 2006 Differential protective effects of palmitoleic acid and cAMP on caspase activation and cell viability in pancreatic β-cells exposed to palmitate. Apoptosis 11 1231–1238.

    • Search Google Scholar
    • Export Citation
  • Zhou YP & Grill VE 1994 Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle. Journal of Clinical Investigation 93 870–876.

    • Search Google Scholar
    • Export Citation

 

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    Mode of β-cell death following exposure to palmitate. BRIN-BD11 cells were treated for 18 h in the absence (control) or presence of 0.1 mM palmitate. The numbers of live (white bars), apoptotic (solid grey bars) and necrotic (cross-hatched bars) cells were estimated by fluorescence microscopy after staining with propidium iodide and Hoechst 33342. *Significantly reduced (P < 0.001) when compared with vehicle-treated cells; **significantly increased (P < 0.001) when compared with vehicle-treated cells.

  • View in gallery

    Effects of fatty acids on β-cell proliferation. BRIN-BD11 cells were initially incubated either in the absence of fatty acids (squares) or in the presence of 0.25 mM palmitate alone (triangles), 0.25 mM palmitoleate alone (circles) or the combination of 0.25 mM palmitate plus 0.25 mM palmitoleate (inverted triangles) for 18 h. After this time, the fatty acids were removed, the culture medium replaced and cell growth monitored over the subsequent 72 h. *P < 0.05 when compared with untreated cells; **P < 0.01 when compared with untreated cells.

  • View in gallery

    Measurement of DNA synthesis in cells exposed to fatty acids. BRIN-BD11 cells were cultured with vehicle (white bars) 0.25 mM palmitate (solid grey bars), 0.25 mM palmitoleate (hatched bars) or 0.25 mM palmitate plus 0.25 mM palmitoleate (cross-hatched bars) for 18 h. The level of cell proliferation was estimated via the incorporation of BrdU during the incubation period. *P < 0.01 when compared with untreated cells.

  • View in gallery

    Insulin secretory responses of BRIN-BD11 cells following exposure to fatty acids. Cells were cultured for 18 h either without fatty acids (UT) or in the presence of 0.25 mM palmitoleate alone (PO) or 0.25 mM palmitoleate plus 0.25 mM palmitate (P+PO). They were then washed and incubated in buffer containing 4 mM glucose alone (control; white bars) or 4 mM glucose plus 10 mM α-ketoisocaproate (KIC; grey bars) for 1 h. After this time, the medium was sampled and insulin was measured by RIA. *P < 0.01 relative to the absence of KIC.

  • View in gallery

    Fatty acid species present in untreated BRIN-BD11 cells. BRIN-BD11 cells were grown in complete medium and their total fatty acid composition analysed by gas chromatography after lipid extraction.

  • View in gallery

    Fatty acid composition of the phosphatidylcholine fraction of BRIN-BD11 cells following incubation with exogenous fatty acids. BRIN-BD11 cells were treated with vehicle (C) 0.25 mM palmitate (P) 0.25 mM palmitoleate (PO) or 0.25 mM palmitate plus 0.25 mM palmitoleate (P+PO) for 18 h. Phosphatidylcholine (PC) was extracted as a representative phospholipid and quantified by gas chromatography. Results are presented as the percentage of each fatty acid measured within the PC fraction under the relevant experimental conditions.

  • View in gallery

    Triglyceride (TG) content (a) and cell viability (b) of fatty acid treated β-cells. BRIN-BD11 cells were treated for 18 h with vehicle (white bars) 0.25 mM palmitoleate (solid grey bars), 0.25 mM 2-bromopalmitate (right-hatched bars) 0.25 mM methyl-palmitoleate (left-hatched bars) or 200 mg/ml etomoxir (cross-hatched bars) in the absence (control) or presence of 0.25 mM palmitate. The cells were then either lysed for analysis of intracellular triglyceride (a) or stained with trypan blue to assess their viability (b). (a)*P < 0.01 when compared with vehicle-treated cells. **P < 0.01 when compared with cells exposed to palmitate alone. ***P < 0.05 when compared with cells exposed to palmitate alone. (b) *P < 0.01 when compared with cells exposed to palmitate alone.

  • View in gallery

    Dose-dependent inhibition of palmitate-induced β-cell death by palmitoleate and methyl-palmitoleate. BRIN-BD11 cells were incubated with increasing concentrations of palmitoleate (squares) or methyl-palmitoleate (circles) in the presence of 0.25 mM palmitate for 18 h. The extent of cell death was estimated after staining with trypan blue. Significant inhibition (P < 0.05) of palmitate-induced cytotoxicity was achieved with concentrations of 0.025 mM or greater for both palmitoleate and methyl-palmitoleate.

  • Ahren B 2005 Type 2 diabetes, insulin secretion and β cell mass. Current Molecular Medicine 5 275–286.

  • Azevedo-Martins AK, Monteiro AP, Lima CL, Lenzen S & Curi R 2006 Fatty acid-induced toxicity and neutral lipid accumulation in insulin-producing RINm5F cells. Toxicology In Vitro 20 1106–1113.

    • Search Google Scholar
    • Export Citation
  • Bollheimer LC, Skelly RH, Chester MW, McGarry JD & Rhodes CJ 1998 Chronic exposure to free fatty acid reduces pancreatic β cell insulin content by increasing basal insulin secretion that is not compensated for by a corresponding increase in proinsulin biosynthesis translation. Journal of Clinical Investigation 101 1094–1101.

    • Search Google Scholar
    • Export Citation
  • Briaud I, Harmon JS, Kelpe CL, Segu VB & Poitout V 2001 Lipotoxicity of the pancreatic β-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids. Diabetes 50 315–321.

    • Search Google Scholar
    • Export Citation
  • Burdge GC, Wright P, Jones AE & Wootton SA 2000 A method for separation of phosphatidylcholine, triacylglycerol, non-esterified fatty acids and cholesterol esters from plasma by solid-phase extraction. British Journal of Nutrition 84 781–787.

    • Search Google Scholar
    • Export Citation
  • Chase JF & Tubbs PK 1972 Specific inhibition of mitochondrial fatty acid oxidation by 2-bromopalmitate and its coenzyme A and carnitine esters. Biochemical Journal 129 55–65.

    • Search Google Scholar
    • Export Citation
  • Chen S, Ogawa A, Ohneda M, Unger RH, Foster DW & McGarry JD 1994 More direct evidence for a malonyl-CoA-carnitine palmitoyltransferase I interaction as a key event in pancreatic β-cell signaling. Diabetes 43 878–883.

    • Search Google Scholar
    • Export Citation
  • Cnop M, Hannaert JC, Hoorens A, Eizirik DL & Pipeleers DG 2001 Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation. Diabetes 50 1771–1777.

    • Search Google Scholar
    • Export Citation
  • Dixon G, Nolan J, McClenaghan NH, Flatt PR & Newsholme P 2004 Arachidonic acid, palmitic acid and glucose are important for the modulation of clonal pancreatic β-cell insulin secretion, growth and functional integrity. Clinical Science 106 191–199.

    • Search Google Scholar
    • Export Citation
  • Eitel K, Staiger H, Brendel MD, Brandhorst D, Bretzel RG, Haring HU & Kellerer M 2002 Different role of saturated and unsaturated fatty acids in β-cell apoptosis. Biochemical and Biophysical Research Communications 299 853–856.

    • Search Google Scholar
    • Export Citation
  • El-Assaad W, Buteau J, Peyot ML, Nolan C, Roduit R, Hardy S, Joly E, Dbaibo G, Rosenberg L & Prentki M 2003 Saturated fatty acids synergize with elevated glucose to cause pancreatic β-cell death. Endocrinology 144 4154–4163.

    • Search Google Scholar
    • Export Citation
  • Folch J, Lees M & Sloane Stanley GH 1957 A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226 497–509.

    • Search Google Scholar
    • Export Citation
  • Fujiwara K, Maekawa F & Yada T 2005 Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet β-cells: mediation by PLC and L-type Ca2+ channel and link to insulin release. American Journal of Physiology 289 E670–E677.

    • Search Google Scholar
    • Export Citation
  • Gey GO & Gey MK 1936 Maintenance of human normal cells in continuous culture; preliminary report; cultivation of mesoblastic tumors and normal cells and notes on methods of cultivation. American Journal of Cancer 27 45–76.

    • Search Google Scholar
    • Export Citation
  • Gromada J 2006 The free fatty acid receptor GPR40 generates excitement in pancreatic β-cells. Endocrinology 147 672–673.

  • Haber EP, Ximenes HM, Procopio J, Carvalho CR, Curi R & Carpinelli AR 2003 Pleiotropic effects of fatty acids on pancreatic β-cells. Journal of Cellular Physiology 194 1–12.

    • Search Google Scholar
    • Export Citation
  • Haber EP, Procopio J, Carvalho CR, Carpinelli AR, Newsholme P & Curi R 2006 New insights into fatty acid modulation of pancreatic β-cell function. International Review of Cytology 248 1–41.

    • Search Google Scholar
    • Export Citation
  • Hardy S, St-Onge GG, Joly E, Langelier Y & Prentki M 2005 Oleate promotes the proliferation of breast cancer cells via the G-protein-coupled receptor GPR40. Journal of Biological Chemistry 280 13285–13291.

    • Search Google Scholar
    • Export Citation
  • Itoh Y, Kawamata Y, Harada M, Kobayashi M, Fujii R, Fukusumi S, Ogi K, Hosoya M, Tanaka Y, Uejima H et al.2003 Free fatty acids regulate insulin secretion from pancreatic β cells through GPR40. Nature 422 173–176.

    • Search Google Scholar
    • Export Citation
  • Jankowska A, Skonieczna D, Rommerts FF & Warchol JB 1997 Investigation on apoptosis in Leydig cells cultured in vitro. Folia Histochemica et Cytobiologica 35 97–98.

    • Search Google Scholar
    • Export Citation
  • Kahn SE, Hull RL & Utzschneider KM 2006 Mechanisms linking obesity to insulin resistance in type 2 diabetes. Nature 444 840–846.

  • Katsuma S, Hatae N, Yano T, Ruike Y, Kimura M, Hirasawa A & Tsujimoto G 2005 Free fatty acids inhibit serum deprivation-induced apoptosis through GPR120 in a murine enteroendocrine cell line STC-1. Journal of Biological Chemistry 280 19507–19515.

    • Search Google Scholar
    • Export Citation
  • Kotarsky K, Nilsson NE, Olde B & Owman C 2003 Progress in methodology. Improved reporter gene assays used to identify ligands acting on orphan seven-transmembrane receptors. Pharmacology and Toxicology 93 249–258.

    • Search Google Scholar
    • Export Citation
  • Listenberger LL, Han X, Lewis SE, Cases S, Farese RV Jr, Ory DS & Schaffer JE 2003 Triglyceride accumulation protects against fatty acid-induced lipotoxicity. PNAS 100 3077–3082.

    • Search Google Scholar
    • Export Citation
  • Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M, Santangelo C, Patane G, Boggi U, Piro S, Anello M et al.2002 Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that β-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated. Diabetes 51 1437–1442.

    • Search Google Scholar
    • Export Citation
  • Maedler K, Oberholzer J, Bucher P, Spinas GA & Donath MY 2003 Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic β-cell turnover and function. Diabetes 52 726–733.

    • Search Google Scholar
    • Export Citation
  • Maestre I, Jordan J, Calvo S, Reig JA, Cena V, Soria B, Prentki M & Roche E 2003 Mitochondrial dysfunction is involved in apoptosis induced by serum withdrawal and fattyacids in the β-cell line INS-1. Endocrinology 144 335–345.

    • Search Google Scholar
    • Export Citation
  • Marchetti P, Del Prato S, Lupi R & Del Guerra S 2006 The pancreatic β-cell in human Type 2 diabetes. Nutrition, Metabolism, and Cardiovascular Diseases 16 (Suppl 1) S3–S6.

    • Search Google Scholar
    • Export Citation
  • McGarry JD & Dobbins RL 1999 Fatty acids, lipotoxicity and insulin secretion. Diabetologia 42 128–138.

  • Milburn JL Jr, Hirose H, Lee YH, Nagasawa Y, Ogawa A, Ohneda M, BeltrandelRio H, Newgard CB, Johnson JH & Unger RH 1995 Pancreatic β-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids. Journal of Biological Chemistry 270 1295–1299.

    • Search Google Scholar
    • Export Citation
  • Moffitt JH, Fielding BA, Evershed R, Berstan R, Currie JM & Clark A 2005 Adverse physicochemical properties of tripalmitin in β cells lead to morphological changes and lipotoxicity in vitro. Diabetologia 48 1819–1829.

    • Search Google Scholar
    • Export Citation
  • Newsholme P, Keane D, Welters HJ & Morgan NG 2007 Life and death decisions for the pancreatic β-cell – the role of fatty acids. Clinical Science 112 27–42.

    • Search Google Scholar
    • Export Citation
  • Oakes ND, Kjellstedt A, Forsberg GB, Clementz T, Camejo G, Furler SM, Kraegen EW, Olwegard-Halvarsson M, Jenkins AB & Ljung B 1999 Development and initial evaluation of a novel method for assessing tissue-specific plasma free fatty acid utilization in vivo using (R)-2-bromopalmitate tracer. Journal of Lipid Research 40 1155–1169.

    • Search Google Scholar
    • Export Citation
  • Piro S, Anello M, Di Pietro C, Lizzio MN, Patane G, Rabuazzo AM, Vigneri R, Purrello M & Purrello F 2002 Chronic exposure to free fatty acids or high glucose induces apoptosis in rat pancreatic islets: possible role of oxidative stress. Metabolism 51 1340–1347.

    • Search Google Scholar
    • Export Citation
  • Rhodes CJ 2005 Type 2 diabetes – a matter of β-cell life and death? Science 307 380–384.

  • Sako Y & Grill VE 1990 A 48-h lipid infusion in the rat time-dependently inhibits glucose-induced insulin secretion and B cell oxidation through a process likely coupled to fatty acid oxidation. Endocrinology 127 1580–1589.

    • Search Google Scholar
    • Export Citation
  • Salehi A, Flodgren E, Nilsson NE, Jimenez-Feltstrom J, Miyazaki J, Owman C & Olde B 2005 Free fatty acid receptor 1 (FFA(1)R/GPR40) and its involvement in fatty-acid-stimulated insulin secretion. Cell and Tissue Research 322 207–215.

    • Search Google Scholar
    • Export Citation
  • Schnell S, Schaefer M & Schofl C 2007 Free fatty acids increase cytosolic free calcium and stimulate insulin secretion from β-cells through activation of GPR40. Molecular and Cellular Endocrinology 263 173–180.

    • Search Google Scholar
    • Export Citation
  • Shimabukuro M, Wang MY, Zhou YT, Newgard CB & Unger RH 1998a Protection against lipoapoptosis of β cells through leptin-dependent maintenance of Bcl-2 expression. PNAS 95 9558–9561.

    • Search Google Scholar
    • Export Citation
  • Shimabukuro M, Zhou YT, Levi M & Unger RH 1998b Fatty acid-induced β cell apoptosis: a link between obesity and diabetes. PNAS 95 2498–2502.

    • Search Google Scholar
    • Export Citation
  • Steneberg P, Rubins N, Bartoov-Shifman R, Walker MD & Edlund H 2005 The FFA receptor GPR40 links hyperinsulinemia, hepatic steatosis, and impaired glucose homeostasis in mouse. Cell Metabolism 1 245–258.

    • Search Google Scholar
    • Export Citation
  • Unger RH 1995 Lipotoxicity in the pathogenesis of obesity-dependent NIDDM. Genetic and clinical implications. Diabetes 44 863–870.

  • Unger RH & Orci L 2002 Lipoapoptosis: its mechanism and its diseases. Biochimica et Biophysica Acta 1585 202–212.

  • Unger RH & Zhou YT 2001 Lipotoxicity of β-cells in obesity and in other causes of fatty acid spillover. Diabetes 50 (Suppl 1) S118–S121.

    • Search Google Scholar
    • Export Citation
  • Welters HJ, Tadayyon M, Scarpello JH, Smith SA & Morgan NG 2004 Monounsaturated fatty acids protect against β-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure. FEBS Letters 560 103–108.

    • Search Google Scholar
    • Export Citation
  • Welters HJ, Diakogiannaki E, Mordue JM, Tadayyon M, Smith SA & Morgan NG 2006 Differential protective effects of palmitoleic acid and cAMP on caspase activation and cell viability in pancreatic β-cells exposed to palmitate. Apoptosis 11 1231–1238.

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    • Export Citation
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