Dietary virgin olive oil enhances secretagogue-evoked calcium signaling in rat pancreatic acinar cells

APPLIED NUTRITIONAL INVESTIGATION

Dietary Virgin Olive Oil Enhances Secretagogue-Evoked Calcium Signaling in Rat Pancreatic Acinar Cells Marı´a A. Martı´nez, PhD, Ana I. Lajas, PhD, Marı´a D. Yago, PhD, Pedro C. Redondo, BSc, Marı´a P. Granados, BSc, Antonio Gonza´lez, PhD, Juan A. Rosado, PhD, Emilio Martı´nez-Victoria, PhD, Mariano Man˜as, PhD, and Jose´ A. Pariente, PhD From the Institute of Nutrition and Food Technology, Department of Physiology, University of Granada, Granada, Spain; and the Department of Physiology, Faculty of Veterinary Sciences, University of Extremadura, Ca´ceres, Spain OBJECTIVE: We evaluated the long-term effects of a fat-enriched diet (virgin olive oil) on calcium mobilization and amylase secretion induced by cholecystokinin-octapeptide (CCK-8) in rat pancreatic acinar cells. Olive oil is a major component of the Mediterranean diet, and its role in human health is actively being debated. METHODS: Weaning male Wistar rats (21 d old) were assigned to one of two experimental groups and fed for 8 wk with a commercial chow (control group) or an experimental diet (olive group) containing 100 g/kg of virgin olive oil as dietary fat. Intracellular free calcium [Ca2⫹]i levels were determined by loading the pancreatic cells with the fluorescent ratio-metric calcium indicator Fura-2 on an inverted fluorescent microscope. For measurement of amylase secretion, cells were incubated with the appropriate secretagogue for 30 min, and amylase activities in the supernatant were determined by the Phadebas blue starch method. Analysis of variance was used to test differences between groups. RESULTS: Compared with the control group, the CCK-8 –induced increase in [Ca2⫹]i occurred in cells from rats in the olive group (P ⬍ 0.05). This stimulatory effect of dietary virgin olive oil was observed in calcium oscillations and large [Ca2⫹]i transients induced by low (20 pM/L) and high (10 nM/L) concentrations of CCK-8, respectively. In addition to the effects of dietary virgin olive oil on calcium mobilization, it increased (P ⬍ 0.05) amylase secretion in response to CCK-8. Olive oil treatment did not significantly alter resting [Ca2⫹]i or amylase release values compared with the control group. Similar results were obtained when pancreatic acinar cells were stimulated with a high concentration of acetylcholine (10 ␮M/L). CONCLUSION: The present results demonstrate that a diet supplemented with virgin olive oil can modify pancreatic cell function as assessed by [Ca2⫹]i mobilization and amylase release evoked by secretagogues in rat pancreatic acinar cells. A role for fatty acids in calcium signaling is suggested. Nutrition 2004;20: 536 –541. ©Elsevier Inc. 2004 KEY WORDS: dietary fat, virgin olive oil, pancreatic acinar cell, calcium signaling, amylase release

INTRODUCTION Calcium is an ubiquitous second messenger involved in a variety of physiologic functions such as muscle contraction, metabolism, secretion, and even cell differentiation and apoptosis. The main function of pancreatic acinar cells is to synthesize, store, and then undergo regulated secretion of digestive enzymes. The pancreatic secretagogue cholecystokinin-octapeptide (CCK-8) activates, by a guanine nucleotide-binding protein– dependent mechanism, the

This study was supported by the Ministerio de Ciencia y Tecnologı´a (grants BFI 2001-0624 and BFI 2002-02772). M. A. Martı´nez and M. D. Yago received a postdoctoral fellowship and a research contract from the University of Granada. P. C. Redondo and M. P. Granados are supported by a DGI fellowship (BFI2001-0624) and a Junta de Extremadura fellowship, respectively. Correspondence to: Jose´ A. Pariente, PhD, Department of Physiology, Faculty of Veterinary Sciences, University of Extremadura, PO Box 643, 10071 Ca´ceres, Spain. E-mail: [email protected] Nutrition 20:536 –541, 2004 ©Elsevier Inc., 2004. Printed in the United States. All rights reserved.

plasma membrane phospholipase C, which generates inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. IP3 in turn releases calcium from intracellular pools, thereby initiating the calcium signal. The release of calcium from intracellular pools also activates calcium entry across the plasma membrane, an influx that is necessary for the maintenance of the calcium signal and for enzyme secretion. In addition, diacylglycerol activates several isoforms of protein kinase C, an enzyme involved in the control of exocytosis mechanisms.1 Intracellular calcium mobilization also can be induced by exogenous free fatty acids such as arachidonic acid, oleic acid, and linoleic acid.2– 6 Most studies on intracellular calcium have been carried out in various cell lines such as Jurkat T lymphocytes,2– 4 thyroid follicular rat thyroid line-5 (FRTL-5) cells,5 and fibroblasts epidermal growth factor receptor (EGFR)-T17.6 Moreover, these studies have demonstrated that short acute exposure to free fatty acids has an acute effect on calcium signal. However, only a few studies have examined the long-term effect of fatty acids on calcium signaling. A recent study by Peck et al.7 showed that olive oil in diets increase intracellular free calcium ([Ca2⫹]i) stimulated 0899-9007/04/$30.00 doi:10.1016/j.nut.2004.03.018

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TABLE I. FATTY ACID COMPOSITION OF THE CHOW AND EXPERIMENTAL DIETS* Fatty acid

Standard chow

Virgin olive oil

16:0 16:1 (␻-7) 18:0 18:1 (␻-9) 18:2 (␻-6) 18:3 (␻-3) SFA MUFA PUFA

15.45 1.16 4.15 21.59 45.67 3.98 19.6 22.75 49.75

11.44 0.85 4.38 74.88 7.72 0.62 15.82 75.84 8.34

* Data are expressed as the mean proportion (g/100 g) of total fatty acid methyl esters for four replicates. MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid.

537

the University of Granada (Granada, Spain), packed in plastic bags, sealed, and stored at 4°C in the dark. During the 8-wk adaptation period to the diets, animals were housed individually in a temperature-controlled room (22 ⫾ 1°C) and kept on a 12-h light-and-dark cycle with free access to water and food. During the feeding period, all rats received fresh food daily. Feeding diets differing in the fat source did not affect daily food intake. The body weight gain was greater in rats fed with the 10% fat diet (data not shown). The protocols were approved by ethical committee of the University of Extremadura. At the end of the study, rats were killed according to the guidelines of the Spanish Society for Laboratory Animal Sciences for the care and use of laboratory animals, and pancreatic acinar cell suspensions were obtained. Materials Chemicals were purchased from Sigma (Madrid, Spain) except collagenase CLSPA, which was obtained from Worthington Biochemical Corporation (Freehold, NJ, USA), and Fura-2 AM, which was purchased from Molecular Probes Europe (Leiden, Netherlands). Preparation of Pancreatic Acinar Cells

by agonists in lymphocytes from mice fed with the olive oil diet for 2 wk. In contrast, it has been accepted for many years that the type of dietary fat influences the exocrine function of the pancreas. The approach most frequently used to tackle this task has been the analysis of the enzyme content of the pancreas after feeding animals with different dietary lipids.8,9 The exact mechanisms by which this adaptive effect is brought about are not clear. Moreover, the enzyme content does not necessarily reflect the secretory activity of the organ. It has been shown in vivo that pancreatic response to endogenous or exogenous stimulation after mediumor long-term intake of diets differ by the type of fat source.10,11 In addition, it is well known that the release of the gastrointestinal hormone CCK from specialized enteroendocrine cells is stimulated by dietary lipids through intraduodenal free fatty acids,12 which is accompanied by an increase in [Ca2⫹]i.13 We evaluated the long-term effects of an oil-enriched diet (virgin olive oil) on calcium mobilization and amylase secretion induced by CCK-8 in rat pancreatic acinar cells. Olive oil was chosen because of its preferential use in our geographic area. Further, olive oil is a major component of the Mediterranean diet, and its role in human health is being actively debated.

MATERIALS AND METHODS Animals and Diets Weaning male Wistar rats (40 to 55 g) were used throughout this study and obtained from the Animal Farm of the Faculty of Veterinary Medicine, University of Extremadura (Ca´ ceres, Spain). Animals were randomly assigned to one of two treatment groups. In the control group (n ⫽ 24), rats were fed for 8 wk with a standard non-purified diet (A04, Panlab Laboratories, Barcelona, Spain). The olive group (n ⫽ 24) was fed over the same 8-wk period with a semipurified diet that was essentially the AIN-93G diet14 except that total fat content was increased from 70 to 100 g/kg at the expense of carbohydrate. The composition of the diet (grams per kilogram) was as follows: casein, 200; cornstarch, 367.5; dextrose, 132; sucrose, 100; cellulose, 50; fat, 100; L-cystine, 3; choline bitartrate, 2.5; AIN-93G mineral mixture, 35; and AIN-93G vitamin mixture, 10. The fat source was virgin olive oil (Fedeoliva SA, Jae´ n, Spain). The fatty acid composition of the experimental and control diets was determined by gas chromatography (Table I). The diets were prepared at the Nutrition Unit of

A suspension of single cells and small acini was prepared from rat pancreas by previously described methods.15 Briefly, overnightfasted Wistar rats were killed by severance of the vertebral column. The abdomen was excised and the pancreas was rapidly removed and placed in Na-HEPES solution containing 1.0 mL/L of an essential amino acid mixture, 0.1 mg/mL of soybean trypsin inhibitor, and (in millimoles per liter) 140 NaCl, 4.7 KCl, 2 CaCl2, 1.1 MgCl2, 10 glucose, and 10 HEPES. The pH was adjusted to 7.4 and equilibrated with 100% oxygen. The pancreas was injected through the connective tissue with 13 mL of Na-HEPES solution containing 0.2% (w/v) bovine serum albumin and collagenase (40 U/mL). The pancreas was oxygenated and digested at 37°C in a shaking bath (200 cycles/min) for 20 min and washed with fresh collagenase solution every 5 min. The incubation was followed by vigorous manual agitation to disintegrate the pancreas into acini. The dispersed acini were placed in 0.2% bovine serum albumin in Na-HEPES solution and centrifuged (1000 rpm for 3 min at 4°C). The supernatant was discarded and the pellet was resuspended in 0.2% bovine serum albumin in Na-HEPES solution. After gentle pipetting through tips of decreasing diameter (from 3 to 1 mm), cells were filtered through a double layer of muslin gauze and centrifuged (1000 rpm for 2 min at 4°C). The supernatant was removed and the pellet was resuspended in Na-HEPES solution. Cell viability, monitored with trypan blue, was always greater than 95% and was not significantly decreased by secretagogues. Dye Loading and [Ca2ⴙ]i Determination After isolation, cells were suspended in physiologic solution (same composition as before) and loaded with the fluorescent ratio-metric calcium indicator Fura-2 by incubation with 4 ␮M/L Fura-2 AM in the presence of 0.025% Pluronic acid at room temperature for 30 min. Once loaded, cells were washed and resuspended in fresh physiologic salt solution and used within the next 2 to 4 h. Cells were kept on ice until use. For quantification of fluorescence, small aliquots of cell suspension were placed on a thin glass coverslip attached to a Perspex perfusion chamber. Perfusion (approximately 1.5 mL/min) at room temperature was started after 5 min to allow spontaneous attachment of the cell to the coverslip. No coating treatment was necessary to immobilize the cells. The chamber was placed on the stage of an inverted fluorescence microscope (Nikon Diaphot 200, Kawasaki, Japan). Cells were alternatively excited at 340 and 380

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FIG. 1. Mobilization of calcium in response to secretagogues in pancreatic acinar cells (control group). Cells were perfused with 10 nM/L of CCK-8 (A), 20 pM/L of CCK-8 (B), or 10 ␮M/L of ACh (C). Traces represent 100 cells taken from five to nine animals (pancreata). ACh, acetylcholine; [Ca2⫹]i, intracellular free calcium; CCK-8, cholecystokinin-octapeptide.

nm by a computer-controlled filter wheel (Lambda-2, Sutter Instruments, California, USA), and the emitted images (⬎515 nm) were captured by a high-speed cooled digital CCD camera (C-

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FIG. 2. Mobilization of calcium in response to secretagogues in pancreatic acinar cells (olive group). Cells were perfused with 10 nM/L of CCK-8 (A), 20 pM/L of CCK-8 (B), or 10 ␮M/L of ACh (C). Traces represent 120 cells taken from 6 to 10 animals (pancreata). ACh, acetylcholine; [Ca2⫹]i, intracellular free calcium; CCK-8, cholecystokinin-octapeptide.

4880-81, Hamamatsu Photonics, Hamamatsu, Japan) and recorded with dedicated software (Argus-HiSCa, Hamamatsu Photonics). [Ca2⫹]i values were calculated after calibration according to a standard method.16

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TABLE II. INTRACELLULAR FREE CALCIUM LEVELS IN PANCREATIC ACINAR CELLS FROM THE CONTROL AND OLIVE GROUPS*

Group

Secretagogue

⌬[Ca2⫹]i (nM/L)†

n Cells

Control

CCK-8, 10 nM/L CCK-8, 20 pM/L ACh, 10 ␮M/L CCK-8, 10 nM/L CCK-8, 20 pM/L ACh, 10 ␮M/L

574.7 ⫾ 17.7

31

463.2 ⫾ 30.7 932.6 ⫾ 37.2‡

41 38

822.7 ⫾ 59.6‡

26

Olive

Amplitude (nM)

Frequency (cycles/min)

n Cells

301.9 ⫾ 33.5

0.56 ⫾ 0.03

29

699.9 ⫾ 35.9‡

0.49 ⫾ 0.02‡

57

* Data are presented as mean ⫾ standard error of the mean. † ⌬[Ca2⫹]i ⫽ peak value ⫺ basal value. ‡ P ⬍ 0.05 versus control group. ACh, acetylcholine; [Ca2⫹]i, intracellular free calcium; CCK-8, cholecystokinin-octapeptide.

Measurement of Amylase Release For measurement of amylase secretion, aliquots (500 ␮L) of fresh acinar cells were incubated with an appropriate secretagogue at 37°C for 30 min followed by centrifugation at 1000g for 30 s. Amylase release was measured as described previously.17 Amylase activities in the supernatant were determined with the Phadebas blue starch method18 and expressed as a percentage of the total content of amylase at the beginning of the incubation. Statistical Analysis Values are expressed as mean ⫾ standard error of the mean. Statistical significance was calculated by one-way analysis of variance. P ⬍ 0.05 was considered statistically significant.

RESULTS The basal [Ca2⫹]i value in pancreatic acinar cells from rats fed for 8 wk with the standard laboratory diet averaged 143.8 ⫾ 6.5 nM/L (n ⫽ 101 cells). Olive oil treatment did not significantly modify the resting [Ca2⫹]i (133.1 ⫾ 3.8 nM/L, n ⫽ 121 cells) compared with the control group. As expected, perfusion of cells with a high concentration of the calcium-mobilizing secretagogue CCK-8 (10 nM/L) induced an initial large transient increase in [Ca2⫹]i in the two groups (Figures 1A and 2A). After 6 to 8 min, [Ca2⫹]i had decreased to a sustained [Ca2⫹]i plateau and was maintained as long as the stimulus was applied. However, in the control group, the [Ca2⫹]i increase (⌬[Ca2⫹]i ⫽ peak ⫺ basal) in response to CCK-8 was significantly smaller when compared with olive group (574.7 ⫾ 17.7 nM/L versus 932.6 ⫾ 37.2 nM/L, P ⬍ 0.05; Table II). This increase was observed in all 38 cells studied and was estimated to be 63%. Similar results were obtained when the cells were stimulated with a high concentration of acetylcholine (ACh; 10 ␮M/L; 463.2 ⫾ 30.7 nM/L versus 822.7 ⫾ 59.6 nM/L, P ⬍ 0.05; Figures 1C and 2C, Table II). This increase averaged 77%. Stimulation of pancreatic acinar with low (physiologic) concentrations of CCK-8 (20 pM/L) generated an oscillating [Ca2⫹]i signal.19 The time-course changes in [Ca2⫹]i evoked by 20 pM/L CCK-8 is shown in Figures 1B and 2B. In the control group, [Ca2⫹]i increased from basal value to a peak (amplitude) averaging 301.9 ⫾ 33.5 nM/L (n ⫽ 29 cells), and oscillation frequency resulted in 0.56 ⫾ 0.03 cycles/min (Table II). This pattern of calcium oscillation was increased by the olive oil diet. In these cells, perfusion of 20 pM/L CCK-8 resulted in oscillations of [Ca2⫹]i with an amplitude of 699.9 ⫾ 35.9 nM/L and a mean

frequency of 0.49 ⫾ 0.02 cycles/min; parameters differed significantly (P ⬍ 0.05) from those observed in control cells (Table II). Because the function of pancreatic acinar cells is exocytosis of digestive enzymes, it was relevant to examine the effect of dietary fat on secretagogue-stimulated amylase secretion. CCK-8 stimulation induces a biphasic increase in amylase release.20 The effect of stimulation of pancreatic acinar cells with CCK-8 on amylase secretion in the control and olive groups are shown in Figure 3. Under our conditions, stimulation of pancreatic acinar cells from the control group with CCK-8 induced a dose-dependent amylase release, reaching a maximum at 0.1 nM/L of 16.4 ⫾ 2.9% of the total (Figure 3). Larger concentrations resulted in a decrease in amylase release. As for the calcium experiments, similar values of basal amylase secretion were obtained in the two groups (4.6 ⫾ 1.6% in the control group and 4.9 ⫾ 0.9% in the olive group). Figure 3 also shows the effect of dietary virgin olive oil on the CCK-8 – evoked secretory response. Similar to the calcium experiments, the olive oil diet increased the secretory effect of CCK-8 compared with the response of CCK-8 in the control group. This increase proved to be significantly different at the maximal concentration of CCK-8. The sensitivity of acini to CCK-8 was not

FIG. 3. Dose–response curve of CCK-8 –induced amylase secretion. Acini from the control group (solid squares) and the olive group (open squares) were incubated with various concentrations of CCK-8. Results show the mean ⫾ standard error of the mean of 6 to 12 animals (pancreata). *Significantly different from the control group. CCK-8, cholecystokinin-octapeptide.

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changed by olive oil, inasmuch as the dose-response curve did not shift. Similar results were obtained when the cells were stimulated with ACh (data not shown).

are required to elucidate the precise mechanisms underlying the observed effects.

DISCUSSION

ACKNOWLEDGMENTS

In the present study we tested the effect of dietary fat on CCKinduced [Ca2⫹]i mobilization and amylase release in pancreatic acinar cells from rats fed for 8 wk with a virgin olive oil diet. Our results demonstrated that the virgin olive oil diet can increase the calcium mobilization stimulated by the physiologic agonists CCK-8 and ACh. The data showed that the oscillatory pattern in [Ca2⫹]i and large [Ca2⫹]i transients in response to a low (physiologic) and high concentration of CCK-8, respectively, was significantly increased by the olive oil diet compared with the control diet. In addition, we found that these effects of virgin olive oil consumption on calcium mobilization correlate, to a great extent, to CCK-8 –induced amylase secretion. However, feeding diets rich in virgin olive oil did not significantly alter the resting [Ca2⫹]i values or basal amylase secretion. Our findings are consistent with results obtained by Peck et al.7 who found that consumption of virgin olive oil for 2 wk increased changes in [Ca2⫹]i stimulated by agonists in murine lymphocytes. Further, short acute administration of free fatty acids has been reported to cause calcium mobilization in various cell lines.2– 6 The differences in acinar secretory activity and [Ca2⫹]i mobilization in our study likely can be associated with the dietaryinduced changes in cell membrane composition. Many steps of the stimulus–secretion coupling process are membrane dependent. Dietary fats differing in the degree of unsaturation have been found to modify insulin binding in rat adipocytes21 and adenylate cyclase activity in the liver22 and the submandibular gland.23 Because previous results have shown that the fatty acid composition of rat pancreatic membranes is profoundly altered by an olive oil diet,24 differential enrichment in certain fatty acids may influence the accessibility of the CCK receptor, the interaction with guanine nucleotide-binding proteins, or the functionality of enzymes such as phospholipases and protein kinase C, which interact with cell membranes during their activation. Apart from their structural role, membrane fatty acids participate as mediators in signal transduction.2–7 In the exocrine pancreas, the existence of CCK and ACh receptors is well established,20 and they are linked to phospholipase C. Phospholipase C activation leads to hydrolysis of phosphatidyl-inositol 4,5bisphosphate and subsequent production of IP3, which initiates the calcium signal, and diacylglycerol. In addition, oleic acid has been shown to modify the physicochemical membrane state6 without perturbing the binding of ligands to receptors.25 These membrane modifications could reasonably involve an alteration in the phosphoinositide turnover and a change in the supply of inositol lipid precursors of IP3. The increased production of IP3 in acini from rats fed with olive oil may explain the enhancement of intracellular calcium mobilization in response to CCK-8 and ACh, because the initial increase in [Ca2⫹]i transients is due mainly to calcium released from IP3-sensitive internal stores. Alternatively, it is tempting to speculate that diacylglycerol, abundantly generated by phospholipase C and possibly with different acyl moieties as a consequence of changes in the membrane, in our study may have resulted in differential activation of protein kinase C, a crucial modulator of the secretory machinery of acinar cells. This notion is strongly supported by the finding that unsaturated fatty acids, found in large proportion in olive oil, enhance the activity of protein kinase C in pancreatic acinar cells,26 guinea pig neutrophils,27 human platelets,28 and rat intact hippocampus.29 In conclusion, the present results demonstrate that a diet including virgin olive oil can modify pancreatic cell function as assessed by intracellular calcium mobilization and amylase release in response to the gastrointestinal hormone CCK. Further studies

The authors thank Mercedes Go´ mez Bla´ zquez for technical assistance.

REFERENCES 1. Petersen OH, Petersen CCH, Kasai H. Calcium and hormone action. Annu Rev Physiol 1994;56:297 2. Chow SC, Jondal M. Polyunsaturated free fatty acids stimulate an increase in cytosolic Ca2⫹ by mobilizing the inositol 1,4,5-trisphosphate-sensitive Ca2⫹ pool in T cells through a mechanism independent of phosphoinositide turnover. J Biol Chem 1990;265:902 3. Chow SC, Sisfontes L, Jondal M, Bjorkhem I. Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2⫹ increase. Biochim Biophys Acta 1991;1092:358 4. Gamberucci A, Fulceri R, Benedetti A. Inhibition of store-dependent capacitative Ca2⫹ influx by unsaturated fatty acids. Cell Calcium 1997;21:375 5. Ekokoski E, Forss L, Tornquist K. Inhibitory action of fatty acids on calcium fluxes in thyroid FRTL-5 cells. Mol Cell Endocrinol 1994;103:125 6. Zugaza JL, Casabiell XA, Bokser L, Casanueva FF. Oleic acid blocks EGFinduced [Ca2⫹]i release without altering cellular metabolism in fibroblast EGFR T17. Biochem Biophys Res Commun 1995;207:105 7. Peck MD, Spalding PB, Moffat FL, Jr, Han T, Jy W. Dietary olive oil enhances murine lymphocyte calcium uptake. J Trauma 2000;49:109 8. Sabb JE, Godfrey PM, Brannon PM. Adaptive response of rat pancreatic lipase to dietary fat: effects of amount and type of fat. J Nutr 1986;116:892 9. Ricketts J, Brannon PM. Amount and type of dietary fat regulate pancreatic lipase gene expression in rats. J Nutr 1994;124:1166 10. Ballesta MC, Man˜ as M, Mata´ ix FJ, Martı´nez-Victoria E, Seiquer I. Long-term adaptation of pancreatic response by dogs to dietary fats of different degrees of saturation: olive and sunflower oil. Br J Nutr 1990;64:487 11. Yago MD, Martı´nez-Victoria E, Huertas JR, Man˜ as M. Effects of the amount and type of dietary fat on exocrine pancreatic secretion in dogs after different periods of adaptation. Arch Physiol Biochem 1997;105:78 12. Beardshall K, Frost G, Morarji Y, Domin J, Blomm SR, Calam J. Saturation of fat and cholecystokinin release: implications for pancreatic carcinogenesis. Lancet 1989;2:1008 13. McLaughlin JT, Lomax RB, Hall L, Dockray GJ, Thompson DG, Warhurst G. Fatty acids stimulate cholecystokinin secretion via an acyl chain length-specific, Ca2⫹-dependent mechanism in the enteroendocrine cell line STC-1. J Physiol 1998;513:11 14. Reeves PG, Nielsen FH, Fahey GC. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition Ad Hoc Writing Committee on the reformulation of the AIN-76A rodent diet. J Nutr 1993;123:1939 15. Lajas AI, Sierra V, Camello PJ, Salido GM, Pariente JA. Vanadate inhibits the calcium extrusion in rat pancreatic acinar cells. Cell Signal 2001;13:451 16. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2⫹ indicators with greatly improved fluorescence properties. J Biol Chem 1985;260:3440 17. Gardner JD, Jackson MJ. Regulation of amylase release from dispersed pancreatic acinar cells. J Physiol 1977;270:439 18. Ceska M, Birath K, Brown B. A new and rapid method for the clinical determination of alpha-amylase activities in human serum and urine. Optimal conditions. Clin Chim Acta 1969;26:437 19. Petersen CCH, Toescu EC, Petersen OH. Different patterns of receptor-activated cytoplasmic Ca2⫹ oscillations in single pancreatic acinar cells: dependence on receptor type, agonist concentration and intracellular Ca2⫹ buffering. EMBO J 1997;10:527 20. Jensen RT. Receptors on pancreatic acinar cells. In: Johnson LR, Jacobsen ED, Christensen J, Alpers DH, Walsh JH, eds. Physiology of the gastrointestinal tract, 3rd ed. New York: Raven Press, 1994:1377 21. Clandinin MT, Cheema S, Field CJ, Garg ML, Venkatraman J, Clandinin TR. Dietary fat: exogenous determination of membrane structure and cell function. FASEB J 1991;5:2761 22. Neelands PJ, Clandinin MT. Diet fat influences liver plasma-membrane lipid composition and glucagon-stimulated adenylate cyclase activity. Biochem J 1983;212:573

Nutrition Volume 20, Number 6, 2004 23. Alam SQ, Mannino SJ, Alam BS. Reversal of diet-induced changes in adenylate cyclase activity and fatty acid composition of rat submandibular salivary gland lipids. Arch Oral Biol 1993;38:387 24. Martı´nez-Victoria E, Dı´az R, Yago MD, Martinez MA, Vilchat JR, Singh B, Marias M. Modulation of amylase release and intracellular Ca2⫹ mobilization by dietary fat in isolated rat pancreatic acinar cells. J Physiol 2003;548P:P183 25. Perez FR, Casabiell X, Camina JP, Zugaza JL, Casanueva FF. Cis-unsaturated free fatty acids block growth hormone and prolactin secretion in thyrotropinreleasing hormone-stimulated GH3 cells by perturbing the function of plasma membrane integral proteins. Endocrinology 1997;138:264 26. Wooten MW, Wrenn RW. Linoleic acid is a potent activator of protein kinase C

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type III-alpha isoform in pancreatic acinar cells; its role in amylase secretion. Biochem Biophys Res Commun 1988;153:67 27. Morimoto YM, Sato E, Nobori K, Takahashi R, Utsumi K. Effect of calcium ion on fatty acid-induced generation of superoxide in guinea pig neutrophils. Cell Struct Funct 1987;12:143 28. Nishikawa M, Hidaka H, Shirakawa S. Possible involvement of direct stimulation of protein kinase C by unsaturated fatty acids in platelet activation. Biochem Pharmacol 1988;37:3079 29. Linden DJ, Murakami K, Routtenberg A. A newly discovered protein kinase C activator (oleic acid) enhances long-term potentiation in the intact hippocampus. Brain Res 1986;379:358