Membrane lipid composition of pancreatic AR42J cells: modification by exposure to different fatty acids.

Dietary fat type influences fatty acids in rat pancreatic membranes, in association with modulation of secretory activity and cell signalling in viable acini. We aimed to confirm whether AR42J cells are a valid model to study the interactions between lipids and pancreatic acinar cell function. For this purpose we have (i) compared the baseline fatty acid composition of AR42J cells with that of pancreatic membranes from rats fed a standard chow; (ii) investigated if fatty acids in AR42J membranes can be modified in culture; and (iii) studied if similar compositional variations that can be evoked in rats when dietary fat type is altered occur in AR42J cells. Weaning Wistar rats were fed for 8 weeks either a commercial chow (C) or semi-purified diets containing virgin olive oil (VOO) or sunflower oil (SO) as fat source. AR42J cells were incubated for 72 hrs in medium containing unmodified fetal calf serum (FCS, AR42J-C cells), FCS enriched with 18:1 n-9 (AR42J-O cells), or FCS enriched with 18:2 n-6 (AR42J-L cells). Fatty acids in crude membranes from rat pancreas and AR42J cells were determined by gas-liquid chromatography. Differences in membrane fatty acids between C rats and AR42J-C cells can be explained in part by variations in the amount of fatty acids in the extracellular environment. Supplementation of FCS with 18:1 n-9 or 18:2 n-6 changed the fatty acid spectrum of AR42J cells in a manner that resembles the pattern found, respectively, in VOO and SO rats, although AR42J-L cells were unable to accumulate 20:4 n-6. The AR42J cell line can be a useful tool to assess the effect of membrane compositional changes on acinar cell function. However, differences in baseline characteristics, and perhaps fatty acid metabolism, indicate that results obtained in AR42J cells should be confirmed with experiments in the whole animal.

Effects of the type of dietary fat on acetylcholine-evoked amylase secretion and calcium mobilization in isolated rat pancreatic acinar cells.

Olive oil is a major component of the Mediterranean diet, and its role in human health is being actively debated. This study aimed to clarify the mechanism of pancreatic adaptation to dietary fat. For this purpose, we examined whether dietary-induced modification of pancreatic membranes affects acinar cell function in response to the secretagogue acetylcholine (ACh). Weaning male Wistar rats were assigned to one of two experimental groups and fed for 8 weeks with a commercial chow (C) or a semisynthetic diet containing virgin olive oil as dietary fat (OO). The fatty acid composition of pancreatic plasma membranes was determined by gas-liquid chromatography. For assessment of secretory function, viable acini were incubated with ACh and amylase of supernatant was further assayed with a substrate reagent. Changes in cytosolic Ca(2+) concentration in response to ACh were measured by fura-2 AM fluorimetry. Compared to C rats, pancreatic cell membranes of OO rats had a higher level of monounsaturated fatty acids and a lower level of both saturated and polyunsaturated fatty acids, thus, reflecting the type of dietary fat given. Net amylase secretion in response to ACh was greatly enhanced after OO feeding, although this was not paralleled by enhancement of ACh-evoked Ca(2+) peak increases. In conclusion, chronic intake of diets that differ in the fat type influences not only the fatty acid composition of rat pancreatic membranes but also the responsiveness of acinar cells to ACh. This mechanism may be, at least in part, responsible for the adaptation of the exocrine pancreas to the type of fat available.

Effect of the type of dietary fat on biliary lipid composition and bile lithogenicity in humans with cholesterol gallstone disease.

OBJECTIVE: The effect of the type of dietary fat on bile lipids and lithogenicity is unclear. This study compared the effects of two dietary oils that differed in fatty acid profile on biliary lipid composition in humans. METHODS: Female patients who had cholesterol gallstones and were scheduled for elective cholecystectomy were studied. For 30 d before surgery, subjects were kept on diets that contained olive oil (olive oil group, n = 9) or sunflower oil (sunflower oil group, n = 9) as the main source of fat. Gallbladder bile and stones were sampled at surgery. After cholecystectomy, duodenal samples were collected by nasoduodenal intubation during fasting and after administration of mixed liquid meals that included the corresponding dietary oil. Duodenal and gallbladder bile samples were analyzed for cholesterol, phospholipids, and total bile acids by established methods. Individual bile acid conjugates in gallbladder bile were measured by high-performance liquid chromatography. Gallstones were analyzed by semiquantitative polarizing light microscopy. RESULTS: Despite marked differences in the absolute concentration of biliary lipids and total lipid content, manipulation of dietary fat ingestion did not influence the cholesterol saturation or the profile of individual bile acids in gallbladder bile obtained from patients who had gallstones. All but one subject had mixed cholesterol stones. A cholesterol saturation index of hepatic bile in fasted cholecystectomized patients was similar in both dietary groups and indicative of supersaturation. In response to the test meal, the cholesterol saturation index decreased significantly in patients given the olive oil diet, reaching values lower than one at 120 min postprandially. In contrast, hepatic bile secreted by patients who consumed sunflower oil appeared supersaturated (cholesterol saturation index >1.5) throughout the experiment. CONCLUSIONS: Our results suggest that the type of dietary fat habitually consumed can influence bile composition in humans. In gallbladder, this influence was noted in the presence of more concentrated bile in the olive oil group. However, this was not translated into a modification of cholesterol saturation, which is likely due to the fact that cholesterol gallstones were present by the time the dietary intervention started. The finding that a typical postprandial variation in hepatic bile lithogenicity occurred only in olive oil patients was revealing. While keeping in mind the methodologic limitations of this part of the study, some gastrointestinal and metabolic mechanisms for this effect are discussed.

Effect of sodium nitroprusside and 8-bromo cyclic GMP on nerve-mediated and acetylcholine-evoked secretory responses in the rat pancreas.

The effects of sodium nitroprusside (SNP) and 8-bromo-guanosine 3'5' cyclic monophosphate (8-Br-cyclic GMP) on nerve-mediated and acetylcholine (ACh)-evoked amylase secretion, tritiated choline ([3H]-choline) release and on intracellular free calcium concentration ([Ca2+]i) in the isolated rat pancreas were investigated. Electrical field stimulation (EFS; 10 Hz) and ACh (1 x 10(-5) M) caused large increases in amylase output from pancreatic segments. The response to ACh was blocked by atropine (1 x 10(-5) M) whereas the EFS-evoked response was markedly reduced but not abolished. In contrast, pretreatment with tetrodotoxin (1 x 10(-6) M) abolished the secretory effect of EFS. Either SNP (1 x 10(-3) M) or 8-Br-cyclic GMP (1 x 10(-4) M) inhibited amylase secretion compared to basal. Combining either SNP or 8-Br-cyclic GMP with EFS resulted in a marked decrease in amylase output compared to EFS alone. In contrast, either SNP or 8-Br-cyclic GMP had no significant effect on the amylase response to ACh. When extracellular Ca2+ concentration ([Ca2+]o) was elevated from 2.56 mM to 5.12 mM, SNP failed to inhibit the response to EFS. EFS stimulated the release of 3H from pancreatic segments preloaded with [3H]-choline. Either SNP or 8-Br-cyclic GMP had no effect on basal 3H release but significantly reduced the EFS-evoked response. In fura-2 loaded acinar cells, SNP elicited a small decrease in [Ca2+]i compared to basal and had no effect on the ACh-induced [Ca2+]i peak response. Nitric oxide may modulate the release of endogenous neural ACh in response to EFS in the rat pancreas.

Dietary fat (virgin olive oil or sunflower oil) and physical training interactions on blood lipids in the rat.

OBJECTIVE: We investigated whether the intake of virgin olive oil or sunflower oil and performance of physical exercise (at different states) affect plasma levels of triacylglycerols, total cholesterol, and fatty acid profile in rats. METHODS: The study was carried out with six groups of male rats subjected for 8 wk to a diet based on virgin olive oil (three groups) or sunflower oil (three groups) as dietary fat. One group for each diet acted as sedentary control; the other two groups ran in a treadmill for 8 wk at 65% of the maximum oxygen consumption. One group for each diet was killed 24 h after the last bout of exercise and the other was killed immediately after the exercise performance. Triacylglycerols, total cholesterol, and fatty acid profile were analyzed in plasma. Analysis of variance was used to test differences among groups. RESULTS: Animals fed on virgin olive oil had lower triacylglycerol and cholesterol values. Physical exercise reduced these parameters with both dietary treatments. Fatty acid profile showed higher monounsaturated fatty acid proportion in virgin olive fed oil animals and a higher omega-6 polyunsaturated fatty acid proportion in sunflower oil fed animals. Physical exercise reduced the levels of monounsaturated fatty acids with both diets and increased the proportions of omega-3 polyunsaturated fatty acids. CONCLUSIONS: Results from the present study supported the idea that physical exercise and the intake of virgin olive oil are very good ways of reducing plasma triacylglycerols and cholesterol, which is desirable in many pathologic situations. Concerning findings on fatty acid profile, we had results similar to those of other investigators regarding the effect of different sources of dietary fat on plasma. The most interesting results came from the effect of physical exercise, with significant increases in the levels of omega-3 polyunsaturated fatty acids, which may contribute to the antithrombotic state and lower production of proinflammatory prostanoids attributed to physical exercise.

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

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 [Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](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 microM/L). CONCLUSION: The present results demonstrate that a diet supplemented with virgin olive oil can modify pancreatic cell function as assessed by [Ca(2+)](i) mobilization and amylase release evoked by secretagogues in rat pancreatic acinar cells. A role for fatty acids in calcium signaling is suggested.

Comparison of the effects of dietary sunflower oil and virgin olive oil on rat exocrine pancreatic secretion in vivo.

The aim of this study was to investigate the functional consequences in vivo of adapting the rat exocrine pancreas to different dietary fats. Weanling rats were fed diets containing 10 wt% virgin olive oil or sunflower oil for 8 wk. We then examined resting and cholecystokinin-octapeptide (CCK-8)-stimulated pancreatic secretion in the anesthetized animals. To confirm a direct influence of the type of fat upon the gland, the FA composition of pancreatic membranes as well as tissue protein and amylase content were determined in separate rats. The membrane FA profile was profoundly altered by the diets, reflecting the type of dietary fat given, although this was not paralleled by variations in the pancreatic content of protein or amylase. Nevertheless, dietary intake of oils evoked different effects on in vivo secretory activity. Resting flow rate and amylase output were significantly (P < 0.05) enhanced by sunflower oil feeding. Time course changes in response to CCK-8 infusion also showed a different pattern in each group. Secretion of fluid, protein, and amylase increased markedly in all animals, reaching a maximum within 20-40 min of infusion that was followed by a dramatic decline in both groups. In the sunflower oil group, this resulted in values reaching the resting level as soon as 60 min after CCK-8 infusion was begun. However, after the initial decline, olive oil group values showed a prolonged plateau elevation above the baseline (P < 0.05) that was maintained for at least the infusion time. In addition, a positive correlation between flow rate and both protein concentration and amylase activity existed in the olive oil group, but not in the sunflower oil group. The precise mechanism by which these effects are produced remains to be elucidated.

Involvement of ryanodine-operated channels in tert-butylhydroperoxide-evoked Ca2+ mobilisation in pancreatic acinar cells.

Reactive oxygen species and related oxidative damage have been implicated in the initiation of acute pancreatitis, a disease characterised in its earliest stages by disruption of intracellular Ca2+ homeostasis. The present study was carried out in order to establish the effect of the organic pro-oxidant, tert-butylhydroperoxide (tBHP), on the mobilisation of intracellular Ca2+ stores in isolated rat pancreatic acinar cells and the mechanisms underlying this effect. Cytosolic free Ca2+ concentrations ([Ca2+]c) were monitored using a digital microspectrofluorimetric system in fura-2 loaded cells. In the presence of normal extracellular Ca2+ concentrations ([Ca2+]o), perfusion of pancreatic acinar cells with 1 mmol l-1 tBHP caused a slow sustained increase in [Ca2+]c. This increase was also observed in a nominally Ca2+-free medium, indicating a release of Ca2+ from intracellular stores. Pretreatment of cells with tBHP abolished the typical Ca2+ response of both the physiological agonist CCK-8 (1 nmol l-1) and thapsigargin (TPS, 1 micromol l-1), an inhibitor of the SERCA pump, in the absence of extracellular Ca2+. Similar results were observed with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 0.5 micromol l-1), a mitochondrial uncoupler. In addition, depletion of either agonist-sensitive Ca2+ pools by CCK-8 or TPS or mitochondrial Ca2+ pools by FCCP were unable to prevent the tBHP-induced Ca2+ release. By contrast, simultaneous administration of TPS and FCCP clearly abolished the tBHP-induced Ca2+ release. These results show that tBHP releases Ca2+ from agonist-sensitive intracellular stores and from mitochondria. On the other hand, simultaneous application of FCCP and of 2-aminoethoxydiphenylborane (2-APB), a blocker of IP3-mediated Ca2+ release, was unable to suppress the increase in [Ca2+]c induced by tBHP, while the application of 50 micromol l-1 of ryanodine (which is able to block the ryanodine channels) inhibits tBHP-evoked Ca2+ mobilisation. These findings indicate that tBHP releases Ca2+ from non-mitochondrial Ca2+ pools through ryanodine channels.

Dietary-induced changes in the fatty acid profile of rat pancreatic membranes are associated with modifications in acinar cell function and signalling.

The effects of dietary lipids on the fatty acid composition of rat pancreatic membranes and acinar cell function were investigated. Weaning rats were fed for 8 weeks on one of two diets which contained 100 g virgin olive oil (OO) or sunflower-seed oil (SO)/kg. Pancreatic plasma membranes were isolated and fatty acids determined. Amylase secretion and cytosolic concentrations of Ca(2+) and Mg(2+) were measured in pancreatic acini. Membrane fatty acids were profoundly affected by the diets; the rats fed OO had higher levels of 18 : 1n-9 (42.86 (sem 1.99) %) and total MUFA compared with the animals fed SO (25.37 (sem 1.11) %). Reciprocally, the SO diet resulted in greater levels of total and n-6 PUFA than the OO diet. The most striking effect was observed for 18 : 2n-6 (SO 17.88 (sem 1.32) %; OO 4.45 (sem 0.60) %), although the levels of 20 : 4n-6 were also different. The proportion of total saturated fatty acids was similar in both groups, and there was only a slight, not significant (P=0.098), effect on the unsaturation index. Compared with the OO group, acinar cells from the rats fed SO secreted more amylase at rest but less in response to cholecystokinin octapeptide, and this was paralleled by reduced Ca(2+) responses to the secretagogue. The results confirm that rat pancreatic cell membranes are strongly influenced by the type of dietary fat consumed and this is accompanied by a modulation of the secretory activity of pancreatic acinar cells that involves, at least in part, Ca(2+) signalling.

Feeding fried oil changes antioxidant and fatty acid pattern of rat and affects rat liver mitochondrial respiratory chain components.

Fat frying is a popular food preparation method but several components like antioxidant vitamins could be lost due to oxidation and some others with toxic effects could appear. Because of such large consumption of frying oils, the effect of high temperatures on the oils is of major concern both for product quality and nutrition, taking into account that dietary fat source deeply influences several biochemical parameters, especially of mitochondrial membranes. Virgin olive oil possesses specific features for modulating the damages occurred by endogenous and exogenous oxidative stress being particularly rich in antioxidant molecules. We evaluated the extent of modifications suffered by virgin olive oil following a short-time deep fat frying procedure: vitamin E and phenolic compound as well as total antioxidant capacity (measured by ESR) decreased, while polar compounds increased. The intake of such an altered oil mainly affected the hydroperoxide and TBARS contents of mitochondrial membranes which were enhanced after the dietary treatments. Also, several mitochondrial respiratory chain components (Coenzyme Q, cytochrome b, c + c1, and a + a3) were affected.

The intake of fried virgin olive or sunflower oils differentially induces oxidative stress in rat liver microsomes.

The effects of non-fried and fried virgin olive and sunflower oils on rat liver microsomal compositional features have been investigated. In addition, plasma antioxidants (alpha-tocopherol and ubiquinone 9) were investigated as well as the possible oxidative modifications suffered by virgin olive and sunflower oils during the frying process. The frying process decreased the content of alpha-tocopherol and phenolics in the oils and increased total polar materials. Sunflower oil was affected to a greater extent than olive oil. In rats, the intake of fried oil led to higher levels of lipid peroxidation and a lower concentration of plasma antioxidants. Microsomal fatty acid and antioxidant profiles were also altered. It seems that a strong relationship exists between the loss of antioxidants and the production of toxic compounds in the oils after frying and the extent of the peroxidative events in microsomes, which were also different depending on the fat source. The highly unsaturated sunflower oil was less resistant to the oxidative stress produced by frying and led to a higher degree of lipid peroxidation in liver microsomes in vivo than virgin olive oil.