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.     

An amphicrine pancreatic cell line: AR42J cells combine exocrine and neuroendocrine properties.

The permanent cell culture line AR42J, derived from a rat pancreatic acinar carcinoma, is widely used for functional studies of exocrine pancreatic acinar cells. We now present evidence that these cells are amphicrine in that they contain zymogen granules as well as small (40-80 nm) neuroendocrine (NE) vesicles and typical neurotransmitters. Using the small NE vesicle-specific markers synaptophysin and "protein S.V.2", including synaptophysin cDNA probes, we have found that AR42J cells synthesize these proteins and contain vesicles harboring these proteins with biophysical properties similar to those of small NE vesicles. NE properties of these cells are further indicated by the presence of considerable amounts of stored amino acids (gamma-aminobutyric acid (GABA), glycine, glutamate) and by the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase. Finally, intermediate filament (IF) protein typing showed only cytokeratins 8 and 18, indicating that AR42J cells possess an IF protein complement indistinguishable from that of acinar and islet cells. Our results document the unusual case of a permanent cell line with combined exocrine and neuroendocrine properties that may be indicative of a derivation from a cell with multipotential character.     

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells

ATP is released from cells in response to various stimuli. Our previous studies on pancreas indicated that pancreatic acini could be major stores of secreted ATP. In the present study, our aim was to establish the role of the vesicular nucleotide transporter (VNUT), SLC17A9, in storage and release of ATP. Freshly prepared acini from mice and AR42J rat acinar cells were used in this study. We illustrate that in AR42J cells, quinacrine (an ATP store marker) and Bodipy ATP (a fluorescent ATP analog) co-localized with VNUT-mCherry to vesicles/granules. Furthermore, in acini and AR42J cells, a marker of the zymogen granule membranes, Rab3D, and VNUT co-localized. Dexamethasone treatment of AR42J cells promoted formation of acinar structures, paralleled by increased amylase and VNUT expression, and increased ATP release in response to cholinergic stimulation. Mechanical stimulus (pressure) and cell swelling also induced ATP release, but this was not influenced by dexamethasone, most likely indicating different non-zymogen-related release mechanism. In conclusion, we propose that VNUT-dependent ATP release pathway is associated with agonist-induced secretion process and downstream purinergic signalling in pancreatic ducts.     

Effects of cholecystokinin on cytosolic calcium in human pancreatic cancer cells.

Cholecystokinin (CCK) has been shown to increase cytosolic calcium and stimulate enzyme release from pancreatic acinar cells and a rat acinar cell line, AR42J. CCK is also trophic to normal pancreas and pancreatic cancer; however, the cellular mechanisms which regulate CCK-stimulated growth are unknown. The effect of CCK on intracellular calcium was evaluated in four human pancreatic cancer cell lines known to grow in response to CCK but not secrete enzymes (SW-1990, MIA PaCa-2, BXPC-3 and PANC-1) and a rat acinar cell line (AR42J) shown to secrete enzymes but not grow with CCK. By using single cell fluorescence microscopy in fura-2 loaded cells, intracellular calcium [Ca2+]i was measured. After obtaining baseline fluorescent cell images, synthetic CCK-octapeptide (CCK8) was added to the cells and images of cell fluorescence captured. [Ca2+]i of the rat acinar cells increased (603%) over the baseline within the first minute after the addition of CCK (4.10(-13) M to 4.10(-10) M) in 77% of cells tested. In contrast [Ca2+]i failed to significantly change in the human cancer cells treated with CCK. To further localize the defect in hormone signal transduction in cancer cells, cells were suspended in low calcium media and the plasma membranes were selectively permeabilized with digitonin. Media free calcium concentration was continuously monitored by fura-2 fluorescence. Addition of inositol 1,4,5-trisphosphate (IP3) resulted in a marked increase in medium calcium concentration indicating IP3 was capable of releasing calcium from intracellular stores in both the AR42J rat acinar cell line and in the human pancreas cancer cell lines. In conclusion, CCK does not increase cytosolic calcium in human pancreatic cancer cells in contrast to rat acinar cells although all contain IP3-sensitive intracellular Ca2+ pools. Our results suggest that growth promoting and secretory effects of CCK on pancreatic cells may occur via two independent signalling pathways.     

Distribution of fatty acids from dietary oils into phospholipid classes of triacylglycerol-rich lipoproteins in healthy subjects

Several studies have suggested that lipoprotein metabolism can be affected by lipoprotein phospholipid composition. We investigated the effect of virgin olive oil (VOO) and high-oleic sunflower oil (HOSO) intake on the distribution of fatty acids in triacylglycerols (TG), cholesteryl esters (CE) and phospholipid (PL) classes of triacylglycerol-rich lipoproteins (TRL) from normolipidemic males throughout a 7 h postprandial metabolism. Particularly, changes in oleic acid (18:1n-9) concentration of PL were used as a marker of in vivo hydrolysis of TRL external monolayer. Both oils equally promoted the incorporation of oleic acid into the TG and CE of postprandial TRL. However, PL was enriched in oleic acid (18:1n-9) and n-3 polyunsaturated fatty acids (PUFA) after VOO meal, whereas in stearic (18:0) and linoleic (18:2n-6) acids after HOSO meal. We also found that VOO produced TRL which PL 18:1n-9 content was dramatically reduced along the postprandial period. We conclude that the fatty acid composition of PL can be a crucial determinant for the clearance of TRL during the postprandial metabolism of fats.     

The Fat-1 Mouse has Brain Docosahexaenoic Acid Levels Achievable Through Fish Oil Feeding

Fat-1 transgenic mice endogenously convert n-6 to n-3 polyunsaturated fatty acids (PUFA). The aims of this study were to test whether a) fish oil feeding can attain similar brain n-3 PUFA levels as the fat-1 mouse, and b) fat-1 mouse brain docosahexaenoic acid (22:6n-3; DHA) levels can be potentiated by fish oil feeding. Fat-1 mice and their wildtype littermates consumed either a 10% safflower oil (SO) or a 2% fish oil and 8% safflower oil chow (FO). Brain total lipid and phospholipid fraction fatty acids were analyzed using GC-FID. Wildtype mice fed FO chow had similar brain levels of DHA as fat-1 mice fed SO chow. Fat-1 mice fed FO chow had similar brain n-3 PUFA levels as fat-1 mice fed SO chow. In conclusion, brain levels of DHA in the fat-1 mouse can be obtained by and were not further augmented with fish oil feeding.     

Alterations of Na,K-ATPase Isoenzymes in the Rat Diabetic Neuropathy: Protective Effect of Dietary Supplementation with n-3 Fatty Acids

Abstract: Diabetic neuropathy is a degenerative complication of diabetes accompanied by an alteration of nerve conduction velocity (NCV) and Na,K-ATPase activity. The present study in rats was designed first to measure diabetes-induced abnormalities in Na,K-ATPase activity, isoenzyme expression, fatty acid content in sciatic nerve membranes, and NCV and second to assess the preventive ability of a fish oil-rich diet (rich in n-3 fatty acids) on these abnormalities. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (D) and nondiabetic control animals (C) were fed the standard rat chow either without supplementation or supplemented with either fish oil (DM, CM) or olive oil (DO, CO) at a daily dose of 0.5 g/kg by gavage during 8 weeks. Analysis of the fatty acid composition of purified sciatic nerve membranes from diabetic animals showed a decreased incorporation of C16:1(n-7) fatty acids and arachidonic acids. Fish oil supplementation changed the fatty acid content of sciatic nerve membranes, decreasing C18:2(n-6) fatty acids and preventing the decreases of arachidonic acids and C18:1(n-9) fatty acids. Protein expression of Na,K-ATPase α subunits, Na,K-ATPase activity, and ouabain affinity were assayed in purified sciatic nerve membranes from CO, DO, and DM. Na,K-ATPase activity was significantly lower in sciatic nerve membranes of diabetic rats and significantly restored in diabetic animals that received fish oil supplementation. Diabetes induced a specific decrease of α1- and α3-isoform activity and protein expression in sciatic nerve membranes. Fish oil supplementation restored partial activity and expression to varying degrees depending on the isoenzyme. These effects were associated with a significant beneficial effect on NCV. This study indicates that fish oil has beneficial effects on diabetes-induced alterations in sciatic nerve Na,K-ATPase activity and function.     

Pancreatic acinar cells utilize tyrosine to synthesize L-dihydroxyphenylalanine

The pancreatic β cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet β cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet β cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet β cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.     

不同类型脂肪酸对SD大鼠血清脂肪酸及胰岛素抵抗的影响

目的分析中链饱和脂肪酸（MC-SFA,MCF组）、长链饱和脂肪酸（LC-SFA,LCF组）、n-6多不饱和脂肪酸（n-6 PUFA,SUF组）和n-3多不饱和脂肪酸（n-3 PUFA,TUF组）四种脂肪酸对大鼠血清脂肪酸及胰岛素抵抗的影响。方法雄性SD大鼠40只随机分为5组,对照组给予普通日粮,高脂组给予脂肪热量比相同的高脂日粮。喂养10周,每18 d测定空腹血糖（GLU）、血清脂肪酸、血清胰岛素水平,根据胰岛素敏感性指数（ISI）=ln1/（FPG×FINS）评定大鼠的胰岛素敏感性。结果10周后,LCF组和SUF组大鼠体重显著高于对照组和其它高脂组;LCF组血清胰岛素显著高于对照组（P﹤0.05）;LCF组、TUF组ISI显著低于对照组（P﹤0.05）;各组间血糖无明显差异（P〉0.05）。SUF组、TUF组血清LC-SFA浓度显著低于LCF组（P﹤0.05）;TUF组血清（n-3 PUFA）显著高于对照组和其它高脂组（P﹤0.05）。结论不同类型脂肪酸的高脂饲料对SD大鼠的血清脂肪酸组成和含量有显著的影响,SD大鼠脂肪沉积及胰岛素抵抗程度随血清脂肪酸代谢作用的不同而变化。     

Glucagon-like peptide-1 does not mediate amylase release from AR42J cells.

In this study, AR42J pancreatic acinar cells were used to investigate if glucagon-like peptide-1 (GLP-1) or glucagon might influence amylase release and acinar cell function. We first confirmed the presence of GLP-1 receptors on AR42J cells by reverse trasncriptase-polymerase chain reaction (RT-PCR), Western blotting, and partial sequencing analysis. While cholecystokinin (CCK) increased amylase release from AR42J cells, GLP-1, alone or in the presence of CCK, had no effect on amylase release but both CCK and GLP-1 increased intracellular calcium. Similar to GLP-1, glucagon increased both cyclic adenosine monophosphate (cAMP) and intracellular calcium in AR42J cells but it actually decreased CCK-mediated amylase release (n = 20, P < 0.01). CCK stimulation resulted in an increase in tyrosine phosphorylation of several cellular proteins, unlike GLP-1 treatment, where no such increased phosphorylation was seen. Instead, GLP-1 decreased such protein phosphorylations. Genestein blocked CCK-induced phosphorylation events and amylase secretion while vanadate increased amylase secretion. These results provide evidence that tyrosine phosphorylation is necessary for amylase release and that signaling through GLP-1 receptors does not mediate amylase release in AR42J cells. J. Cell. Physiol. 181:470-478, 1999. Published 1999 Wiley-Liss, Inc.     

Phosphatidic acid production,required for cholecystokinin octapeptide-stimulated amylase secretion from pancreatic acinar AR42J cells,is regulated by a wortmannin-sensitive process

To investigate the role of phospholipids in exocytotic secretory events, we utilized rat pancreatic acinar AR42J cells that secreted amylase in response to cholecystokinin octapeptide (CCK-8). Wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), was found to inhibit the secretion in a dose-dependent manner. When changes in cell membrane phospholipids were investigated before and after CCK-8 stimulation using [32P]orthophosphoric acid-labeled AR42J cells, we observed a rapid increase in phosphatidic acid (PtdOH) levels right after stimulation, which was not observed in non-stimulated cells. The increase, however, was suppressed by wortmannin pre-treatment, which also inhibited amylase secretion. Changes in other major phospholipids were not significant. These results indicate that CCK-8 induces amylase secretion through PI3K-regulated production of PtdOH in cell membranes.     

Response of rat heart membranes and associated ion-transporting ATPases to dietary lipid

The effects of different dietary fat intake on the lipid composition and enzyme behaviour of sarcolemmal (Na+ + K+)ATPase and sarcoplasmic reticulum Ca2+-ATPase from rat heart were investigated. Rat diets were supplemented with either sunflower seed oil (unsatd./satd. 5.6) or sheep kidney fat (unsatd./satd. 0.8). Significant changes in the phospholipid fatty acid composition were observed in both membranes after 9 weeks dietary lipid treatment. For both membranes, the total saturated/unsaturated fatty acid levels were unaffected by the dietary lipid treatment, however the proportions of the major unsaturated fatty acids were altered. Animals fed the sunflower seed oil diet exhibited an increase in n-6 fatty acids, including linoleic (18:2(n-6] and arachidonic (20:4(n-6] while the sheep kidney fat dietary rats were higher in n-3 fatty acids, principally docosahexaenoic (22:6), with the net result being a higher n-6/n-3 ratio in the sunflower seed oil group compared to sheep kidney fat dietary animals. Fluorescence polarization indicated that the fluidity of sarcoplasmic reticular membrane was greater than that of sarcolemmal membrane, with a dietary lipid-induced decrease in fluidity being observed in the sarcoplasmic reticular membrane from sheep kidney fat dietary animals. Despite these significant changes in membrane composition and physical properties, neither the specific activity nor the temperature-activity relationship (Arrhenius profile) of the associated ATPases were altered. These results suggest that with regard to the parameters measured in this study, the two ion-transporting ATPases are not modulated by changes which occur in the membrane lipid composition as a result of the diet.     

Saturated, but not n-6 polyunsaturated, fatty acids induce insulin resistance: role of intramuscular accumulation of lipid metabolites.

Consumption of a Western diet rich in saturated fats is associated with obesity and insulin resistance. In some insulin-resistant phenotypes this is associated with accumulation of skeletal muscle fatty acids. We examined the effects of diets high in saturated fatty acids (Sat) or n-6 polyunsaturated fatty acids (PUFA) on skeletal muscle fatty acid metabolite accumulation and whole-body insulin sensitivity. Male Sprague-Dawley rats were fed a chow diet (16% calories from fat, Con) or a diet high (53%) in Sat or PUFA for 8 wk. Insulin sensitivity was assessed by fasting plasma glucose and insulin and glucose tolerance via an oral glucose tolerance test. Muscle ceramide and diacylglycerol (DAG) levels and triacylglycerol (TAG) fatty acids were also measured. Both high-fat diets increased plasma free fatty acid levels by 30%. Compared with Con, Sat-fed rats were insulin resistant, whereas PUFA-treated rats showed improved insulin sensitivity. Sat caused a 125% increase in muscle DAG and a small increase in TAG. Although PUFA also resulted in a small increase in DAG, the excess fatty acids were primarily directed toward TAG storage (105% above Con). Ceramide content was unaffected by either high-fat diet. To examine the effects of fatty acids on cellular lipid storage and glucose uptake in vitro, rat L6 myotubes were incubated for 5 h with saturated and polyunsaturated fatty acids. After treatment of L6 myotubes with palmitate (C16:0), the ceramide and DAG content were increased by two- and fivefold, respectively, concomitant with reduced insulin-stimulated glucose uptake. In contrast, treatment of these cells with linoleate (C18:2) did not alter DAG, ceramide levels, and glucose uptake compared with controls (no added fatty acids). Both 16:0 and 18:2 treatments increased myotube TAG levels (C18:2 vs. C16:0, P < 0.05). These results indicate that increasing dietary Sat induces insulin resistance with concomitant increases in muscle DAG. Diets rich in n-6 PUFA appear to prevent insulin resistance by directing fat into TAG, rather than other lipid metabolites.     

Bombesin binding and biological effects on pancreatic acinar AR42J cells

The effects of bombesin on amylase release and the receptor binding of 125I-[Tyr4]bombesin in the rat pancreatic acinar carcinoma cell line AR42J were examined. Bombesin-like peptides stimulated amylase release from AR42J cells in a dose-dependent manner; a maximal 2-fold stimulation occurred at a bombesin concentration of 300 pM. Binding of 125I-[Tyr4]-bombesin to AR42J cells was specific, saturable and temperature dependent. The relative potencies with which various structurally related peptides stimulated amylase release correlated well with their relative abilities to compete for the bombesin receptor.     

Increase of heat shock protein gene expression by melatonin in AR42J cells.

Heat shock proteins (HSPs) have been reported to protect the pancreatic cells from the acute damage produced by caerulein overstimulation. However the effects of caerulein, melatonin or hyperthermia preconditioning on mRNA signal for HSP60 in the pancreatic acinar cells has not been examined yet. The aims of this study were: 1. To investigate the gene expression for HSP60 in the pancreatic AR42J cells stimulated by melatonin, caerulein or combination of both these substances. 2. To compare above changes with mRNA signal for HSP60 in pancreatic AR42J cells subjected to hyperthermia preconditioning. AR42J cells were incubated in standard medium at 37 degrees C for: 0, 1, 3, 5, 12 or 24 h, under basal conditions. Above cells were then subjected to heat shock (42 degrees C) for 0, 1 or 3 h. In the next part of the study AR42J cells were incubated in presence of caerulein (10(-11), 10(-9) or 10( -7) M), melatonin (10(-8) or 10(-6) M), or combination of above under basal conditions or following heat shock pretreatment. Gene expression for HSP60 was determined by RT-PCR. The mRNA signal for HSP60 has been observed in AR42J cells under basal conditions, and this signal was markedly and time-dependently increased in these cells subjected to hyperthermia preconditioning. Incubation of AR42J cells in presence of melatonin (10(-8) or 10(-6) M) resulted in the significant and dose-dependent increase of gene expression for HSP60 in both groups of AR42J cells: preconditioned and in those, which were not subjected to hyperthermia. Caerulein stimulation reduced mRNA signal for HSP60. The strongest signal has been observed after the exposition of AR42J cells to hyperthermia preconditioning, combined with melatonin and caerulein. We conclude that: 1. Gene expression for HSP60 has been detected in pancreatic AR42J cells under basal conditions. 2. Hyperthermia preconditioning resulted in a significant and time-dependent increase of HSP60 signal in pancreatic AR42J cells. 3. HSP60 gene expression was significantly increased in pancreatic AR42J cells stimulated by melatonin whereas caerulein reduced this signal. 4. The strongest gene expression for HSP60 has been found in the cells subjected to the combination of hyperthermia preconditioning, caerulein and melatonin.