Amylase mRNA Research Articles

Effects of age on diabetes- and insulin-induced changes in pancreatic levels of α-amylase and its mRNA

During aging, protein synthesis undergoes decremental changes in many organs and tissues. In the pancreas, as well as in other exocrine glands, the rate of protein synthesis declines with age. However, it is unknown whether this decline is related to intrinsic aging changes which affect the secretory cell function. In this study, we compared the ability of pancreatic acinar cells to synthesize amylase and its messenger RNA (mRNA) in response to insulin treatment of young and old rats rendered diabetic with streptozotocin (STZ). In STZ-induced diabetic rats, amylase protein and its mRNA levels were reduced drastically in the pancreas of young and old groups. Injections of these diabetic rats with insulin increased pancreatic amylase mRNA contents significantly in both young and old rats. Insulin also increased proportions of amylase protein synthesis in the pancreas of both age groups. These results indicate that the pancreatic cells remain effective during aging in their transcriptional activity for functional amylase mRNA in response to an exogenous stimulus of insulin.

Changes in pancreatic trophism and gene expression during a prolonged fasting period in rats

To examine the effects of fasting on trophism and gene expression in pancreas, adult male rats were deprived of food from 0-6 d. Total DNA, RNA, and proteins, and specific mRNAs for rat amylase, chymotrypsinogen B, trypsinogen I, proinsulin I, and actin (assessed by employing cloned cDNAs and dot-blot hybridization) were quantitated in pancreas. Body and pancreatic wt diminished progressively to reach 65 and 75% of initial values at the 6th d of fasting. Protein/DNA and total RNA/DNA ratios decreased 2.04 and 2.31-fold, respectively, during 6 d of fasting. The concentration of amylase, chymotrypsinogen B, trypsinogen I, and actin mRNA, expressed as cpm/microgram RNA, decreased significantly throughout the study period, whereas the decrease observed in Proinsulin I mRNA concentration was not significantly different. When mRNA concentrations were refereed to the total content of DNA, however, the decrease was significant for all messengers tested. It is concluded that the prolonged absence of nutrients in the digestive tract exerts negative trophic influence on pancreas and triggers differential changes in pancreatic gene expression. These changes are gradual, asynchronic, and nonparallel.

Effects of Dietary Manganese Deficiency on Rat Pancreatic Amylase mRNA Levels

Manganese deficiency is associated with increased pancreatic amylase activity in rats. The present study investigated whether this increase in amylase activity is associated with increased pancreatic amylase messenger RNA (mRNA) levels. Weanling rats were fed a high carbohydrate diet containing either 40 micrograms Mn/g (control) or 0.5 microgram Mn/g (deficient) for 4 to 8 wk. Mn deficiency was confirmed by determining hepatic Mn content, which was significantly lower in Mn-deficient rats than in controls. Pancreatic RNA from both groups of rats was hybridized with 32P-labeled complementary DNAs for amylase and trypsinogen. Amylase mRNA levels were increased in rats fed Mn-deficient diets for both 4 wk (200%) and 8 wk (250%) when compared with respective control levels. In contrast, Mn deficiency was not associated with alterations in trypsinogen mRNA levels. Serum levels of insulin and corticosterone, hormones known to increase pancreatic amylase mRNA, were not affected by Mn deficiency. These observations suggest that Mn may participate in the regulation of amylase gene expression in a manner that is independent of insulin and corticosterone.

Genetic Analysis of Amylase-producing Cell Lines: Ectopic Activation of the Amylase Gene by Translocation

Two amylase-producing cell lines have been established, KMK-2 from a patient with gastric cancer, and KHM-1B from a patient with IgA lambda-type multiple myeloma. Both patients exhibited extremely high levels of serum amylase. The production of S-type amylase m-RNA by KMK-2 and KHM-1B was demonstrated by Northern blot analysis. Chromosome analysis showed many qualitative and quantitative abnormalities in both cell lines. In KHM-1B, a translocation was found between 1p13 or 21, near the amylase genes locus, and 9q34, the abl oncogene locus. These findings suggest the amylase gene in KHM-1B to be activated by translocation. A rearranged amylase gene was demonstrated by Southern blot analysis with only one enzyme, Accl.

Effects of Diabetes and Insulin on α-amylase Messenger RNA Levels in Rat Parotid Glands

Previous studies have shown that amylase levels are reduced significantly in the pancreas and parotid gland of diabetic rats and that insulin reverses this effect and increases the secretory protein levels. In the pancreas, these changes in amylase protein levels are accompanied by parallel changes in amylase mRNA levels. In the present study, the effects of diabetes and subsequent insulin treatments on contents (per cell) of amylase protein and its mRNA in parotid glands were compared in rats rendered diabetic with an injection of a beta-cell toxin, streptozotocin (STZ). Both amylase protein and its mRNA contents were reduced significantly in diabetic rats, compared with control rats, and this reduction was reversed following insulin injections of diabetic rats. In insulin-injected diabetic rats, amylase protein contents increased before a detectable increase in amylase mRNA levels was seen. The mRNA contents of a non-secretory protein, actin, did not change during diabetogenesis or subsequent insulin treatments. The reductions in parotid contents of amylase and its mRNA in diabetic rats and the reversal of these changes by insulin are similar to those changes that occur in the pancreas under the same conditions. However, the magnitude of these changes in parotid glands was much smaller than in the pancreas, and the effect of insulin on amylase mRNA synthesis was not as immediate as in the latter gland.

Expression of the amylase gene in the rat exocrine pancreas during postnatal development: Effect of dexamethasone

Pancreatic amylase enzyme activity starts to increase rapidly around weaning (17–21 days) and reaches the adult level during postnatal development in the rat. To see whether the maturation of amylase involves changes in amylase gene expression, total pancreatic RNA was prepared from rats of various ages (term-fetus, 5, 10, 15, 20, 28 days and adult). Northern blots of these RNAs were probed with amylase cDNA. Levels of amylase mRNA peaked around 10 days i.e., about 1 week prior to peak amylase enzyme activity. The role of glucocorticoid in pancreatic amylase development was studied by giving rat pups at ages 5, 10 and 30 days a single injection (i.p.) of dexamethasone (DX). They and their littermates (controls) were killed 24 and 48 h after the injection. Increases in amylase mRNA levels were seen in the DX treated 5- and 10-day-old groups with corresponding increases in amylase enzyme activities. A slight decrease in amylase mRNA level was, however, observed in the DX treated 30-day-old pups which also had a slight decrease in amylase enzyme activity suggesting an age dependent differential responsiveness to DX. A time sequence study with 10-day-old pups killed after a single injection of DX at 6, 12, 24 and 48 h showed a rapid increase in mRNA levels which peaked around 12 h. Amylase enzyme activity, however, did not peak until 24 h after DX injection. These results suggest that pancreatic amylase is regulated at the level of gene expression in both normal- and DX-induced maturation. Regulation appears to occur at the transcription level as both increases to amylase activity and mRNA were blocked by actinomycin D.

Dynamic changes of pancreatic structure and function in rats treated chronically with nicotine

Adult, male Sprague-Dawley rats weighing initially 185–225 g, were treated with 5, 15, or 50 mg nicotine or placebo 3-week-release pellets by sc implantation, for 1.5, 3, 6, and 12 weeks. These doses of nicotine correspond to infusion rates of 9.9, 29.8, and 99.2 μg/hr, respectively. At the highest nicotine dose trypsin and chymotrypsin activities were markedly higher in pancreas from 12-week nicotine-treated rats compared with controls. This was associated with a fourfold increase in steady-state amylase mRNA levels in comparison to placebo controls. In addition, secretagogue-stimulated enzyme release from pancreatic acini isolated from rats treated with 50 mg nicotine pellets was significantly higher than controls at 1.5 and 3 weeks and declined below control levels after 12 weeks of treatment. In rats treated with 15-mg nicotine pellets, maximal secretagogue-stimulated enzyme release from isolated acini occurred at 1.5 weeks, declining thereafter to control levels. Electron microscopy of pancreas from rats treated with the 50 mg nicotine dose revealed intracytoplasmic vacuoles appearing after 3 weeks of treatment, and persisting throughout the remaining experimental period. It is concluded that 12-week nicotine treatment results in increased pancreatic enzyme biosynthesis and accumulation of digestive enzymes within the pancreas. This is associated with altered responsiveness to secretagogues and evidence of morphological damage.

Changes in Growth and Pancreatic mRNA Concentrations During Postnatal Development of Rat Pancreas

Changes in pancreatic growth and in mRNA concentrations in rat pancreas were monitored by dot-blot hybridization with cloned cDNAs of rat amylase, chymotrypsinogen B, proinsulin I, and actin during the pre- and postnatal period in the rat. Wistar rats were killed at the 18th day of gestation and at the 1st, 10th, 20th, 35th, and 87th day of postnatal life. It was concluded from the ratio of pancreatic weight/body weight that pancreatic growth preceded body growth. Pancreatic protein and total RNA concentration increased 2.9 times during the period studies. All studied mRNAs increased in concentration during the postnatal development period. Messenger RNA for chymotrypsinogen B and proinsulin I exhibited a significant increase after birth, decreased by the 10th day of life, and increased thereafter. For amylase mRNA, no significant changes were observed around birth, a progressive increase occurring thereafter up to the 87th day of life. The mRNA for actin showed a progressive increase between the 18th day of gestation and the 20th postnatal day, after which it remained stable. We concluded that each mRNA showed a singular profile of increase during postnatal development.

Vanadate Induction of Pancreatic Amylase mRNA in Diabetic Rats

Amylase activity and mRNA abundance are reduced to less than 1% of normal levels in diabetic rat pancreas. Administration of vanadate in drinking water restored normal amylase activity and amylase mRNA levels. These results demonstrate an effect of vanadate on pancreatic acinar cells and suggest that vanadate can mimic the effect of insulin on transcription of the pancreatic amylase gene.

Vanadate induction of pancreatic amylase mRNA in diabetic rats

Vanadate induction of pancreatic amylase mRNA in diabetic rats

Expression of rat pancreatic lipase gene is modulated by a lipid-rich diet at a transcriptional level

The amount of cytoplasmic mRNAs specific for pancreatic lipase and amylase as well as transcription of the corresponding genes were investigated in rats fed a diet containing 25 % sunflower oil. Concentration of lipase mRNA was actually increased by the lipid-rich carbohydrate-low diet and reached a maximum level after 2 days, but further remained constant for at least 10 days. In contrast, about a two-fold decrease in the concentration of pancreatic amylase mRNA was only observed after rats were fed the high-lipid diet for 10 days. Transcription rate measurements on isolated nuclei from pancreatic tissue indicated that the concentration of nascent lipase and amylase transcripts was consistent with a transcriptional regulation of expression of these two genes.

Establishment of a New Human Pancreatic Adenocarcinoma Cell Line, MDAPanc-3

A new cell line was established from a liver metastasis of a human pancreatic adenocarcinoma. The cell line, MDAPanc-3, which arose from a moderately differentiated adenocarcinoma, produces carbonic anhydrase II mRNA, but no detectable levels of insulin or alpha amylase mRNA. The stem line chromosome number was determined to be 43, with six marker chromosomes. Growth of MDAPanc-3 is stimulated by cholecystokinin (CCK) fragment 26-33. The cell line will be useful in further studies on the mechanism(s) by which CCK stimulates growth of certain human pancreatic adenocarcinomas and normal human pancreatic exocrine tissue.

Pancreatic digestive enzyme gene expression: effects of CCK and soybean trypsin inhibitor

Regulation of pancreatic gene expression by cholecystokinin (CCK) was examined in the rat using cloned cDNA probes to quantify changes in specific mRNAs (amylase, trypsinogen I, chymotrypsinogen B, and ribonuclease). Rats were administered intraduodenally an elemental liquid diet. Plasma CCK levels were raised to levels comparable to physiological postprandial levels either by intraduodenal perfusion with soybean trypsin inhibitor (SBTI) (6.9 +/- 1.0 pM, n = 8) or by continuous intravenous infusion with cholecystokinin octapeptide (CCK-8, 6.0 +/- 0.9 pM, n = 6). SBTI infusion resulted in fivefold increases in trypsinogen I and chymotrypsinogen B mRNA levels after 48 h. In contrast SBTI infusion had no effect on amylase mRNA levels and led to a decrease in ribonuclease mRNA levels to approximately 50% of control after 48 h. Intravenous infusion with CCK-8 for 24 h resulted in plasma levels of CCK comparable to those obtained with SBTI and had similar effects on digestive enzyme mRNA levels. These data suggested that SBTI acted via its ability to raise plasma CCK levels. To further test the specificity of these effects, animals were infused intraduodenally with the specific CCK receptor antagonist L364,718. Although the antagonist itself had no effect on digestive enzyme mRNA levels, antagonist treatment totally abolished the effects of both CCK infusion and SBTI treatment. These data therefore indicate that CCK regulates digestive enzyme gene expression at plasma concentrations comparable to physiological postprandial levels. Furthermore, the ability of SBTI infusion to increase plasma CCK accounts for its effects on pancreatic digestive enzyme mRNA levels.

Changes in mRNA levels of rat pancreatic lipase in the early days of consumption of a high‐lipid diet

The time-course response of rat pancreatic enzymes to a diet containing 25% sunflower oil was investigated. A 1.2-fold enhancement in lipase specific activity was observed as early as the first day of diet consumption and was further increased up to 1.9-fold on the 5th day. On the other hand, colipase activity was slightly decreased during the first two days of high-lipid diet intake and then increased. An immediate and direct effect was also exerted by the 25% lipid diet on lipase biosynthesis. Both fractional synthetic rate and specific activity of lipase were comparably induced. Due to a 1.6-fold increase in the overall protein synthesis following 5 days of lipid diet consumption, the absolute synthesis of lipase and amylase was increased by 3.5-fold and 0.98-fold, respectively, as compared to control animals. By contrast, the synthesis of procarboxypeptidases and serine proteases did not increase before day 5, probably as the result of a distinct adaptive mechanism. The pancreatic mRNA levels in control and adapted animals, which were determined by dot-blot hybridization with amylase and lipase cDNAs, were consistent with a biphasic induction of lipase synthesis since a first increase in the level of the enzyme-specific mRNA during the first two days of diet intake (4-fold on day 1) was followed by a second increase after the fourth day (6.5-fold on day 5). On the other hand, amylase mRNA level was unchanged during the dietary manipulation. Thus, hyperlipidic diets exerted an both lipase activity and synthesis but a delayed effect on procarboxypeptidase and serine protease synthesis. In a similar manner, the immediate induction of lipase mRNA level by dietary fat, followed by another increase a few days later, suggested that at least two different mechanisms are involved in lipase mRNA induction.

Protein synthesis and amylase messenger RNA content in rat parotid salivary glands after total or partial stimulation with isoproterenol

The rate of synthesis of secretory proteins increases significantly in rat parotid glands after stimulated discharge of stored proteins. How any difference in the amount of secretory protein discharge affects the rate of subsequent protein synthesis, and whether this post-secretory synthesis is regulated at the level of messenger RNA, was now examined. One group of rats was stimulated to secrete 97% of stored secretory proteins by an intraperitoneal injection of isoproterenol. The other group received a much smaller dose to induce the discharge of about 40% of the proteins. Despite this difference in secretion, the subsequent rates of total protein synthesis, as well as of amylase, were increased to about the same extent. The amylase messenger RNA (mRNA) was identified and quantified by hybridization with a 32P-labelled amylase complementary DNA (cDNA) probe. The amylase mRNA in stimulated and unstimulated rats was of the same molecular size (Northern blot analyses). The amount of amylase mRNA, determined by dot blot analyses, were also increased in stimulated rats, although this increase was not as great as that in the rate of amylase protein synthesis. The implications of this discrepancy concern the possibility that the mechanism of regulation of secretory protein synthesis in parotid glands is at the translational level.

DNA rearrangement causes multiple changes in gene expression at the amylase locus inDrosophila melanogaster

A spontaneous null mutation at the alpha-amylase locus in Drosophila melanogaster was recovered from a laboratory population. The mutant strain was found to lack amylase enzyme production and to produce low, but detectable, levels of amylase mRNA. Moreover, the null strain is also lacking the glucose repression of amylase mRNA production which is seen in wild-type strains. The mutant phenotype correlates with a rearrangement in genomic DNA which, in turn, corresponds to a simple inversion in the arrangement observed most frequently in North American populations of D. melanogaster, including the common laboratory strain, Oregon-R. These results have implications for our understanding of both the evolution of the duplicated amylase gene structure and the regulation of amylase gene expression.

Pancreatic adaptation to dietary lipids is mediated by changes in lipase mRNA

Lipase activity, rates of biosynthesis of lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) and amylase (1,4-α- d-glucan glucanohydrolase, EC 3.2.1.1) as well as concentrations of their corresponding mRNAs were measured in the pancreatic tissue of rats fed isocaloric and isoprotein diets with inverse changes in the amounts of lipids and carbohydrates. A control diet (3% sunflower oil-62% starch) and three lipid-rich diets (10% sunflower oil-46.2% starch, 25% sunflower oil-12.5% starch and 30% sunflower oil-1.25% starch) were fed to rats for 10 days. Ingestion of the 10% lipid diet already resulted in a 1.4-fold increase in lipase activity while a 2.4-fold increase was observed with the other 2 high-lipid low-carbohydrate diets. Similarly, 1.3- and 3.1-fold increases in the total rate of protein synthesis were measured in pancreatic lobules of rats fed 10 and 25% or 30% lipid diets, respectively, as compared with control animals. While absolute lipase synthesis showed an important increase during the dietary manipulation (1.7- and 5.9-fold, respectively), amylase synthesis was significantly lower (1.1- and 1.5-fold, respectively). The level of lipase mRNA, as measured by dot-blot hybridization with the corresponding specific cDNA, showed a 2.2-fold increase (10% lipid diet) and a 3.9-fold increase (25% lipid diet), whereas the level of amylase mRNA showed only 1.1- and 1.3-fold increases under the same experimental conditions. These data demonstrated that protein-specific synthesis rates more accurately reflected pancreatic adaptive states than tissue levels of enzymes. The overall results: 1) are consistent with the conclusion that pancreatic adaptation to lipid-rich diets involves a selective enhancement in lipase synthesis, 2) indicate that the observed adaptive process is mediated to a great extent by changes in the corresponding cytoplasmic mRNA concentration and 3) suggest, for the first time, that adaptation of pancreatic lipase to dietary lipid is genetically regulated.

Translational control of anionic trypsinogen and amylase synthesis in rat pancreas in response to caerulein stimulation.

Infusion of rats with optimal doses of caerulein for up to 24 hr resulted in divergent changes in protein synthesis in the exocrine pancreas: a 3-fold increase in synthesis of anionic trypsinogen and a 75% decrease in synthesis of amylase. Lipase synthesis showed no change. Rates of total protein synthesis increased 2-fold, while DNA, RNA, and poly(A)+ mRNA concentrations were unchanged during hormonal stimulation. mRNA concentrations for anionic trypsinogen, lipase, and amylase were determined by dot blot hybridization analysis with cDNA and cRNA probes. Despite 12-fold changes in the ratio of synthesis of anionic trypsinogen to amylase at 24 hr of caerulein stimulation, changes in levels of mRNA encoding these two proteins were not observed. The slight decreases observed in amylase mRNA concentrations were found in both hormone and saline-infused animals. In vitro pulse-chase experiments after 12 hr of saline or caerulein infusion indicated that differential turnover of anionic trypsinogen and amylase did not occur during hormone stimulation. These data demonstrate that the differential regulation observed in protein synthesis that results from a single period of hormone stimulation is mediated by differential regulation of mRNA translation. The high degree of conservation observed in the 5' terminal sequences of both amylase and anionic trypsinogen mRNAs between mouse, rat, and dog suggests that sequence-specific mechanisms and secondary structure may play a role in the translational control of these two mRNAs.

Isolation and characterization of mutants which show an oversecretion phenotype in Saccharomyces cerevisiae.

We have isolated mutants responsible for an oversecretion phenotype in Saccharomyces cerevisiae, using a promoter of SUC2 and the gene coding for alpha-amylase from mouse as a marker of secretion. These mutations defined two complementation groups, designated as ose1 (over secretion) and rgr1 (resistant to glucose repression). The ose1 mutant produced an oversecretion of amylase by 12- to 15-fold under derepressing conditions; however, the amylase mRNA was present at nearly the same amount as it was in the parent cells. No expression of the amylase gene was detected under repressing conditions. The rgr1 mutant oversecreted amylase by 11- to 13-fold under repressing conditions by 15- to 18-fold under derepressing conditions. The rgr1 mutant showed pleiotropic effects on the following cellular functions: (1) resistance to glucose repression, (2) temperature-sensitive lethality, (3) sporulation deficieny in homozygous diploid cells, and (4) abnormal cell morphology. The rgr1 mutation was not allelic with ssn6 and cyc9, and failed to suppress snf1.

Amylase expression in human parotid neoplasms: evidence by in situ hybridization for lack of transcription of the amylase gene.

Salivary alpha-amylase (EC 3.2.1.1) is the major protein component of human parotid gland secretion. We studied amylase gene structure and expression in tissue from a series of normal and neoplastic parotid glands by Southern blot analysis, in situ hybridization, and immunohistochemistry. Thirty-two tumors were examined. Southern blot analysis of DNA extracted from a Warthin tumor, an adenoid cystic carcinoma, and a mucoepidermoid carcinoma showed no evidence of structural rearrangement of amylase genes. Eleven parotid Warthin tumors were negative for amylase protein and mRNA by immunocytochemistry and in situ hybridization. One pleomorphic adenoma in the group of 10 examined showed focal staining for amylase protein, although amylase mRNA could not be demonstrated in the same population of cells by in situ hybridization in serial tissue sections. Five mucoepidermoid carcinomas and three acinar cell carcinomas were devoid of amylase protein and mRNA. Normal parotid tissue obtained from all patients studied revealed abundant acinar cell amylase mRNA and protein. In situ hybridization, in conjunction with immunocytochemistry, allows precise cellular localization of mRNA and protein, thereby establishing the site of production of specific transcripts. We conclude that the interruption in amylase gene expression in parotid gland neoplasms occurs at the transcriptional level.