Amylase mRNA Levels Research Articles

Thyroxine control of pancreatic amylase gene expression: modulation of PTF1 binding activity

The role of pancreas specific transcription factor (PTF1) in thyroxine (T4) modulation of amylase gene expression in suckling rats was evaluated. Electrophoretic mobility shift assay (EMSA) was used to determine the PTF1 binding activity by the amount of a synthetic oligonucleotide containing the amylase enhancer sequence bound by nuclear protein extracts. Nuclear protein from rat pancreata showed a developmental increase of PTFI activity correlated with age. To study the action of T4, pups were made hyperthyroid by T4 injection and hypothyroid by feeding propylthiouracil (PTU) to the lactating dams. EMSA of nuclear proteins isolated from these groups showed an increase in PTF1 binding activity in the T4 group and a decrease in the PTU group. Concomitantly, T4 increased, while PTU decreased both amylase enzyme and mRNA concentrations. T4 replacement reversed the effect of PTU on PTF1 binding, amylase enzyme activity and mRNA levels. To examine the age dependence of T4 effects, T4 was injected to pups for 5 days prior to killing at the age of 15, and 25 days. T4 was effective when given at an earlier age (15 days) but not at a later stage (25 days) in increasing amylase activity and amylase mRNA levels. Nuclear proteins isolated from pancreata of these groups showed an increase in PTF1 binding activity in the T4-treated 15-day-olds but not in the 25-day-olds in comparison to their corresponding age matched littermates. These results suggest that PTF1 is an important intermediary in T4 modulation of amylase gene expression during ontogeny of the rat exocrine pancreas.

Effects of hypercholecystokininemia produced by pancreaticobiliary diversion on pancreatic growth and enzyme mRNA levels in starved rats.

The purpose of the present study was to examine the weight, DNA content, and enzyme mRNA levels in the pancreas in response to endogenous hypercholecystokininemia produced by pancreaticobiliary diversion (PBD) in starved rats. The results showed that PBD, which is known to increase the circulating cholecystokinin (CCK) concentration, prevented the reduction in the weight and DNA content of the pancreas after 3 days of starving, and that PBD increased the mRNA levels of amylase, chymotrypsinogen B, and procarboxypeptidase A in the pancreas of starved rats. The findings support the view that endogenous CCK plays an important role in maintaining the weight of the normal pancreas of starved rats and that it stimulates the transcription of genes coding for pancreatic exocrine enzymes.

The effect of pretranslational regulation on synthesis of pancreatic colipase in streptozotocin-induced diabetes in rats.

A significant increase in synthesis of pancreatic colipase in streptozotocin (STZ)-induced diabetes in rats has been demonstrated previously. The aim of the present study was to identify whether this change in colipase synthesis was related to a pretranslational or translational regulation. The levels of colipase, lipase, and amylase mRNA were determined by Northern blot hybridization. The enzymatic activities and synthesis rates for these proteins were determined. One week after injection of STZ, the mRNA levels for both colipase and lipase were increased by about 100% over control, with accompanying increases in enzyme synthesis rates and enzymatic activities. The amylase mRNA, amylase synthesis rates, and amylase activity decreased by 95%. Insulin injection at a dose of 2 U/100 g/day for 5 days restored enzyme mRNA levels as well as enzyme activities. Kinetic studies revealed that lipase mRNA rapidly increased after induction of diabetes, closely followed by increases in lipase synthesis rates and lipase content. Colipase mRNA also rapidly increased, with values 60, 85, and 82% over control 1, 2, and 3 days after STZ injection, respectively. But the colipase synthesis rate increased slowly, being only 10, 20, and 40% over control 1, 2, and 3 days after STZ treatment, respectively. Colipase content did not increase until 4 days after STZ injection (3 days after the increase in colipase mRNA). The decrease in amylase mRNA was paralleled by decreases in amylase synthesis rates and amylase content. In conclusion, the increase in colipase content in STZ-induced diabetes in rats is a consequence of enhanced transcriptional or pretranslational regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular characteristics of long-term cultured rat parotid acinar cells

We have successfully maintained and biochemically characterized differentiated rat parotid acinar cells cultured for long periods (6 mo.). The cells were cultured on a reconstituted basement membrane matrix in a medium containing a variety of agents that promote cellular proliferation and differentiation. The cultured cells retain the characteristics of the parental parotid acinar cells. They exhibit both secretory granules and abundant cellular organelles required for protein synthesis and secretion. In situ hybridization and immunocytochemistry demonstrate high levels of proline-rich protein mRNA and protein, and lower levels of amylase mRNA and protein, in their cytoplasm. These findings suggest that rat parotid acinar cells can be maintained in a differentiated state in vitro for long periods, and can serve as a useful model system for studying the regulation of exocrine secretory processes.

Gastrin affects enzyme activity and gene expression in the aging rat pancreas

The structural and functional properties of the pancreas are known to be affected by a number of hormones, particularly those of the gastrin-CCK family, yet little is known about the responsiveness of the pancreas to gastrin-CCK peptides during the latter stages of the steady-state mRNA levels of some of the digestive enzymes during advancing age and after administration of gastrin. Groups of 3-,6-,12-, and 16-month-old male Fischer-344 rats were infused (osmotic minipump) with either gastrin G-17 (250 ng/kg/h) or saline (controls) for 14 days. In control pancreas, aging resulted in slight progressive reduction in pancreatic DNA, RNA, and protein concentrations. This decrease was markedly enhanced by gastrin treatment in 16-month-old rats. Pancreatic amylase and trypsin (TRP) activities in these animals were also slightly decreased with aging, whereas the steady-state mRNA levels of both enzymes were significantly higher in 16-month-old rats than in their 3-month-old counterparts. However, in 16-month-old rats, the steady-state mRNA levels of amylase and TRP were significantly reduced after gastrin administration, when compared with the corresponding controls. Chymotrypsin (CHY) activity in the pancreas remained essentially unchanged between 3- and 12-month-old rat, but in 16-month-old animals it was markedly decreased. CHY activity was further reduced by gastrin treatment only in the 16-month-old group. We conclude that a) both age-associated and gastrin induced changes in the activity of pancreatic enzymes are not accompanied by parallel alterations in the steady-statte mRNA levels of these enzymes, and b) gastrin caused an antiproliferative effect on the rat pancreas with advancing age.

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.

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.

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.

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.

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.

Mechanism of glucocorticoid-induced increase in pancreatic amylase gene transcription.

To determine the mechanism(s) responsible for glucocorticoid-induced increases in amylase content in pancreatic acinar AR42J cells, we examined the effects of dexamethasone on amylase protein biosynthesis, steady-state mRNA levels, and gene transcription. Dexamethasone treatment led to a dose-dependent increase in amylase synthesis which was one-half maximal at 2 nM and maximal at 100 nM where a 6-fold increase was achieved. This dexamethasone-induced increase in amylase synthesis was detectable after 12 h, one-half maximal after 19 h, and approached maximal after 72 h. Dexamethasone treatment also increased amylase mRNA levels in a time- and dose-dependent manner in parallel with the changes in amylase synthesis. Nuclear RNA transcript elongation (run-on) assays indicated that amylase gene transcription was also increased in a time- and dose-dependent manner. Glucocorticoid enhancement of amylase gene transcription occurred relatively slowly, with a 6-fold increase occurring after 48 h of treatment with 100 nM dexamethasone. Thus, the effects of glucocorticoids on pancreatic amylase gene transcription fully accounted for the increased levels of amylase mRNA, synthesis, and content. However, due to the slow time course of dexamethasone induction of amylase gene expression we evaluated the possibility of glucocorticoid induction of a regulatory protein. We found that inclusion of cycloheximide or puromycin during dexamethasone treatment blocked the induction of amylase mRNA. These data suggest that the glucocorticoid-induced increase in amylase gene transcription requires induction of an unidentified regulatory protein(s).

A Drosophila gene is subject to glucose repression

Amylase-specific cDNA probes were used to assay amylase mRNA levels in third-instar larvae of Drosophila melanogaster. It is shown that there is a difference, of the order of 100-fold, in the mRNA levels between larvae that are fed 10% glucose and larvae of the same wild-type strain that are fed an equivalent diet lacking glucose. In fact, the glucose-fed larvae have barely detectable levels of amylase mRNA. This large difference in transcript abundance indicates that the glucose effect, which we previously characterized at the level of enzyme activity, probably reflects a change in the transcriptional activity of the amylase gene.

Glucocorticoids increase amylase mRNA levels, secretory organelles, and secretion in pancreatic acinar AR42J cells.

Previous studies have suggested a role for glucocorticoids in the differentiation of the acinar pancreas. We have now used the rat tumor cell line AR42J, derived from the acinar pancreas, to directly study this effect of glucocorticoids in vitro. The steroid hormones dexamethasone, corticosterone, aldosterone, and progesterone, but not estrogen, increased both the amylase content and the number of secretory granules of these cells. The potencies of the steroids were directly related to their effectiveness as glucocorticoids; dexamethasone was the most potent hormone and gave maximal effects at 100 nM. Morphometric analyses revealed that dexamethasone increased the volume density of granules 5.5-fold from 0.20 +/- 0.08 to 1.10 +/- 0.20% (n = 4) of the cytoplasmic volume. Dexamethasone treatment also increased the volume density of rough endoplasmic reticulum 2.4-fold from 1.20 +/- 0.09 to 2.86 +/- 0.30% (n = 5) of the cytoplasmic volume. After 48 h of dexamethasone treatment the cellular content of amylase increase eightfold from 2.8 +/- 0.4 to 22.6 +/- 3.8 U/mg protein (n = 6). This effect of dexamethasone was discernible after 12 h of incubation and approached maximal stimulation after 72 h of incubation. The increases in cellular amylase content were due to increased amylase synthesis as shown by specific immunoprecipitation of [35S]methionine-labeled proteins. Moreover, in vitro translation of cellular mRNA indicated that dexamethasone treatment increased amylase mRNA. Dexamethasone treatment also led to increased secretion of amylase in response to the secretagogue cholecystokinin. These data indicate, therefore, that glucocorticoids induce a more highly differentiated phenotype in AR42J pancreatic cells, and they suggest that glucocorticoids act via the enhanced transcription of specific mRNAs for acinar cell proteins.

The effects of gibberellic acid and abscisic acid on ?-amylase mRNA levels in barley aleurone layers studies using an ?-amylase cDNA clone

Two cDNA clones were characterized which correspond to different RNA species whose level is increased by gibberellic acid (GA3) in barley (Hordeum vulgare L.) aleurone layers. On the criteria of amino terminal sequencing, amino acid composition and DNA sequencing it is likely that one of these clones (pHV19) corresponds to the mRNA for α-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1.), in particular for the B family of α-amylase isozymes (Jacobsen JV, Higgins TJV: Plant Physiol 70:1647-1653, 1982). Sequence analysis of PHV19 revealed a probable 23 amino acid signal peptide. Southern hybridization of this clone to barley DNA digested with restriction endonucleases indicated approximately eight gene-equivalents per haploid genome.The identity of the other clone (pHV14) is unknown, but from hybridization studies and sequence analysis it is apparently unrelated to the α-amylase clone.Both clones hybridize to RNAs that are similar in size (∼1500b), but which accumulate to different extents following GA3 treatment: α-amylase mRNA increases approximately 50-fold in abundance over control levels, whereas the RNA hybridizing to pHV14 increases approximately 10-fold. In the presence of abscisic acid (ABA) the response to GA3 is largely, but not entirely, abolished. These results suggest that GA3 and ABA regulate synthesis of α-amylase in barley aleurone layers primarily through the accumulation of α-amylase mRNA.

On the mechanism of variation of pancreatic amylase levels in mouse strains.

The relative levels of pancreatic amylase mRNA closely parallel the 3-fold variation of pancreatic amylase protein levels observed in several mouse strains studied. In contrast pancreatic elastase mRNA levels were similar in these strains. Each of the strains contained the same number of amylase-like genes (about 8). The selective variation in the pancreatic amylase gene expression could be due either to differences in the numbers of active amylase genes or to different rates of synthesis and/or degradation of the amylase mRNA from a constant number of genes.

Accumulation of the predominant pancreatic mRNAs during embryonic development.

We have examined the pattern of accumulation of specific adult pancreas mRNAs during embryonic de- velopment by comparative analysis of the in vitro translation products of RNAs isolated from embryonic and adult pancreases. Changes in the concentration of individual mRNAs were inferred from the sodium do- decyl sulfate-polyacrylamide gel profiles of the in vitro translation products. The concentrations of several mRNAs coding for predominant adult translation prod- ucts increased dramatically between 14 and 20 days of gestation, coincident with pancreatic cytodifferentia- tion. A few prominent adult mRNAs were not detected in RNA isolated from embryonic pancreases, indicating that further qualitative changes in pancreatic gene expression occur after birth. The accumulation of the mRNA for the major secre- tory enzyme, amylase, was measured by specific im- munoprecipitation of the in vitro translation products. In vitro synthesized amylase, identified by immunopre- cipitation and by sodium dodecyl sulfate-polyacryl- amide gel separation of peptides generated by protease digestion, appeared approximately 2000 daltons larger than amylase isolated from adult pancreatic secretions. Amylase mRNA levels measured by immunoprecipita- tion of in vitro translation products increased a mini- mum of loo-fold between Days 14 and 20 of gestation, coincident with the increase in amylase synthesis. These results indicate that the rate of amylase synthe- sis during this developmental period is controlled in large part by the rate of accumulation of amylase mRNA. Enzymatic analyses have shown a biphasic accumulation of the exocrine digestive enzymes during the development of the rat pancreas (l-3). These data suggested that the developing exocrine pancreas undergoes at least two differentiative tran-