Catherine Wicker

Rat pancreatic colipase mRNA: nucleotide sequence of a cDNA clone and nutritional regulation by a lipidic diet

A cDNA clone encoding rat pancreatic colipase was isolated using as a probe a synthetic deoxyoligonucleotide corresponding to a highly conserved amino acid sequence region in colipases from other species. The cloned messenger codes for a protein of 95 amino acids plus a signal peptide of 17 amino acids. The structure of the full-length cDNA was also determined and the corresponding amino acid sequence showed a high degree of homology with those of other known colipases. Quantification of the homologous mRNA in the pancreas of animals fed a high-lipid diet was consistent with a specific though moderate induction of colipase messenger by the nutritional manipulation.

Specific regulation of the gene expression of some pancreatic enzymes during postnatal development and weaning in the calf.

The construction of cDNA library from calf pancreas allowed us to examine the mRNA levels of four pancreatic hydrolases (chymotrypsin, lipase, trypsin and amylase) during postnatal development in preruminant and ruminant animals. The lack of parallel variations in the levels of the enzyme specific activities suggested that protein synthesis was not coordinately regulated. In preruminant calves, the change in chymotrypsin and lipase mRNA concentrations (0-28 day period) and in trypsin mRNA concentrations (0-119 day period) was opposite to that in the corresponding specific activities. In contrast, both the activity and mRNA profiles of amylase during the latter period, on the one hand, and those of chymotrypsin and lipase during the 28-119 day period, on the other hand, were comparable. However, the extent to which the specific activity and mRNA concentration of each enzyme were increased did not necessarily coincide. The observed changes in mRNA levels probably resulted from some transcriptional control of the gene expression and/or variation in mRNA stability. Moreover, a translational regulation of the messengers could explain the existence of non-parallel mRNA and specific activity profiles. In sharp contrast with the multiple control of protein synthesis during postnatal development in preruminant calves, weaning was found to induce the same increase in enzyme activity and corresponding mRNA for each of the four pancreatic enzymes, suggesting that pretranslational modulation of gene expression was mainly, if not exclusively, concerned.

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-alpha-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.(ABSTRACT TRUNCATED AT 250 WORDS)

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 measurement 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.

Autolysis of proproteinase E in bovine procarboxypeptidase A ternary complex gives rise to subunit III.

Extracts of bovine pancreatic tissue are shown by HPLC to contain two distinct ternary complexes of procarboxypeptidase A (subunit I), chymotrypsinogen C (subunit II) and either proproteinase E or subunit III. It is shown that proproteinase E in the complex generates subunit III by removal of 13 N‐terminal residues when the former is allowed to autolyze in solution or when catalytic amounts of isolated active proteinase E are added to it. Autolysis of proproteinase E was accompanied by the loss of potential activity towards specific synthetic substrates and occurred at a higher rate in pancreatic juice than in pancreatic tissue extracts, even when both were processed in the presence of serine protease inhibitors. We conclude that subunit III (also called truncated protease E) is an autolytic product of proproteinase E and not an ab initio component of the native ternary complex.

Further studies on subunit III of bovine procarboxypeptidase A: Structure and reactivity of the weakly functional active site

The activation of pancreatic zymogens results from the tryptic cleavage of the first basic bond in the N-terminal part of the molecules [ 11. As indicated by crystallographic data [2,3], the major event induced by this cleavage is the appearance at the newly formed N-terminus of the chain of a positively charged hydrophobic residue (Ile 16 in bovine chymotrypsinogen A) which moves into the interior of the molecule and forms an ion pair with the buried carboxylate of Asp 194. The concomitant repositioning of other residues, has been proposed to improve the structure of the primary substrate binding site and that of the oxyanion hole [4]. However, the charge relay system between Asp 102, His 57 and Ser 195 which plays a key role in catalysis is largely if not fully developed in the zymogens. The resulting latent active sites are able to react at a measurable rate with titrants and substrates ]51. Bovine procarboxypeptidase A is composed of 2 or 3 subunits [6,7] which can be dissociated under nondenaturing conditions after dimethylmaleylation of the complex [8]. Subunit I is the immediate precursor of carboxypeptidase A whereas subunit 11 is a chymotrypsinogen of the C type [9]. In contrast, no activity has ever been found in subunit III prior to or following tryptic treatment. This subunit has been assumed to be a new type of a non-activatable zymogen for the following 2 reasons:

Modulation de l'ARN messager de la chymotrypsine pancréatique au cours du développement post-natal et du sevrage chez le veau

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Modulation de l’ARN messager de la chymotrypsine pancréatique au cours du développement post-natal et du sevrage chez le veau I Le Huerou, C Wicker, P Guilloteau, R Toullec, A Puigserver