Diabetes and pancreatic exocrine dysfunction due to mutations in the carboxyl ester lipase gene-maturity onset diabetes of the young (CEL-MODY): a protein misfolding disease

CEL-maturity onset diabetes of the young (MODY), diabetes with pancreatic lipomatosis and exocrine dysfunction, is due to dominant frameshift mutations in the acinar cell carboxyl ester lipase gene (CEL). As Cel knock-out mice do not express the phenotype and the mutant protein has an altered and intrinsically disordered tandem repeat domain, we hypothesized that the disease mechanism might involve a negative effect of the mutant protein. In silico analysis showed that the pI of the tandem repeat was markedly increased from pH 3.3 in wild-type (WT) to 11.8 in mutant (MUT) human CEL. By stably overexpressing CEL-WT and CEL-MUT in HEK293 cells, we found similar glycosylation, ubiquitination, constitutive secretion, and quality control of the two proteins. The CEL-MUT protein demonstrated, however, a high propensity to form aggregates found intracellularly and extracellularly. Different physicochemical properties of the intrinsically disordered tandem repeat domains of WT and MUT proteins may contribute to different short and long range interactions with the globular core domain and other macromolecules, including cell membranes. Thus, we propose that CEL-MODY is a protein misfolding disease caused by a negative gain-of-function effect of the mutant proteins in pancreatic tissues.     

Absence of Diabetes and Pancreatic Exocrine Dysfunction in a Transgenic Model of Carboxyl-Ester Lipase-MODY (Maturity-Onset Diabetes of the Young)

Background

CEL-MODY is a monogenic form of diabetes with exocrine pancreatic insufficiency caused by mutations in CARBOXYL-ESTER LIPASE (CEL). The pathogenic processes underlying CEL-MODY are poorly understood, and the global knockout mouse model of the CEL gene (CELKO) did not recapitulate the disease. We therefore aimed to create and phenotype a mouse model specifically over-expressing mutated CEL in the pancreas.

Methods

We established a monotransgenic floxed (flanking LOX sequences) mouse line carrying the human CEL mutation c.1686delT and crossed it with an elastase-Cre mouse to derive a bitransgenic mouse line with pancreas-specific over-expression of CEL carrying this disease-associated mutation (TgCEL). Following confirmation of murine pancreatic expression of the human transgene by real-time quantitative PCR, we phenotyped the mouse model fed a normal chow and compared it with mice fed a 60% high fat diet (HFD) as well as the effects of short-term and long-term cerulein exposure.

Results

Pancreatic exocrine function was normal in TgCEL mice on normal chow as assessed by serum lipid and lipid-soluble vitamin levels, fecal elastase and fecal fat absorption, and the normoglycemic mice exhibited normal pancreatic morphology. On 60% HFD, the mice gained weight to the same extent as controls, had normal pancreatic exocrine function and comparable glucose tolerance even after resuming normal diet and follow up up to 22 months of age. The cerulein-exposed TgCEL mice gained weight and remained glucose tolerant, and there were no detectable mutation-specific differences in serum amylase, islet hormones or the extent of pancreatic tissue inflammation.

Conclusions

In this murine model of human CEL-MODY diabetes, we did not detect mutation-specific endocrine or exocrine pancreatic phenotypes, in response to altered diets or exposure to cerulein.     

Pioglitazone reverses insulin resistance and impaired CCK-stimulated pancreatic secretion in eNOS(-/-) mice: therapy for exocrine pancreatic disorders?

In mice, eNOS (endothelial nitric oxide synthase) maintains in vivo pancreatic secretory responses to carbachol or cholecystokinin octapeptide (CCK-8), maintains insulin sensitivity, and modulates pancreatic microvascular blood flow (PMBF). eNOS(-/-) mice are insulin resistant, and their exocrine pancreatic secretion is impaired. We hypothesized that the reduced exocrine pancreatic secretion in eNOS(-/-) mice is due to insulin resistance or impaired PMBF. To test this hypothesis, we gave eNOS(-/-) and wild-type (WT) mice pioglitazone (20 or 50 mg.kg(-1).day(-1)), an insulin-sensitizing peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activator, and measured pancreatic protein secretion evoked by CCK-8 (160 pmol.kg(-1).h(-1), a maximal stimulus). We also measured insulin resistance, serum glucose, C-peptide, insulin, pancreatic RNA digestive enzyme expression, and PMBF (microsphere technique). In WT mice, pioglitazone did not increase CCK-8-stimulated protein output over baseline. In eNOS(-/-) mice, however, pioglitazone substantially increased the low CCK-8-stimulated protein output that is characteristic of these mutant mice (P < 0.005). Pioglitazone abolished the CCK-8-evoked hyperinsulinemia (P < 0.005) and increased insulin sensitivity of eNOS(-/-) mice (P < 0.05), the latter based on hyperinsulinemic-euglycemic clamp studies. Pioglitazone had no effect on PMBF or pancreas mRNA expression of insulin or digestive enzymes. We conclude that in hyperinsulinemic eNOS(-/-) mice, a nonobese model of insulin resistance relevant to diabetes mellitus and possibly chronic pancreatitis, reduced pancreatic secretion is caused, at least in part, by insulin resistance. Insulin-sensitizing PPAR-gamma agonists such as pioglitazone may thus simultaneously correct endocrine and exocrine pancreatic disorders.     

Studies on the secretory process in exocrine pancreas cells. II. C57 black and beige mice

Published electron microscopic and cytochemical studies (thiamine pyrophosphatase and acid phosphatase) on exocrine pancreas cells of guinea pig, hamster, rat and rabbit have demonstrated that the nascent secretory granules, or condensing vacuoles, are part of GERL. The studies reported here show this to be true of the mouse pancreatic exocrine cells as well, thus permitting comparison of this cell type in the C57 black mouse and its "beige" mutant. This is of considerable interest because GERL is very much enlarged in these cells of the beige mouse. Most of GERL consists of wide dilated portions filled with electron-opaque materials that appear to be packaged into huge residual body-type lysosomes ("anomalous granules"). Acid phosphatase activity is demonstrable not only in these portions of GERL, but also in the condensing vacuoles as in pancreatic acinar cells in the black mouse where these dilated lysosome-producing regions are not present.     

Exocrine Pancreas Dysfunction in Type 1 Diabetes

Objective: Type 1 diabetes (T1D) is characterized by autoimmune β-cell destruction, but exocrine pancreas abnormalities may also play a role in the disease pathophysiology. Herein, we review the current evidence of exocrine damage in T1D and discuss its underlying pathophysiology, clinical evaluation, and treatment.Method: Extensive literature search was performed for “type 1 diabetes” and “exocrine dysfunction” on PubMed and Google Scholar databases.Results: T1D pancreata are significantly smaller than controls, both in weight and volume. T cells, dendritic cells, neutrophils, and products of complement activation are seen in T1D exocrine tissues. Exocrine pancreas fibrosis, arteriosclerosis, fatty infiltration, and acinar atrophy are also observed on histology. Pancreatic exocrine insufficiency (PEI) can be assessed through direct exocrine testing, fecal elastase concentration, and measurement of serum exocrine enzymes. The prevalence of PEI in T1D varies by modality and study but is consistently greater than controls. The clinical relevance of PEI in T1D is debatable, as many patients with laboratory evidence of PEI are asymptomatic. However, in PEI-symptomatic patients reported benefits of pancreatic enzyme replacement therapy (PERT) include relief of gastrointestinal symptoms, improved quality of life, better glycemic control, and optimal nutrition.Conclusion: Exocrine pancreas abnormalities often occur in T1D. Whether exocrine dysfunction occurs simultaneously with β-cell destruction, as a result of β-cell loss, or as a combination of both remains to be definitively answered. In T1D with gastrointestinal complaints, PEI should be evaluated, usually via fecal elastase measurements. PERT is recommended for T1D patients with symptoms and laboratory evidence of PEI.Abbreviations: AAb+ = autoantibody positive; AAb- = autoantibody negative; FEC = fecal elastase concentration; PEI = pancreatic exocrine insufficiency; PERT = pancreatic enzyme replacement therapy; PP = pancreatic polypep-tide; T1D = type 1 diabetes     

Secretin-stimulated amylase release into blood is impaired in type 1 diabetes mellitus.

BACKGROUND: Prior studies have provided data indicating the existence of close interaction between pancreatic endocrine and exocrine function, but few clinical studies have explored this relationship in depth. We compared pancreatic exocrine function non-endoscopically in individuals with type 1 diabetes mellitus, type 2 diabetes mellitus, and normal glucose tolerant controls, to assess the importance of local insulin production to pancreatic exocrine function. METHODS: The plasma amylase response to intravenous secretin challenge was measured in men with type 1 diabetes mellitus (n = 5), type 2 diabetes mellitus (n = 5), and normal controls (n = 3). Patients were characterized by their urinary excretion of c-peptide and albumin over 24 hours. Autonomic neuropathy was non-invasively assessed by measuring RR variation (with deep respiration on EKG). RESULTS: Post-secretin amylase responses were generally absent with low baseline levels in the patients with type 1 diabetes mellitus. Patients with type 2 diabetes mellitus and controls showed similar twofold increases over baseline after secretin administration. When normal glucose tolerant and type 2 diabetic patients were pooled and compared against type 1 diabetes mellitus, the differences were statistically significant (p < 0.03). Total amylase response correlated positively, but weakly, with 24 h urinary C-peptide excretion (r = 0.507; p < 0.112), but not with glycemic control, duration of diabetes, or indices of autonomic neuropathy. CONCLUSIONS: Patients with type 1 diabetes mellitus, but not type 2 diabetes mellitus, have reduced pancreatic exocrine function, supporting the concept of a local paracrine effect of insulin on pancreatic acinar cells. Further studies are needed to determine the clinical impact of this deficiency, and whether such patients with type 1 diabetes mellitus would benefit from therapy with pancreatic enzyme supplementation.     

Concerted action of human carboxyl ester lipase and pancreatic lipase during lipid digestion in vitro: importance of the physicochemical state of the substrate

The pancreatic enzyme carboxyl ester lipase (CEL) has been shown to hydrolyse a large number of different esters, including triacylglycerols, cholesteryl esters and retinyl esters with an absolute requirement for bile salts. Some of the lipids that are substrates for CEL can also be hydrolysed by pancreatic lipase. In order to investigate the relative roles of human CEL and pancreatic lipase, the two enzymes were incubated on a pH-stat with isotope-labelled lipid substrate mixtures in physicochemical forms resembling the state of the dietary lipids in human intestinal contents. In the first set of experiments, cholesteryl oleate (CO) and retinyl palmitate (RP) were solubilised in an emulsion of triolein (TO) stabilised by egg phosphatidylcholine and bile salts. Lipase (always added together with its cofactor, colipase) hydrolysed TO, with monoolein and oleic acid as end-products, whereas CEL alone could not hydrolyse TO in the presence of phosphatidylcholine (PC). Lipase alone did not hydrolyse CO or RP, but CEL did hydrolyse these esters if lipase was present. Release of [3H]glycerol from labelled TO increased only slightly if CEL was added compared to lipase alone, suggesting that monoolein hydrolysis was slow under these conditions. In the second set of experiments, CO and RP were dissolved in bile salt/monoolein/oleic acid dispersions with varying bile salt concentrations. CEL hydrolysed CO and RP more rapidly in a system with a high bile salt concentration containing mixed micelles than in a system with a low bile salt concentration, where the lipids were dispersed in the form of mixed micellar and non-micellar aggregates; both types of aggregate have been reported to exist in human intestinal contents. In conclusion, these data suggest that the main function of CEL under physiological conditions is to hydrolyse cholesteryl and retinyl esters, provided that the triacylglycerol oil phase is hydrolysed by pancreatic lipase, which probably causes a transfer of the substrate lipids of CEL from the oil emulsion phase to an aqueous bile salt/lipolytic product phase. Depending on the bile salt/lipolytic product ratio, the substrate will reside in either micellar or non-micellar lipid aggregates, of which the micellar state is preferred by CEL.     

Vesicle-associated Membrane Protein (VAMP) Cleavage by a New Metalloprotease from the Brazilian Scorpion Tityus serrulatus

We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction ν, an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction ν that cleaves VAMP2, and report its amino acid sequence.     

Immunocytochemical localization of amylase and chymotrypsinogen in the exocrine pancreatic cell with special attention to the Golgi complex

Affinity-purified, monospecific rabbit antibodies against rat pancreatic alpha-amylase and bovine pancreatic alpha-chymotrypsinogen were used for immunoferritin observations of ultrathin frozen sections of mildly fixed exocrine pancreatic tissue from secretion-stimulated (pilocarpine) rats and from overnight-fasted rats and guinea pigs. The labeling patterns for both antibodies were qualitatively alike: Labeling occurred in (a) the cisternae of the rough endoplasmic reticulum (RER) including the perinuclear cisterna, in (b) the peripheral area between the RER and cis-Golgi face, and (c) all Golgi cisternae, condensing vacuoles, and secretory granules. Labeling of cytoplasmic matrix was negligible. Structures that appeared to correspond to rigid lamellae were unlabeled. Differences in labeling intensities indicated that concentration of the zymogens starts at the boundary of the RER and cis-side of the Golgi complex. These data support the view that the Golgi cisternae are involved in protein processing in both stimulated and unstimulated cells and that Golgi cisternae and condensing vacuoles constitute a functional unit.     

Pdk1 activity controls proliferation, survival, and growth of developing pancreatic cells

The formation of adequate masses of endocrine and exocrine pancreatic tissues during embryogenesis is essential to ensure proper nutrition and glucose homeostasis at postnatal stages. We generated mice with pancreas-specific ablation of the 3-phosphoinositide-dependent protein kinase 1 (Pdk1) to investigate how signaling downstream of the phosphatidylinositol-3-OH kinase (PI3K) pathway controls pancreas development. Pdk1-conditional knock-out mice were born with conspicuous pancreas hypoplasia, and within a few weeks, they developed severe hyperglycemia. Our detailed characterization of the mutant embryonic pancreas also revealed distinct temporal, cell type-specific requirements of Pdk1 activity in the control of cell proliferation, cell survival, and cell size during pancreas development. These results thus uncover Pdk1 as a novel, crucial regulator of pancreatic growth during embryogenesis. In addition, we provide evidence that Pdk1 activity is required differently in mature pancreatic cell types, since compensatory proliferation and possible mTORC2 activation occurred in exocrine cells but not in β cells of the Pdk1-deficient postnatal pancreas.     

VAMP8/endobrevin as a general vesicular SNARE for regulated exocytosis of the exocrine system

The molecular mechanism governing the regulated secretion of most exocrine tissues remains elusive, although VAMP8/endobrevin has recently been shown to be the major vesicular SNARE (v-SNARE) of zymogen granules of pancreatic exocrine acinar cells. In this article, we have characterized the role of VAMP8 in the entire exocrine system. Immunohistochemical studies showed that VAMP8 is expressed in all examined exocrine tissues such as salivary glands, lacrimal (tear) glands, sweat glands, sebaceous glands, mammary glands, and the prostate. Severe anomalies were observed in the salivary and lacrimal glands of VAMP8-null mice. Mutant salivary glands accumulated amylase and carbonic anhydrase VI. Electron microscopy revealed an accumulation of secretory granules in the acinar cells of mutant parotid and lacrimal glands. Pilocarpine-stimulated secretion of saliva proteins was compromised in the absence of VAMP8. Protein aggregates were observed in mutant lacrimal glands. VAMP8 may interact with syntaxin 4 and SNAP-23. These results suggest that VAMP8 may act as a v-SNARE for regulated secretion of the entire exocrine system.     

Effect of pancreatic polypeptide-antiserum on bombesin-stimulated pancreatic exocrine secretion in dogs

Bombesin is a potent stimulus of both pancreatic protein secretion and plasma pancreatic polypeptide (PP) release in dogs. Physiological plasma levels of PP have been shown to inhibit pancreatic exocrine secretion in dogs. We examined the question whether the concomitant release of PP exerts a suppressive action on the pancreatic exocrine response to bombesin in dogs by measuring pancreatic exocrine secretion with and without in vivo immunoneutralization of PP with a high affinity PP-antiserum. Bombesin was infused in a dose of 150 ng/kg·hr, resulting in a rise of plasma PP from 24±5 to 224±25 pM (p<0.01). Before this bombesin infusion, 7 ml of normal rabbit serum had been administered to the dogs (n=8). At a later stage, the study was repeated after administration of 7 ml of PP-antiserum to the same animals. The bombesin induced increase in pancreatic exocrine secretion during administration of PP-antiserum (flow rate 24±10 ml/hr, protein output 1.35±0.43 g/hr, and bicarbonate output 3.25±1.42 mmol/hr) was not significantly different from that during control rabbit serum (flow rate 21±7 ml/hr, protein output 1.26±0.38 g/hr, and bicarbonate output 3.18±1.10 mmol/hr). It is therefore concluded that the pancreatic exocrine response to bombesin is not affected by the concomitant secretion of PP.     

Importance of arginines 63 and 423 in modulating the bile salt-dependent and bile salt-independent hydrolytic activities of rat carboxyl ester lipase

Previous studies using chemical modification approach have shown the importance of arginine residues in bile salt activation of carboxyl ester lipase (CEL) activity. However, the x-ray crystal structure of CEL failed to show the involvement of arginine residues in CEL-bile salt interaction. The current study used a site-specific mutagenesis approach to determine the role of arginine residues 63 and 423 in bile salt-dependent and bile salt-independent hydrolytic activities of rat CEL. Mutations of Arg(63) to Ala(63) (R63A) and Arg(423) to Gly(423) (R423G) resulted in enzymes with increased bile salt-independent hydrolytic activity against lysophosphatidylcholine, having 6.5- and 2-fold higher k(cat) values, respectively, in comparison to wild type CEL. In contrast, the R63A and R423A mutant enzymes displayed 5- and 11-fold decreases in k(cat), in comparison with wild type CEL, for bile salt-dependent cholesteryl ester hydrolysis. Although taurocholate induced similar changes in circular dichroism spectra for wild type, R63A, and R423G proteins, this bile salt was less efficient in protecting the mutant enzymes against thermal inactivation in comparison with control CEL. Lipid binding studies revealed less R63A and R423G mutant CEL were bound to 1,2-diolein monolayer at saturation compared with wild type CEL. These results, along with computer modeling of the CEL protein, indicated that Arg(63) and Arg(423) are not involved directly with monomeric bile salt binding. However, these residues participate in micellar bile salt modulation of CEL enzymatic activity through intramolecular hydrogen bonding with the C-terminal domain. These residues are also important, probably through similar intramolecular hydrogen bond formation, in stabilizing the enzyme in solution and at the lipid-water interface.     

Effect of a new CCK-A receptor antagonist,dexloxiglumide, on the exocrine pancreas in the rat

The effect of dexloxiglumide, a new potent cholecystokinin (CCK) antagonist, on pancreatic enzyme secretion and growth was studied in the rat. Pancreatic exocrine secretion was studied both in vitro (isolated and perfused pancreatic segments) and in vivo (anaesthetized animals with cannulation of the common bile duct) whereas the trophic effect was investigated after short-term (7 days) administration of the CCK-agonist, caerulein, or camostate (a potent trypsin inhibitor), with or without dexloxiglumide. CCK-8 stimulated amylase release from in vitro pancreatic segments in a concentration-dependent manner. Dexloxiglumide displaced the concentration response curves to CCK-8 to the right without affecting the maximum response, suggesting a competitive antagonism. The Schild plot analysis of data gave a straight line with a slope (0.90±0.36) not significantly different from unity. The calculated pA₂ for dexloxiglumide was 6.41 ± 0.38. In vivo experiments confirmed results from in vitro studies since intravenous dexloxiglumide reduced pancreatic exocrine secretion induced by submaximal CCK-8 stimulation (0.5 nmol/kg/h) in a dose-dependent manner, the ID₅₀ being 0.64 mg/kg. Both exogenous and endogenous (released by camostate) CCK increased the weight of the pancreas, the total pancreatic protein and DNA, trypsin and amylase content. Dexloxiglumide (25 mg/kg), administered together with caerulein (1 μg/kg), reduced the peptide-induced increase in pancreatic weight, protein and enzyme content. Similarly, when dexloxiglumide was given together with camostate (200 mg/kg), all the observed changes were reduced by concomitant administration of the antagonist. These results demonstrate the ability of dexloxiglumide to antagonize the effects of CCK on pancreatic secretion and growth, suggesting that this compound is a potent and selective antagonist of CCK-A-receptors in the pancreas.     

Chromogranin B (secretogranin I), a neuroendocrine-regulated secretory protein, is sorted to exocrine secretory granules in transgenic mice.

Chromogranin B (CgB, secretogranin I) is a secretory granule matrix protein expressed in a wide variety of endocrine cells and neurons. Here we generated transgenic mice expressing CgB under the control of the human cytomegalovirus promoter. Northern and immunoblot analyses, in situ hybridization and immunocytochemistry revealed that the exocrine pancreas was the tissue with the highest level of ectopic CgB expression. Upon subcellular fractionation of the exocrine pancreas, the distribution of CgB in the various fractions was indistinguishable from that of amylase, an endogenous constituent of zymogen granules. Immunogold electron microscopy of pancreatic acinar cells showed co-localization of CgB with zymogens in Golgi cisternae, condensing vacuoles/immature granules and mature zymogen granules; the ratio of immunoreactivity of CgB to zymogens being highest in condensing vacuoles/immature granules. CgB isolated from zymogen granules of the pancreas of the transgenic mice aggregated in a mildly acidic (pH 5.5) milieu in vitro, suggesting that low pH-induced aggregation contributed to the observed concentration of CgB in condensing vacuoles. Our results show that a neuroendocrine-regulated secretory protein can be sorted to exocrine secretory granules in vivo, and imply that a key feature of CgB sorting in the trans-Golgi network of neuroendocrine cells, i.e. its aggregation-mediated concentration in the course of immature secretory granule formation, also occurs in exocrine cells although secretory protein sorting in these cells is thought to occur largely in the course of secretory granule maturation.     

Three-day continuous drainage of pancreatic juice in the cortisone-treated conscious rat: Analysis of enzyme activities and ultrastructural changes

Summary We have investigated the short-term effects of hydrocortisone (60 mg/kg per day) and placebo on basal and stimulated pancreatic secretion in the conscious rat. Volume and enzyme secretion were determined; fine structural changes were examined simultaneously.The pancreatic and bile ducts were cannulated separately; pancreatic juice was drained via an isolated fistula, and bile was recirculated into the duodenum. The application of hydrocortisone led to an almost complete inhibition of the secretory response of the exocrine pancreas when stimulated with 0.25 U secretin in combination with 5 × 10^-8 g caerulein per h. It strongly affected the secretion rates of volume, protein, lipase, chymotrypsin, trypsin and carboxypeptidase, whereas the secretion rate of alpha-amylase continued to show a slight increase after stimulation.After stimulation with secretin and caerulein, the hydrocortisone-treated animals showed a higher density of zymogen granules in the acinar cell and an increase in the number of autophagic vacuoles in comparison to the equally stimulated placebo-treated rats.It is concluded that the short-term inhibition of pancreatic secretion by hydrocortisone occurs largely as a result of an inhibition of cellular enzyme discharge.Supported by the Deutsche Forschungsgemeinschaft, Ga 279     

STUDIES ON DISPERSED PANCREATIC EXOCRINE CELLS : II. Functional Characteristics of Separated Cells

The functional characteristics of separated guinea pig pancreatic exocrine cells have been examined following dissociation of the gland by a procedure described in the previous paper (J. Cell Biol. 1974. 63:1037). The ability of isolated cells to incorporate labeled amino acids into secretory proteins was assessed biochemically and by quantitative electron microscope autoradiography. Incorporation remained linear for up to 4-h incubation at levels equivalent to those of pancreatic slices; over 95% of the exocrine cells in the population were viable, and all appeared to be equally active in incorporating amino acids. The capacity of separated cells to transport, concentrate, and store exportable proteins was monitored by electron microscope autoradiography on populations pulse labeled with [³H]leucine and chase incubated for 4 h. The same overall pathway previously mapped in pancreatic slices was followed by secretory proteins in separated cells although in quantitative studies a defect was noted in the rate of conversion of condensing vacuoles to zymogen granules. Secretogogue responsiveness was assessed by monitoring discharge of labeled secretory proteins or of amylase in response to carbamylcholine and caerulein to the medium. While the separated cells released secretory proteins linearly for up to 4 h in response to both secretogogues, the net release was ~50% less than previously noted for pancreatic slices and required a ten times higher concentration of stimulant. The defect may represent alteration in receptors due to the protease used for dissociation. Our data indicate, however, that separated exocrine cells retain their ability to process secretory proteins stepwise and vectorially which is consistent with preservation of structural polarity.     

Cell fractionation studies on the guinea pig pancreas. Redistribution of exocrine proteins during tissue homogenization

A double-label protocol was used to estimate the extent of leakage and relocation artifacts that affect exocrine pancreatic proteins in cell fractionation experiments. Guinea pig pancreatic lobules were pulsed in vitro with a mixture of 14C-amino acids to enable the lobules to produce and process endogenously labeled exocrine proteins. At the end of the pulse (10 min) or after an appropriate chase interval, the lobules were homogenized in 0.3 M sucrose to which a complete mixture of 3H-labeled exocrine pancreatic proteins was added as an exogenous tracer. The distribution of both labels was studied in each cell fraction of interest at the level of TCA-insoluble proteins and individual exocrine proteins resolved by using a two-dimensional gel system. Based on the premises that the exogenous and endogenous label behave identically during homogenization-fractionation and that all endogenously labeled exocrine proteins found in the postmicrosomal supernate come from intracellular compartments ruptured during tissue homogenization, a series of equations was derived to quantitate leakage and adsorption and to define the ratio of endogenous label still in its primary location to total label (primary location index or PLI) for each cell fraction. Leakage was found to be uniform for all exocrine proteins, but unequal in extent from different cell compartments (condensing vacuoles is greater than zymogen granules is greater than rough endoplasmic reticulum) ; it increased with exposure to shearing forces especially in the case of zymogen granules and condensing vacuoles, and was substantially reduced from rough microsomes by adding 10 mM KCl to the homogenization media. Relocation of exogenous label by adsorption to other subcellular components was extensive (approximately 55%), uneven (free polysomes is greater than rough microsomes is greater than smooth microsomes and zymogen granules), preferential (cationic proteins are massively adsorbed to ribosomes and membranes, resulting in a complementary enrichment of the post-microsomal supernate with anionic exocrine proteins), and reversible (with successive 50-100 mM KCl washes). After correction for adsorption and leakage, the kinetics of intracellular transport derived from cell fractionation data were found to be nearly identical to those obtained from quantitative autoradiographic studies.     

Lipid and exocrine pancreatic ultrastructural changes due to experimental diabetes.

The aim of this study was to establish if the changes in the ultrastructure of the exocrine part of the pancreas are correlated with changes in serum glucose, cholesterol and lipoprotein fractions during the progression of diabetes in rabbits. Diabetes mellitus was induced in male New Zealand rabbits by a single injection of alloxan into the auricular vein. On the day 7th the glucose level in the whole blood was measured and this day was designated as the first day of diabetes. Rabbits were divided into 5 groups: untreated control, 21-day diabetes, 42-day diabetes, 90-day diabetes and 180-day diabetes. The cholesterol, HDL (high-density lipoprotein) and LDL (low-density lipoprotein) levels were examined in the serum. The total pancreatic lipase activity was measured spectrophotometrically in the pancreatic homogenate. Histological specimens were examined under an electron microscopy. The glucose level increased significantly in all of the alloxan exposed animals. The significant elevation of cholesterol level was observed on day 21 and 180. The HDL level was increased (P<0.05) only on the day 21st. The LDL level and the total activity of pancreatic lysosomal lipase increased significantly on day 21, 42 and 90. Further dilation of granular endoplasmic reticular ducts and decrease in the number of zymogen granules were observed amongst exocrine cells. Fragmented mitochondrial and translucent matrix were also seen. Intensification of the pancreatic fibrosis was found on day 90. Microvascular changes were reported in exocrine cells after 180 days. Their nuclei were smaller with large bulges on the nuclear membrane, and the number of heterogeneous electron granules of zymogen further declined. We concluded that the intensification of ultrastructural changes of the exocrine part of the pancreas correlated with the changes of the pancreatic lipase activity, and glucose and lipoprotein levels.