Detection of cytomegalovirus nucleic acid sequences in pancreas in type 2 diabetes.

Nucleic acid sequences specific for human cytomegalovirus (CMV) were found in samples of pancreatic tissue from patients with non-insulin-dependent (type 2) diabetes mellitus. RNA extracted from paraffin-embedded or fresh-frozen specimens from 14 of 32 (44%) diabetic patients but from none of 49 non-diabetic controls reacted with 10 kb (pJN201) or 6.6 kb (pCM3) probes of human CMV immediate-early or late gene products, respectively. The RNA from the 32 diabetic patients did not react with nucleic acid probes for mumps, rubella, or coxsackie B viruses. In-situ nucleic acid hybridisation on tissues from 5 randomly selected human-CMV-positive patients showed that the human CMV signal was localised primarily in the islets of Langerhans and not in exocrine cells. Despite the clear viral nucleic acid signal in tissues of human-CMV-positive patients, there were no morphological injuries to the islets, no inflammatory cells in the islets, and no perivascular inflammatory cell cuffing. These findings suggest a possible association of human CMV with type 2 diabetes in human beings.

Potentiation of azaserine by cholestyramine in the rat.

In the rat, when pancreatic growth is stimulated there is an increased incidence of spontaneous pancreatic neoplasms and marked potentiation of the pancreatic carcinogen azaserine. Since previous studies showed that cholestyramine caused pancreatic growth in this species we have now studied the effect of azaserine in rats fed soya flour diets containing cholestyramine. Two groups, each of eight rats, were fed either heated soya flour (HSF) or raw soya flour (RSF). Two further groups, each of 12 rats, received the same diets containing 2% cholestyramine (HSF + C, RSF + C). In each group, four rats received azaserine (30 mg/kg i.p.) and the remainder saline, weekly, for the first 5 weeks. Animals were killed after 24 weeks and pancreatic growth and the number and size of pancreatic neoplastic nodules was measured. RSF caused a significant increase in pancreatic weight, protein, RNA and DNA, compared with HSF and cholestyramine caused a further significant increase in pancreatic weight, protein and RNA but not DNA. Azaserine did not affect pancreatic growth. In azaserine-injected rats significantly more nodules were seen and the nodules were larger and the tumour burden greater in rats fed HSF + C than in rats fed HSF alone. However, the nodule count and other nodule parameters were not significantly different in RSF and RSF + C fed rats. It is concluded that 2% cholestyramine enhances pancreatic growth when added to soya flour diets and in rats fed HSF it potentiates the action of azaserine on the pancreas. It does not increase the potentiation of azaserine seen with RSF up to 24 weeks.

Trophic stimulation of the ductal/islet cell axis: a new approach to the treatment of diabetes.

The purpose of this study was to demonstrate that the pancreas of the hamster contains a growth factor(s) that can induce cells associated with the ductular epithelium to differentiate along an endocrine pathway and thereby provide a means of regenerating a functioning islet cell mass. We have shown previously that partial obstruction of the pancreatic duct leads to the induction of nesidioblastosis. A cytosol extract prepared from the partially obstructed hamster pancreas was injected at a dose of 4000 microliters intraperitoneally twice a day for 2 days and produced significant increases in pancreatic weight, protein, and deoxyribonucleic acid of 18%, 18% and 42% respectively, over saline-treated control animals. To assess the effects of this extract on morphology, 150 microliters intraperitoneally twice a day was administered for 21 days. Tissue was processed for histologic, morphometric, and autoradiographic analysis. Budding of endocrine cells from cells of the terminal ductules was observed in cytosol-injected animals and the number of islets per square millimeter was determined to be increased by 100% compared with saline-treated controls (p less than 0.01). Tritiated thymidine uptake by ductal and islet cells was increased tenfold and sixfold, respectively, over that of control animals (p less than 0.01). Cytosol extract was also administered to hamsters rendered diabetic by streptozocin. Survival in these animals was 100% compared with only 60% for saline-treated control animals (p less than 0.05). Furthermore, the blood levels of glucose in cytosol-treated animals was significantly less than the levels in saline-treated controls (p less than 0.05). We conclude that the pancreas does indeed contain a growth factor(s) responsible for the induction of nesidioblastosis and the new islet tissue is functionally capable of stabilizing a diabetic state.

Immune recognition of affinity-labelled cholecystokinin receptor.

The present study was undertaken to characterize the immune recognition of pancreatic cholecystokinin receptor by an anti-cholecystokinin antibody. Cholecystokinin receptor from pancreatic plasma membranes was photoaffinity labelled using the specific, cleavable probe 125I-labelled 2-(p-azidosalicylamido)-1,3-dithiopropionate-[Thr28,Ahx31 ]CCK(25-33) [CCK(25-33) is the C-terminal nonapeptide of the 33-amino-acid form of cholecystokinin]. Labelled receptor was then solubilized and subsequently prepurified on immobilized wheat-germ agglutinin. The C-terminal-directed anti-cholecystokinin serum (8E) specifically immunoprecipitated a fraction of affinity-labelled cholecystokinin receptor which was identified at Mr 85,000 - 100,000 on SDS/PAGE. The binding affinity of antiserum 8E for covalently labelled cholecystokinin receptor was lower (Kd 0.11 +/- 0.02 nM) than for cholecystokinin (Kd 3.65 +/- 0.55 pM). The compound L364-718, an A-subtype cholecystokinin-receptor antagonist did not interfere with the immune recognition of cholecystokinin. However, the recognition of affinity-labelled cholecystokinin receptor was enhanced as a result of an increasing availability of cholecystokinin molecules. Indeed, the amount of immunoprecipitated receptor was doubled in the presence of 10 microM L364-718. This study offers the possibility of using an anti-cholecystokinin antibody for cholecystokinin-receptor purification and demonstrates that prepurified affinity-labelled cholecystokinin receptor retains A-subtype specificity.

Essential fatty acid deficient rats in the study of cystic fibrosis: an X-ray microanalytical and ultrastructural study in chronically reserpinized rats.

Essential fatty acid (EFA) deficiency has been proposed as a major pathogenic mechanism for cystic fibrosis (CF) and EFA-deficient animals have been proposed as an animal model for CF. In the present study, the elemental composition and ultrastructure of the acinar cells of the submandibular and parotid gland of the pancreas of EFA-deficient rats were investigated by X-ray microanalysis and electron microscopy. The effects of EFA-deficiency were compared to changes in these exocrine glands in chronically reserpine-treated rats, an established animal model for CF. EFA-deficiency did not cause any significant changes in the elemental composition of the acinar cells of the submandibular or parotid gland, or of the pancreas. The changes in elemental composition induced by reserpine treatment were only slightly modified by EFA-deficiency, mainly towards normalization. EFA-deficiency resulted in the presence of abnormal, electron translucent, zymogen granules in the parotid gland and in a reduction of the number of zymogen granules in pancreatic acinar cells. Since EFA-deficiency in rats only causes minor changes in structure and elemental composition of salivary glands and pancreas, and does not potentiate the effect of chronic reserpine treatment on these tissues, it is concluded that EFA-deficiency is likely to be of minor importance in the exocrine gland disturbances in CF.

Changes in free amino acid levels in various tissues of common carp in response to insulin injection followed by force-feeding an amino acid diet.

The effects of a single injection of porcine insulin on free amino acid levels in plasma, erythrocytes, hepatopancreas and skeletal muscle were simultaneously monitored in common carp; the fish were force-fed a complete diet composed of crystalline amino acids as the sole protein precursors (amino acid diet) to study the dynamics of amino acid metabolism. The force-feeding of the amino acid diet caused surges in the concentrations of almost all amino acids in fish injected with saline, and amino acid levels reached peaks within 1 h in plasma as well as in hepatopancreas. It took more than 2.5 h for most amino acids to reach maximum levels in erythrocytes and skeletal muscle of the same fish. The injection of insulin stimulated drastic reductions of free amino acid levels in the plasma. At the same time, it facilitated reduction of free amino acid levels without elevating glutamine and ammonia levels in erythrocytes, hepatopancreas or skeletal muscle. These results suggest that exogenous insulin accelerated assimilation of dietary free amino acids and their deposition in these tissues.

Islet amyloid polypeptide, a novel pancreatic peptide, is a circulating hormone secreted under glucose stimulation.

Islet amyloid polypeptide (IAPP) was identified in human plasma using immunoaffinity chromatography, gel filtration and reverse- phase high performance liquid chromatography coupled with radioimmunoassay specific for the peptide. IAPP[1-37], IAPP[17-37], and other two IAPP- related peptides which were putative pro-IAPPs or different processing products of IAPP, were isolated. All of these IAPPs were also found in human pancreatic extract, indicating that they were secreted from B cell secretory granules into the circulation. IAPP[1-37] is a major molecular form of IAPP in the pancreas, but it accounted for 31% of immunoreactive IAPP in the plasma. Plasma concentration of IAPP in normal individuals increased to 3.0 times the basal level in response to oral administration of 75 g glucose. This study indicated that IAPP is a circulating hormone secreted under glucose stimulation.

Regional distribution and molecular forms of rat islet amyloid polypeptide.

Using a highly sensitive and specific radioimmunoassay (RIA) for rat islet amyloid polypeptide (IAPP), we clarified regional distribution and molecular forms of rat IAPP. IAPP[1-37] and IAPP[19-37] were identified in normal rat pancreas by sequence analyses IAPP[19-37], accounting for 57% of IAPP-immunoreactivity in rat pancreas, is a major molecular form of rat IAPP moiety. In human, however, IAPP[1-37] is the major component, with IAPP[17-37] composing as little as 2-6% of IAPP-immunoreactivity in pancreas. This indicates that processing of IAPP in pancreas differs in species. A large amount of IAPP (328.5 +/- 25.0 pmol/g wet weight) was found in rat pancreas and the peptide was also detected in pyloric antrum of the stomach, duodenum, jejunum, ileum, and colon at 0.1-0.8% of the level of pancreas. It was not detected in central nervous system. The content of rat IAPP in pancreas fell to 54% of control after 4 day fasting. The distribution of IAPP suggests its possible endocrine or paracrine function in pancreas and gastrointestinal tract.

Diffrerential cell reactivity to the zinc iodide-osmium tetroxide mixture of rat pancreatic acinar cells / Reactividad diferencial de las células acinares pancreáticas a yoduro de zinc-tetraoxido de osmio

Se investigó la actividad e las células pancreáticas exócrinas de rata a la tinción de yoduro de zinc y tetróxido de osmio (ZIO) en microscópia de luz y electrónica. Claramente se distinguieron tres tipos de células acinares: intensamente reactivas (I-PAC), moderadamente reactivas (M-PAC) y no reactivas a la reacción de ZIO (N-PAC). las I-PAC mostraron finos depósitos electrodensos homogéneamente distribuidos en el cisterna perinuclear, retículo endoplásmico, aparato de Galgi y vesículas pequeñas en la región apical. En las M-PAC, el producto de reacción solamente se observó en la cisterna perinuclear y en vesículas pequeñas entre los gránulos de zimógeno. No obstante que la mayoría de las N-PAC no mostraron producto de reacción en sus componentes, algunas células mostraron material electródenso en escasas vesículas entre los gránulos de zimógeno. Se descartó la posibilidad de que cambios en la permeabilidad de las membranas celulares pudieran ser factor determinante del grado variable de acceso de los reactivos de ZIO al citoplasma, con el empleo de peroxidasa de raíz fuerte como marcador de espacio intercelular. De acuerdo con los elementos intracelulares con producto de reacción, se concluyo que las diferencias en la ZIO-reactividad de las células pancreáticas exócrinas en la rata pudiera ser debido a diferentes estados metabólicos o funcionales de las células acinares, preincipalmente relacionados con la síntesis, transporte, almacenamiento y secreción de proteínas por las células acinares (

Measurement of 5-fluorouracil and its active metabolites in tissue.

The 5-fluorouracil content of serum, bile, pancreatic juice, liver, pancreas and muscle was measured by reversed-phase high-performance liquid chromatography using a mobile phase of 5 mM 1-heptanesulfonic acid in 5 mM acetic acid. Free or unmetabolized 5-fluorouracil was extracted from samples with a mixture of light petroleum-n-propanol (40:60). The active metabolites of 5-fluorouracil were hydrolyzed with hot perchloric acid to free 5-fluorouracil and the combined 5-fluorouracil content was extracted. The active metabolite fraction was calculated from the difference between the combined and the free fractions. A straight line plot of the peak areas against concentration was achieved and the detection limit was 50 ng/ml. Five minutes after stopping an intravenous infusion of 15 mg/kg of 5-fluorouracil in a dog, the serum contained only the free form, but other body fluids and tissues contained both free and metabolite fractions. The method may be useful to determine the amount of total drug in patient samples.

Immunohistochemical localization of the glucocorticoid receptor in pancreatic beta-cells of the rat.

We used a monoclonal antibody against an epitope located in the N-terminal moiety of the rat glucocorticoid receptor to identify the glucocorticoid receptor-containing cells in the rat pancreas. Monospecific polyclonal antisera against insulin, glucagon, somatostatin, and amylase were applied to serial sections in colocalization studies to identify the respective endocrine and exocrine cells. Glucocorticoid receptor immunoreactivity was exclusively present in nuclei and cytoplasm of the beta-cells of pancreatic islets. Western blots using the glucocorticoid receptor antibody resulted in identical 94K immunoreactive proteins in both liver and pancreas. After adrenalectomy, the glucocorticoid receptor immunoreactivity of beta-cells decreased significantly. A computer-assisted method of semiquantitative evaluation of the glucocorticoid receptor immunoreactivity demonstrated a significant decrease in the staining intensity of the beta-cells by 23.5% and in that of insulin antibodies by 10.4%, while amylase immunoreactivity was only slightly decreased. Serum levels of corticosterone determined by RIA decreased from 225 micrograms/ml in sham-operated animals to 55 micrograms/ml in animals 14 days after adrenalectomy, while the tissue content of amylase decreased by 45%. The immunohistochemical findings give circumstantial evidence of the presence of glucocorticoid receptor in beta-cells. We interpret our data as indicating an indirect effect of glucocorticoids on amylase synthesis via a glucocorticoid-insulin-exocrine cell pathway.

Identification of immunoreactive pancreatic stone protein in pancreatic stone, pancreatic tissue, and pancreatic juice.

We first examined whether pancreatic stone protein (PSP) was present in pancreatic stone and normal pancreatic tissue. By using HPLC and Western blotting, a protein of Mr 13.5 kDa that reacted with monoclonal antibody against PSP was detected as a major component in EDTA-soluble fractions of pancreatic stone. In an in vitro experiment, this protein dose-dependently suppressed CaCO3 precipitation. PSP was immunohistochemically stained in the acinar cells of normal pancreatic tissue. Based on these findings, it seemed that PSP in pancreatic stone is probably a physiological secretory protein of the pancreas. We subsequently examined immunoreactive PSP in normal pancreatic juice by the Western blotting method. In all of the specimens, the band for immunoreactive PSP in pancreatic juice was found to correspond to 13.5 kDa, which thus agreed with that of purified PSP from a stone.

Identification of disulfide-containing peptides in endocrine tissue extracts by HPLC-electrochemical detection and mass spectrometry.

A new procedure to selectively identify disulfide-containing peptides in extracts of biological tissues is described. Disulfide-containing peptides are detected by their UV absorbance and electrochemical (EC) activity after chromatographic separation, and subsequently identified by fast atom bombardment mass spectrometry (FABMS). This combination of fractionation by HPLC and selective detection is attractive because it is rapid, highly specific for disulfide-containing peptides, and applicable to all disulfide-containing peptides that may be present in complex biological mixtures. Useful procedures for applying the method are demonstrated with tissue extracts from bovine pituitary and catfish pancreas. In addition to finding the expected disulfide-containing peptides, evidence for two forms of catfish insulin are presented. The merits of this and other methods used to detect peptides in similar tissue extracts are discussed.

Diabetes is not associated with a change in the elemental composition of the pancreatic B cell in diabetic C57BL KsJ-db/db mice.

Freeze-dried pancreas sections from 7-, 17- and 27-week-old genetically diabetic (db/db) and normal (+/-/+/-) mice were subjected to proton bombardment and the concentrations of 15 elements in B cells and exocrine pancreas were calculated from the characteristic X-rays emitted. In the 7-week-old diabetic animals, B cells contained significantly above-normal levels of Na and S, while exocrine pancreas contained subnormal levels of Ca, and excess Mn. The B cells from the 17-week-old diabetic animals contained subnormal levels of Cu and the exocrine pancreas of the 27-week-old diabetic animals was deficient in Cd. The 7-, 17- and 27-week-old, genetically diabetic (db/db) mice were hyperglycemic, hyperinsulinemic and heavier than age-matched normal (+/-/+/-) mice. Although significant changes were found in elemental composition when comparing both B cells and exocrine pancreas at different ages, the changes were not consistent. Therefore, it appears as if the measured elemental changes were random and not related to the onset of diabetes.

DNA adducts in different tissues of smokers and non-smokers.

Purified DNA from human lung, liver, bladder, pancreas, breast and cervix has been analysed for DNA adducts using the nuclease P1 modification of the 32P post-labelling technique. Tissues were obtained at autopsy from 13 men and 6 women. Relatives were asked to provide information on smoking history for deceased subjects. All tissues examined except the breast had detectable adducts. In lung, bladder and pancreatic tissue a characteristic pattern of adducts was seen which has previously been reported as typical of cigarette-smoke-induced damage. Smokers and former smokers tended to have higher adduct levels than non-smokers in the tissues examined but this was only significant for the lung. There appeared to be considerable variation in adduct levels among smokers which could not be accounted for by duration or daily consumption level. Certain smokers had high adduct levels in all tissues examined, whilst in others high levels were only seen in some tissues. All cervical samples examined had detectable adducts. These results confirm the finding that cigarette smoking is associated with DNA damage in the lung and suggest that similar damage may be related to tobacco-induced neoplasms of other tissues.

Mechanism of oxygenation of pancreas during preservation by a two-layer (Euro-Collins' solution/perfluorochemical) cold-storage method.

To clarify the mechanism of oxygenation of the pancreas during preservation by two-layer (Euro-Collins' solution [EC]/perfluorochemical [PFC]) cold-storage method, the pancreas viability in the canine model of the pancreatic autotransplantation and tissue concentration of adenosine triphosphate were examined after 24-hr preservation by original and modified two-layer methods with respect to the position of the pancreas and oxygen bubbling into the PFC. Namely, the pancreas was in EC and on the surface of PFC with (group 1, original method) or without (group 2) oxygen bubbling into PFC. The pancreas was floated in EC with oxygen bubbling into PFC (group 3); compared with simple cold storage of the pancreas in EC (group 4); and nonpreserved pancreas (control, group 5). The preserved pancreas grafts by each method functioned immediately after transplantation and maintained normoglycemia for at least 5 days except that 1 of 5 dogs in group 4 died of a cause unrelated to the pancreas graft. The functional success rates of groups 1, 2, 3, 4, and 5 were 100%, 100%, 100%, 80%, and 100%, respectively. It was clear that mitochondrial function was well-preserved during 24-hr preservation regardless of the preservation method. In the condition that the mitochondrial function is well-preserved the tissue concentration of ATP was mostly dependent on the tissue oxygenation. The tissue concentration of ATP of group 1, 7.92 +/- 1.06 mumol/g dry weight, was significantly higher than that of nonpreserved pancreas (group 5), 4.44 +/- 0.49 mumol/g dry weight (P less than 0.01). It was apparent that the two-layer method was excellent to supply oxygen to the pancreas and maintain high ATP concentration of the pancreas during preservation. In contrast, ATP concentration of the pancreas of group 2 was 1.83 +/- 0.30 mumol/g dry weight, and there was no significant difference between group 2 and group 4, 1.19 +/- 0.33 mumol/g dry weight, thus meaning that PFC was biologically inert without oxygenation. In addition, when the pancreas was not contacted with oxygenated PFC and floated in EC (group 3) ATP concentration of the pancreas, 2.24 +/- 0.90 mumol/g dry weight, was significantly lower than group 1 (P less than 0.01), and no significant different was found as compared to groups 2 and 4. It was essential that the pancreas was contacted with oxygenated PFC to maintain high ATP tissue concentration during preservation by the two-layer method.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunohistochemical demonstration of carcinogen-DNA adducts in target and non-target tissues of rats given a prostate carcinogen, 3,2'-dimethyl-4-aminobiphenyl.

An immunohistochemical procedure was applied which allows accurate localization of DNA lesions within organs and tissues of rats given 3,2'-dimethyl-4-aminobiphenyl (DMAB) using polyclonal antibodies against DMAB-DNA adducts. Dose-related nuclear staining was observed in organs regardless of DMAB-carcinogenic organotropism. In the male accessory sex organs, the lateral lobe of the prostate, a non-target site, demonstrated a similar staining intensity to that found for the ventral prostate and seminal vesicle, target sites. Orchiectomy and pretreatment with ethinyl estradiol resulted in a moderate to slight decrease in binding in the accessory sex organs. No observable decrease in staining intensity was evident in most organs 168 h after the administration of DMAB. These findings suggest that DNA adduct formation itself is not necessarily sufficient for tumor induction.

Galanin in the porcine pancreas.

Galanin, a 29 amino acid neuropeptide, was recently isolated from pig intestine. We studied the localization, nature and effect of galanin in pig pancreas. Galanin immunoreactive nerve fibers were regularly found in the pancreas. A peptide chromatographically similar to synthetic galanin was identified in pancreas extracts. The effect of galanin on the endocrine and exocrine secretion was studied in isolated pancreases, perfused with a synthetic medium containing 3.5, 5 or 8 mmol/l glucose and synthetic galanin (10(-10)-10(-8) mol/l). There was no effect on the basal exocrine secretion. The output of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) was measured in the effluent. There was no effect on PP secretion. At a perfusate glucose concentration of 5 mmol/l, galanin at 10(-9) mol/l increased insulin secretion by 55 +/- 14% (mean +/- S.E.M., n = 5) of basal secretion, and at 10(-8) mol/l by 58 +/- 27% (n = 6). At 8 mmol/l glucose, insulin secretion increased by 25 +/- 10% (n = 6) and 62 +/- 17% (n = 8). At 5 mmol/l glucose glucagon secretion was increased by 15 +/- 3% (n = 5) by galanin at 10(-9) mol/l and by 29 +/- 11% (n = 5) by galanin at 10(-8) mol/l, and at 8 mmol/l glucose by 66 +/- 27% and 41 +/- 25%. Somatostatin secretion was inhibited to 72 +/- 2% (n = 5) of basal secretion by galanin at 10(-9) mol/l and to 65 +/- 7% (n = 7) at galanin at 10(-8) mol/l, both at 5 mmol/l glucose. At 8 mmol/l the figures were 83 +/- 6% and 70 +/- 10%. Insulin secretion in response to square wave increases in glucose concentration from 3.5 to 11 mmol/l (n = 5) increased 2-fold during simultaneous perfusion with galanin (10(-8) mol/l).