摘要:

Effects of green tea catechins onN-nitrosobis(2-oxopropyl)amine (BOP)-induced oxidative stress in pancreas and liver were examined. Hamsters were divided into two groups: one group was given free access to a 0.1% solution of green tea catechins as drinking water (c-ham) and the other to plain tap water (w-ham) for 1 week before subcutaneous injection of BOP 20 mg/kg body weight. Zero, 1, 2, 6, 12, 24, and 48 h after BOP injection, the pancreas and liver were excised and the tissue concentration of lipid peroxides (TBA values) and the amount of 8-hydroxydeoxyguanosine (8-OHdG) in nuclear DNA were measured. The concentration of lipid peroxides and the amount of 8-OHdG in the pancreas showed similar patterns of change between c-and w-ham. Soon after BOP injection, the concentration of lipid peroxides and the amount of 8-OHdG increased with a peak at 1 and 6 h, respectively. Their peak values of c-ham were significantly depressed compared with those of w-ham. Both levels returned to steady-state levels by 24 h. In the liver, the concentration of lipid peroxides and the amount of 8-OHdG were not affected by BOP administration. These results suggest that BOP induces oxidative damages in the target organ and oral intake of green tea catechins has a protective effect on the oxidative stress.

ISSN:0885-3177    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

In non-insulin-dependent diabetes, circulating insulin-related immunoreactivity (IRI) is often composed of a higher fraction of the incompletely converted forms proinsulin and des-31, 32 proinsulin. The present study describes an immunoadsorption method for measuring the proportions of proinsulin, its two split products, and insulin in human pancreatic tissue and for determining their rates of formation in human isolated islets. The method uses two junction-specific monoclonal proinsulin antibodies in a protein G fractionation; it is validated by ≥90% specificity and recovery. The peptide contents measured in tissue extracts were comparable to those determined in a previously developed immunoradiometric assay. In the nine tissue extracts from nondiabetic donor organs, 97% of IRI corresponded to insulin, 1% to proinsulin, 2% to the des-31, 32 proinsulin conversion product, and 0.1% to des-64, 65 proinsulin. Two samples from non-insulin-dependent diabetics under sulfonylurea treatment contained a fourfold lower content of IRI but the peptide distribution was comparable except for a low percentage (0.3) of proinsulin in one case. In pulse-chase experiments on three preparations of human islets isolated from nondiabetic donors, proinsulin represented the major (>90%) IRI that was synthesized at the end of the 30-min pulse; a subsequent 90-min chase at either 2.5 or 10 mMglucose resulted in conversion of 75% of proinsulin to des-31, 32 (20%) and des-64, 65 (2%) intermediates and to insulin (50%); after a 180-min chase, 88% of proinsulin was converted to insulin, but 10% remained present as proinsulin. In a pulse-chase experiment on islets isolated from tissue with a high proportion of des-31, 32 intermediate (5% instead of 2%), the conversion process was slower (45% after 90 min and 70% after 180 min) and resulted in a higher fraction of des-31, 32 intermediate, suggesting that the elevated tissue content in this intermediate is caused by a reduced PC2 converting activity. These data confirm that des-31, 32 proinsulin represents the major conversion intermediate in normal human islets and indicate the existence of slow converters, possibly as a result of decreased enzymatic processing of the prohormone's AC junction.

ISSN:0885-3177    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

To define the functions of retinoids and their receptors in insulin secretion, we tested the effects of all-trans-retinoic acid (ATRA) and retinoic acid receptor (RAR) expression on cell growth, differentiation, and secretion using insulin-secreting RINm5F cells. Wild-type cells with a low abundance of mRNA for RARβ were transfected with RARβ or chloramphenicol acetyltransferase (CAT control). Cells were cultured for 2–7 days in media without (A-def) or with ATRA, 1, 10, 100, and 1,000 nM. At day 2 of culture, ATRA stimulated insulin release in wild-type and transfected cells, and this effect was dose dependent. At 7 days, ATRA stimulated insulin secretion from wild-type cells twofold at glucose concentrations of 0.5 mM(A-def, 5.1 ± 0.27; ATRA, 1,000 nM, 10.5 ± 1.43 ng/106cells) and at 11.0 mM(A-def, 6.9 ± 0.24; ATRA, 1,000 nM, 13.6 ± 1.86 ng/106cells). The cellular insulin content was increased about threefold (A-def, 39.2 ± 2.95; ATRA, 1,000 nM, 118 ± 8.54 ng/106cells). ATRA inhibited growth of wild-type cells as early as 3 days, and this effect was dose dependent. Whereas in the absence of ATRA, the cell number increased over fivefold between day 3 and day 5, ATRA, 1,000 nM, inhibited cell growth completely. ATRA, 1,000 nM, increased apoptotic RINm5F cells (day 3 A-def, 0.53 ± 0.27% of total cells, and ATRA, 2.30 ± 1.44; day 5 A-def, 0.38 ± 0.23, and ATRA, 2.14 ± 0.59; day 7 A-def, 0.90 ± 0.29, and ATRA, 6.02 ± 1.64). RARβ-transfected cells showed overexpression of mRNA to RARβ and dose-dependent inhibition of growth, with almost-complete inhibition at ATRA concentrations as low as 100 nM. Overexpression of RARβ increased insulin secretion at ATRA, 100–1,000 nM. In summary, ATRA increased the insulin secretion and content of RINm5F cells, while inhibiting growth and increasing apoptosis. Increased expression of RARβ facilitated these effects on growth and secretion. These findings may reflect the known effect of ATRA on differentiation of cells and mediation through RARβ.

ISSN:0885-3177    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

In this study we assessed whether conditioned media from a human pancreatic cancer cell line (MIA PaCa 2) can interfere with some intracellular pathways involved in glucose metabolism in isolated rat hepatocytes. The hepatocytes, isolated from Male Wistar rats, were incubated with MIA PaCa 2-conditioned or nonconditioned media. Conditioned and nonconditioned hepatocytes were run for 120 min in the presence or absence of insulin (100 mM) and were sampled at fixed time intervals. Supernatant glucose levels decreased to a similar extent over time in both conditioned and nonconditioned hepatocytes, while lactate levels significantly increased in nonconditioned hepatocytes with respect to conditioned hepatocytes. A pyruvate kinase activity increase was observed only in nonconditioned hepatocytes and was biphasic in nature, since this increased activity was detected both after a few and after 30 min following insulin stimulation. The cyclic AMP level increase was significantly higher in conditioned than in non-conditioned hepatocytes. It appears that MIA PaCa 2 cells produce a factor(s) that may interfere with one of the insulin-mediated intracellular pathways of glucose metabolism, namely, glycolysis. This detrimental effect on glycolysis is supported by the blunted rise in lactate concentration in the medium after the glucose challenge. This substance(s) probably transfers its signal inside the target cells, activating the adenylate cyclase pathway. These results support the hypothesis that pancreatic cancer is the cause rather than the consequence of diabetes mellitus.

ISSN:0885-3177    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

GAD65 is targeted by different patterns of autoantibodies [glutamic acid decarboxylase (GAD)-AAbs] in insulin-dependent diabetes mellitus (IDDM) and stiff-man syndrome (SMS). To study differentiation of the GAD-AAb pattern by immunohistochemistry, we examined the immunostaining of 15 monoclonal GAD antibodies (mc-GAD-Abs), which recognized different epitope regions of the antigen, on human pancreatic sections that were unfixed or fixed with different fixatives. By a competitive sandwich enzymelinked immunosorbent assay (ELISA), three binding patterns of mc-GAD-Abs were identified: 5 of 15 mc-GAD-Abs recognized a linear N-terminal epitope (p1), 5 of 15 were reactive with a conformational GAD65 epitope region (p2), and 5 of 15 were cross-reactive with GAD67 (p3). These patterns of mc-GAD-Abs were tested for islet cell binding by indirect immunofluorescence on pancreatic sections treated with either (1) Bouin's solution, (2) Zamboni's solution, or (3) phosphate-buffered formaldehyde for 0.5, 1, 2, and 18 h at 4°C. After fixation for up to 2 h no differentiation of immunoreactivity of patterns was observed using the three fixatives. mc-GAD-Abs recognizing conformational epitope regions (p2) revealed a marked reduced immunoreactivity on pancreatic sections fixed for 18 h with 4% formaldehyde, while mc-GAD-Abs reactive with linear epitopes (p1, p3) were detectable with strong binding. This fixation procedure was used to compare the immunoreactivity of GAD-AAb+or GAD-AAb−islet cell cytoplasmic antibody-positive (ICA+) sera of IDDM (n= 27) and SMS patients (n= 3). The three SMS sera were reactive with GAD on fixed islets but showed a reduced liter, whereas the majority of IDDM sera (22/27; 81.5%) were not detectable; 70.6% (12/17) of GAD-AAb+IDDM sera were not detectable on fixed islets. Furthermore, all 10 GAD-AAb−IDDM sera tested failed to react with fixed pancreas, which also suggested an alteration of non-GAD-ICA antigens. In conclusion, the fixation of human pancreatic sections with formaldehyde for 18 h allows the differentiation of GAD-AAbs recognizing linear and conformational epitope regions.

ISSN:0885-3177    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

The aim of this study was to examine the effect of total parenteral nutrition (TPN) on the endocrine and exocine function of the pancreas. Endocrine function was investigated using an intravenous glucose tolerance test (IGTT) in rats with TPN for 7 or 14 days. Exocrine function was evaluated by measuring amylase secretion from isolated acini as well as pancreatic weight, water content, protein, and enzymes after 7 days of TPN. When the TPN rats were compared with the controls, the glucose tolerance curve after an IGTT was unchanged, the basal plasma insulin levels were slightly lower and the insulin secretory response to intravenous glucose was markedly impaired. No differences could be seen between the insulin response after 7 days and that after 14 days of TPN. The weight of pancreas, the total content and concentration of pancreatic protein, and the total amylase content of the pancreas were lower, whereas the total content of both chymotrypsin and trypsin was higher. The concentration of DNA remained intact, whereas the total DNA content decreased. The levels of lipolytic enzymes, except for carboxylesterlipase, were unaffected. After TPN treatment, the insulin secretory response to glucose is impaired, the exocrine pancreas is hypoplastic and the storage pattern of pancreatic exocrine enzymes is altered.

ISSN:0885-3177    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Tumor relapse occurs frequently in patients with ductal pancreatic head cancer despite the absence of residual tumor detectable at primary surgery. Therefore it has to be assumed that current tumor staging procedures fail to detect minimal amounts of disseminated tumor cells present in secondary organs, which might be the seed for subsequent meta-static relapse. We evaluated lymph nodes from 18 patients without overt metastases who had undergone radical tumor resection (R0resection). Lymph nodes judged as “tumor-free” by routine histopathology were further examined for the presence of single tumor cells using immunohistochemistry with the antiepithelial monoclonal antibody Ber-EP4. Sixteen of 37 “tumor-free” lymph nodes (43.2%), obtained from 13 of 18 patients (72.2%), displayed Ber-EP4+tumor cells. Twelve of these 18 patients presented at pT2stage. Nine of 12 patients (75%) staged as pN0had these cells. Two of six pN1patients had no Ber-EP4+in histopathologically tumor-free lymph nodes. Using multivariate Cox's regression analysis, the presence of Ber-EP4+cells in “tumor-free” lymph nodes was an independent factor for a significantly reduced relapse-free survival (p= 0.006) and overall survival (p= 0.01). Remarkably, all patients who were restaged as lymph node negative by both histopathology and immunohistochemistry survived the observation period without recurrence. The frequent occurrence of disseminated tumor cells in patients with pancreatic cancer and their prognostic impact support the need for a refined staging system of excised lymph nodes, which should include immunohistochemical examination. Thus patients with a minimal residual tumor load who might benefit from an adjuvant therapy could be selected.

ISSN:0885-3177    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Cholecystokinin (CCK) can stimulate secretion and DNA synthesis in pancreatic acinar cells. Hyperstimulation with cerulein (a CCK analogue) induces acute edematous pancreatitis. To study the effects of in vivo pancreatic stimulation with cerulein, we analyzed the expression of the protooncogenes jun, myc, and fos on the mRNA and protein levels. RNA and protein were extracted from the pancreas of rats administered an infusion of cerulein, 10 μg/kg/h (Group A) or 0.25 μg/kg/h (Group B), or saline (Group C) and sacrificed 2, 4, and 6 h after beginning the infusion and 0, 12, and 24 h and 2, 4, and 6 days after completing the infusion period. Transcript levels were studied using slot-blot analysis. Protein expression was studied using Western blot and immunohistochemistry. No changes were found for the expression of protooncogenes myc and fos on either the transcript or the protein levels. Higher jun mRNA levels were found in Group A than in Group B or C, particularly after 2 h of infusion and 12, 24, and 48 h after the end of a 12-h cerulein infusion. No significant difference was observed in Groups B and C. The jun protein behavior was similar in Groups A and B, revealing two peaks: one early during infusion and a second one after the end of a 12-h cerulein infusion. Jun protein was found mainly in the acinar cells. In conclusion, (1) acinar cells in the rat pancreas respond to cerulein stimulation by increasing the expression of jun; (2) in vivo high doses of cerulein increase the jun mRNA and jun protein levels, whereas low doses raise only the protein levels; and (3) mye and fos are apparently uninfluenced by cerulein administration.

ISSN:0885-3177    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Enhanced synthesis and deposition of extracellular matrix (ECM) components is a characteristic feature during regeneration from acute cerulcin-induced pancreatitis in rats. Transforming growth factor β1 (TGFβ1) has been suggested to be an important modulator of the ECM by interfering with a number of essential processes such as the synthesis of ECM components. To study the involvement of the ECM degrading proteases (matrix metalloproteinases; MMPs) and their specific inhibitors in the process of pancreatic regeneration, we examined the expression of these genes on the transcript level and the activation of the corresponding enzymes by use of zymographies. Pancreatic RNA and protein were extracted from rats sacrificed 1, 2, 3, 5, and 7 days after induction of cerulein pancreatitis. To investigate the influence of TGFβ on gene expression of ECM proteases and their specific inhibitors, we blocked the activity of TGFβ1 during regeneration from acute pancreatitis by use of neutralizing antibodies against TGFβ1. Steady levels of 72-kD type IV collagenase (MMP-2), stromelysin (MMP-3), and tissue inhibitor of metalloproteinase 2 (TIMP-2) mRN A were significantly increased 2 days after induction of pancreatitis. MMP-9 and MMP-3 enzyme activity was elevated 12 h after induction of pancreatitis, whereas MMP-2 activity increased 12 h later. Inhibition of TGFβ1 by neutralizing antibodies only reduced the amount of stromelysin transcripts throughout pancreatic regeneration. In summary, ECM degrading proteases, in particular stromelysin, appear to be involved in ECM remodeling during pancreatic regeneration. TGFβ1 may be responsible for regulation of stromelysin transcription.

ISSN:0885-3177    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Obstruction of the pancreatic duct induces acinar cell deletion followed by duct proliferation and interstitial fibrosis. Apoptosis has been reported to be involved in the induction of acinar cell deletion after pancreatic duct ligation (PDL) in rats, however, the mechanism of pancreatic duct cell proliferation is still unknown. We hypothesized that Bcl-2 (antiapoptosis protein) and PCNA (cell cycle-related protein) could be involved in the mechanism of pancreatic duct cell proliferation after PDL. In PDL rats, acinar cells decreased in number and disappeared completely after duct ligation and duct-lining cells increased in number and formed duct-tubular complexes. Immunohistochemical study showed that PCNA expression appeared in the ductules and centroacinar cells from early stages after duct ligation and that Bcl-2 expression in duct cells, which was faint in normal pancreas, increased significantly when acinar cells were diminishing. Western blotting demonstrated that Bcl-2 was detected as a single band at 26 kDa, and the intensity of Bcl-2 in PDL rats was approximately ninefold stronger than in normal pancreas. Expression of Bcl-2 and PCNA after pancreatic duct ligation may be related to the prevention of apoptosis and cell proliferation of pancreatic duct cells in rats.

ISSN:0885-3177    

出版商:OVID    

年代:1997

数据来源: OVID