Uptake of tritiated D-mannoheptulose by liver, pancreatic exocrine and endocrine cells.

Tritiated D-mannoheptulose, a ketoheptose known to inhibit D-glucose metabolism in hepatocytes and pancreatic islets, but not so in pancreatic acinar cells, was injected intravenously in streptozotocin-induced diabetic mice transplanted under the kidney capsule with islets from control mice of the same strain. One hour after the injection of the tritiated heptose, the radioactive content was 5-8 times higher in the liver and transplanted islets than in the pancreatic gland. It is proposed that suitably radiolabelled D-mannoheptulose could be used to label preferentially the endocrine moiety of the pancreatic gland, e.g., in the perspective of its non-invasive imaging.     

Uptake of 1-deoxy-1-[125I]iodo-D-mannoheptulose by different cell types: in vitro and in vivo experiments

D-mannoheptulose was recently proposed as a tool to label preferentially insulin-producing cells in the pancreatic gland in the perspective of the non-invasive imaging of the endocrine pancreas. In such a perspective, we have now synthesized 1-deoxy-1-[125I]iodo-D-mannoheptulose ([125I]MH) and examined its uptake by different rat cell types. No phosphorylation of [125I]MH by bovine heart hexokinase could be detected. The apparent distribution space of [125I]MH largely exceeded that of [U-14C]sucrose, considered as an extracellular marker, in erythrocytes, parotid cells, hepatocytes, pancreatic pieces and isolated pancreatic islets. Relative to the mean intracellular distribution space of 3HOH, that of [125I]MH was not significantly different in pancreatic pieces from either normal rats or streptozotocin-induced diabetic animals (STZ rats). In pancreatic islets, the uptake of [125I]MH was decreased at low temperature, but failed to be significantly affected by cytochalasin B. Sixty min after the intravenous injection of [125I]MH, the radioactive content of selected organs displayed the following hierarchy: muscle相似文献   

Uptake of D-mannoheptulose by normal and tumoral pancreatic islet cells

D-mannoheptulose was recently proposed as a possible tool to label preferentially insulin-producing cells in the pancreatic gland. In the present study, D-[3H]-mannoheptulose uptake by rat pancreatic islets or dispersed islet cells was found to represent a time-related and temperature-sensitive process inhibited by cytochalasin B. This mould metabolite also inhibited the efflux of D-[3H]-mannoheptulose from prelabelled islets. After 60 min incubation at 37 degrees C, the apparent intracellular distribution space of the tritiated heptose was close to or somewhat higher than that of D-[5-3H]glucose and close to 50% of the intracellular 3HOH space. It was further enhanced by D-glucose and a high concentration of 10 mM of D-mannoheptulose. The uptake of D-[3H]mannoheptulose was much lower however than that of D-[3H]mannoheptulose hexaacetate. As judged from the fate of D-mannoheptulose hexa[2-14C]acetate, the latter ester was efficiently hydrolyzed in the islet cells. The internalization of D-[3H]mannoheptulose (or its ester) coincided with the generation of tritiated acidic metabolites, reflecting phosphorylation of the heptose. The situation found in normal islet cells sharply differed from that found in tumoral islet cells of either the RINm5F or INS-1 line, in which the apparent distribution space of D-[3H]mannoheptulose represented only about 3 and 9%, respectively, of the intracellular 3HOH space. These results indicate that the entry of D-mannoheptulose into islet cells represents a carrier-mediated process, possibly mediated at the intervention of GLUT2 and, hence, provide further support to the possible use of a suitable D-mannoheptulose analog as a tool for the preferential labelling of insulin-producing cells in the pancreatic gland.     

Pancreatic glycogen content in Goto-Kakizaki rats

In situations of sustained hyperglycemia, much larger amounts of glycogen accumulate in islet B-cells than in other pancreatic cells. The labelling of such a glycogen pool could thus conceivably provide a mean for assessing the relative contribution of insulin-producing cells to the total pancreatic mass. In such a perspective, the present study aims at investigating pancreatic glycogen accumulation in hereditarily diabetic Goto-Kakizaki (GK) rats. When cultured at 30 mM D-glucose in the presence of D-[U-14C]glucose, pancreatic islets from GK rats accumulated 14C-labelled glycogen in a manner comparable to that previously documented in islets from normal rats. Likewise, the glycogen content of the pancreatic gland, relative to the plasma D-glucose concentration, was not different in GK and normal rats. The GK rats thus apparently represent a suitable model for further studies on the in vivo labelling of B-cell glycogen in the perspective of the non-invasive imaging and quantification of the endocrine pancreas.     

Fate of 2-deoxy-2-[18F]fluoro-D-glucose in hyperglycemic rats

Sustained hyperglycemia allows the preferential labelling of pancreatic glycogen by D-[U-14C]glucose in control rats, as compared to animals previously injected with streptozotocin (STZ rats). The major aim of the present study was to investigate whether a sizeable difference between control and STZ rats could also be observed in terms of the radioactive content of the pancreatic gland 8 h after the intravenous injection of 2-deoxy-2-[18F]fluoro-D-glucose, both types of animals being examined at the same level of hyperglycemia. Although the radioactive content of muscle, liver and kidney was lower in STZ rats than in control animals, such a difference failed to achieve statistical significance in brain, hypophysis, pancreas and parotid gland. It is proposed, therefore, that 11C-labelled D-glucose, rather than 2-deoxy-2-[18F]fluoro-D-glucose should be used in the perspective of the non-invasive imaging of the endocrine pancreas.     

Fate of 2-deoxy-2-[18F]fluoro-D-glucose in control and diabetic rats

Recent experiments conducted in vitro have documented a marked difference in the time course for D-[U-14C]glucose net uptake by pieces of pancreatic tissue versus isolated pancreatic islets. The present study aimed, therefore, at assessing whether the endocrine pancreas contributes to a detectable extent to the overall net uptake of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) by the pancreatic gland. For this purpose, the radioactive content of the pancreas was compared to that of plasma, erythrocytes, liver, brain, hypophysis and parotid gland 3 min, 15 min and 240 min after the intravenous injection of FDG to both control rats and animals injected with streptozotocin and later treated with insulin or not. In the control rats, the radioactive content (cpm/mg wet wt.) of erythrocytes was always lower than that of liver. In other organs, it displayed the following hierarchy pancreas < parotid < hypophysis < brain, the absolute values being either lower (3 min) or much higher (240 min) than in liver. In the diabetic rats, whether treated with insulin or not, the radioactive content of erythrocytes, pancreas, brain, hypophysis and parotid gland, relative to the paired value found in liver, was equal or lower than that of control rats when the animals were hyperglycemic and equal or higher than that of control rats when the animals became hypoglycemic as the result of intensive insulin treatment. Even only 3 min after the injection of FDG, and despite persistent hyperglycemia in the streptozotocin-injected and insulin-treated rats, the pancreas/ liver paired ratio in radioactive content failed to be significantly lower in the diabetic animals than in control rats. These findings indicate that 2-deoxy-2-[18F]fluoro-D-glucose is not a suitable tool to detect any preferential labelling of insulin-producing cells, relative to acinar cells, at least when considering only the total radioactive content of the pancreatic gland.     

D-mannoheptulose uptake and its metabolic and secretory effects in human pancreatic islets

D-mannoheptulose was recently proposed to be transported into cells at the intervention of GLUT2. Since GLUT1, rather than GLUT2, represents the major carrier system for the transport of monosaccharides across the islet B-cell plasma membrane in human subjects, the uptake of D-mannoheptulose and its metabolic and secretory effects were investigated in human islets. The uptake of D-glucose reached much more rapidly a close-to-equilibrium value in isolated islets than in pieces of pancreas obtained from the same donor. The distribution space of D-[3H]mannoheptulose in the human islets largely exceeded that of [U-14C]sucrose, considered as an extracellular marker, and did not differ significantly from that of 3HOH. In the human islets, the heptose (10.0 mM) inhibited both D-[5-3H]glucose utilization and D-[U-14C] glucose oxidation, and decreased glucose-stimulated insulin release to the same extent as D-mannoheptulose hexaacetate. These findings indicate that a suitable radioactive analog of D-mannoheptulose could be used, in human like in rat islets, for preferential labelling of the endocrine moiety of the pancreatic gland.     

Impairment by cytochalasin B of the inhibitory action of D-mannoheptulose upon D-glucose metabolism in rat pancreatic islets

D-mannoheptulose was recently found to inhibit D-glucose metabolism in hepatocytes and pancreatic islets, whilst failing to do so in parotid cells, erythrocytes and the exocrine pancreas. In the latter three systems, however, the hexaacetate ester of D-mannoheptulose efficiently inhibits D-glucose metabolism. It was proposed, therefore, that the transport of unesterified D-mannoheptulose into cells may be mediated by GLUT2. Since cytochalasin B is known to inhibit D-glucose transport into pancreatic islet cells, it was now investigated whether the mould metabolite (0.02 mM) also impairs the inhibitory action of D-mannoheptulose (1.0 mM) upon D-glucose metabolism in rat pancreatic islets. The relative extent of D-mannoheptulose inhibitory action on D-[5-3H]glucose utilization and D-[U-14C]glucose conversion to 14CO2, as well as radioactive amino acids and acidic metabolites, was indeed much less marked in the presence of cytochalasin B (13+/-4% inhibition) than in its absence (40+/-3% inhibition). A comparable situation was not observed, however, in the case of glucose-stimulated insulin secretion, cytochalasin B augmenting insulin output to the same relative extent in the absence or presence of D-mannoheptulose. These findings support the view that the entry of D-mannoheptulose into cells may be mediated by a cytochalasin B-sensitive transport system, such as the GLUT2 carrier.     

Pancreatic uptake of [2-(14)C]alloxan

The uptake of [2-(14)C]alloxan by the pancreatic gland was investigated in control and streptozotocin-induced diabetic (STZ) rats, using both in vitro and in vivo techniques. Whether after 10 to 60 min incubation of pieces of pancreas in the presence of [2-(14)C]alloxan or 60 min to 24 h after intravenous injection of [2-(14)C]alloxan to control and insulin-treated STZ rats, the radioactive content of the pancreas (dpm/mg wet weight) only represented, in the STZ rats, about two thirds of the reference value found in control animals. These findings indicate that insulin-producing islet B-cells participate to a sizeable extent to the overall uptake of [2-(14)C]- alloxan by the whole pancreatic gland, despite the fact that they account for no more than about one percent of the total pancreas mass. Hence, it should be possible to preferentially label the endocrine moiety of the pancreas, in the perspective of its imaging and quantification by a non-invasive procedure, by use of a suitable radiolabelled molecule selectively taken up by islet, as distinct from acinar, pancreatic cells.     

Expansion of mesenchymal stem cells from human pancreatic ductal epithelium

Fibroblast-like cells emerging from cultured human pancreatic endocrine and exocrine tissue have been reported. Although a thorough phenotypic characterization of these cells has not yet been carried out, these cells have been hypothesized to be contaminating fibroblasts, mesenchyme and/or possibly beta-cell progenitors. In this study, we expanded fibroblast-like cells from adult human exocrine pancreas following islet isolation and characterized these cells as mesenchymal stem cells (MSCs) based on their cell surface antigen expression and ability to differentiate into mesoderm. Analysis by flow cytometry demonstrated that pancreatic MSCs express cell surface antigens used to define MSCs isolated from bone marrow such as CD13, CD29, CD44, CD49b, CD54, CD90 and CD105. In addition, utilizing protocols used to differentiate MSCs isolated from other somatic tissues, we successfully differentiated pancreatic MSCs into: (1) osteocytes that stained positive for alkaline phosphatase, collagen, mineralization (calcification) and expressed osteocalcin, (2) adipocytes that contained lipid inclusions and expressed fatty acid binding protein 4 and (3) chondrocytes that expressed aggrecan. We also demonstrated that pancreatic MSCs are multipotent and capable of deriving cells of endodermal origin. Pancreatic MSCs were differentiated into hepatocytes that stained positive for human serum albumin and expressed endoderm and liver-specific genes such as GATA 4 and tyrosine aminotransferase. In addition, preliminary protocols used to differentiate these cells into insulin-producing cells resulted in the expression of genes necessary for islet and beta-cell development such as Pax4 and neurogenin 3. Therefore, multipotent MSCs residing within the adult exocrine pancreas could represent a progenitor cell, which when further manipulated could result in the production of functional islet beta-cells.     

Immunocytochemical localization of progesterone receptors in endocrine cells of the human pancreas.

Progesterone receptors (PgR) have been immunocytochemically localized in the nuclei of several (40% to 75%) endocrine cells of the human pancreas and in a more variable number of neoplastic cells of 7 of 18 endocrine pancreatic tumors. Conversely the exocrine epithelial cells of the pancreas did not exhibit any PgR immunoreactivity in normal as well as in different pathologic conditions, including pancreatic adenocarcinomas. Estrogen receptors were not detected in any of the pancreatic samples investigated. Double immunocytochemical experiments have documented that PgR immunoreactivity in normal Langerhans islets is a consistent feature of most (75%) glucagon-producing A cells, of approximately 5% to 20% of insulin-producing B cells, and of a variable percentage of pancreatic polypeptide (PP)-producing cells, ranging from 5% to 70%. These figures were not affected by the sex, age, or underlying disease of the patients. The reported findings corroborate previous clinical and experimental evidence indicating that sex steroid hormones may have some regulatory effects on the functional activity of the endocrine pancreas.     

Alterations of pancreatic hepatocytes in rats exposed to carcinogens.

In this study, acute and chronic responses of pancreatic hepatocytes induced in F-344 rats by copper depletion-repletion protocol to certain hepatocarcinogens were examined. Administration of a single dose of tannic acid (subcutaneous), aflatoxin B1 (gavage), or lasiocarpine (intraperitoneally) caused characteristic nucleolar segregation in parenchymal cells of liver as well as in pancreatic hepatocytes. Chronic dietary administration of 2-acetylaminofluorene (0.025%) for 12 to 32 weeks led to the development of glutathione S-transferase-P-positive pancreatic hepatocytes in the pancreas. In addition, oval cell proliferation was observed in close association with pancreatic hepatocytes, but not in other areas of pancreas containing residual acinar cells. Oval cells in the pancreas and in the liver that developed in rats after chronic 2-acetylaminofluorene treatment and pancreatic duct cells stained positively with rat liver oval cell marker OV-6 antibodies by immunoperoxidase. These findings indicate that pancreatic hepatocytes respond to carcinogens in a fashion similar to parenchymal cells of liver.     

Chromatin-remodeling factors allow differentiation of bone marrow cells into insulin-producing cells

Type 1 diabetes is caused by the destruction of pancreatic beta-cells by T cells of the immune system. Islet transplantation is a promising therapy for diabetes mellitus. Bone marrow stem cells (BMSC) have the capacity to differentiate into various cell lineages including endocrine cells of the pancreas. To investigate the conditions that allow BMSC to differentiate into insulin-producing cells, a novel in vitro method was developed by using the histone deacetylase inhibitor, trichostatin A (TSA). BMSC, cultured in presence of TSA, differentiated into islet-like clusters under appropriate culture conditions. These islet-like clusters were similar to the cells of the islets of the pancreas. The islet-like clusters showed endocrine gene expression typical for pancreatic beta-cell development and function, such as insulin (I and II), glucagon, somatostatin, GLUT-2, pancreatic duodenal homeobox-1 (PDX-1), and Pax 4. Immunocytochemistry confirmed islet-like clusters contained pancreatic hormones. The colocalization of insulin and C-peptide was also observed. Enzyme-linked immunosorbent assay analysis demonstrated that insulin secretion was regulated by glucose. Western blot analysis demonstrated the presence of stored insulin. Electron microscopy of the islet-like cells revealed an ultrastructure similar to that of pancreatic beta-cells, which contain insulin granules within secretory vesicles. These findings suggest that histone-deacetylating agents could allow the differentiation of BMSC into insulin-producing beta-cells.     

Insulin-like growth factor-II in endocrine pancreatic tumours. Immunohistochemical, biochemical and in situ hybridization findings

In earlier studies a high-molecular-weight (HMW) insulin-like growth factor-II (IGF-II) peptide was identified in adult human pancreas and localized to the insulin-producing B-cells. This peptide has now been investigated in neoplastic insulin cells. Forty endocrine pancreatic tumours and 17 pancreatic adenocarcinomas of ductal type were included in the study. All cases were investigated with immunohistochemical techniques using antibodies to IGF-II, insulin, pro-insulin, glucagon, somatostatin, pancreatic polypeptide, gastrin and vasoactive intestinal peptide (VIP). Frozen tissue from nine tumours and two normal pancreatic glands was extracted, gel separated, and quantified using radioimmunoassay. The tumours were also investigated by in situ hybridization. IGF-II-immunoreactive cells were found in nearly all the 18 insulin-producing tumours (16/18), in a minority of the other endocrine tumours, but not in pancreatic adenocarcinomas. All extracts from the endocrine tumours showed varying amounts of IGF-II and had different molecular-weight forms. The immunohistochemical and radioimmunoassay findings are both based on immunological binding and were further confirmed by Northern blot and in situ hybridization. These results show that IGF-II is expressed in insulin-producing tumours as well as in pancreatic tumours producing other peptides, in contrast to normal pancreatic islets where IGF-II is found exclusively in insulin-producing cells.     

The growth of liver cells in the pancreas after intra-splenic implantation: the effects of portal perfusion.

A newly developed rat model of portal diversion, in which the pancreas gland is perfused by splanchnic effluent, has been used to monitor the effects of such portal perfusion on the development of isolated hepatocytes within the pancreatic parenchyma. Two groups of animals (control and portally-perfused) received intra-splenic hepatocyte implants (2 x 10(6) cells). The development of liver cells in the pancreas was then assessed histologically 10 days-9 months later. Portal diversion did not alter the incidence of hepatocytes within the pancreas as a whole (70% of all animals). However hepatocyte-islet rosettes (the intimate association between liver cells and endocrine tissue) were found in a significantly higher percentage of portally-perfused pancreases (92%) in comparison to controls (43%) 6 and 9 months after implantation. It appears that liver cell translocation from the spleen to the pancreas is dependent largely on factors associated with the implantation procedure itself. The growth of liver cells specifically around the islets of Langerhans, however, seems to be positively influenced by portal factors. These studies provide further evidence for the theory that extrahepatic liver cells are susceptible to local and systemic growth factors in a similar way to homotopic hepatocytes in the intact liver.     

Pancreas transplantation: an immunohistochemical analysis of pancreatic hormones and HLA-DR expression

The immunohistochemical features of pancreatic grafts in eight patients with pancreatic transplants were analyzed and compared with pancreases from five patients with chronic pancreatitis and with three pancreatic tissues without histological abnormalities. There was a significant increase in glucagon producing cells in patients with transplanted pancreases compared with those with chronic pancreatitis (P less than 0.05). A significant decline in insulin-producing cells was seen in the transplanted pancreases with rejection in comparison with normal pancreatic tissue. Immunohistochemical staining for HLA-DR (Ia) antigens revealed expression of HLA-DR by endothelial cells, mononuclear cells, and by some ductal epithelial cells, but not by the endocrine islet cells. These results suggest that significant changes in insulin and glucagon production occur in the transplanted pancreas with rejection and that HLA-DR is not expressed by islet cells during graft rejection or with chronic inflammation.     

Glycogen accumulation in cultured tumoral or normal pancreatic islet and acinar cells.

Tumoral pancreatic islet cells of either the RINm5F or INS-1 cell lines, when cultured in the presence of 30.0 mM D-glucose, accumulate about 50 times more glycogen than tumoral pancreatic acinar cells of the AR42J line cultured under the same experimental conditions. Expressed per nl of intracellular water, the glycogen content of the RINm5F or INS-1 cells is even higher than that found in rat pancreatic islets also cultured under the same experimental conditions. Moreover, at variance with normal islet cells, tumoral islet cells do not require to be exposed to a high concentration of D-glucose to accumulate large amounts of glycogen. Based on these findings, it is proposed that the labelling of the glycogen pool, e.g. by 11C-labelled D-glucose or 2-deoxy-2-[18F]fluoro-D-glucose, may allow identification and localization of insulinomas in the pancreatic gland by a non-invasive imaging procedure.