Identification and purification of functional human beta-cells by a new specific zinc-fluorescent probe.

Pancreatic beta-cells contain large amounts of zinc. We took advantage of this to try to localize, quantify, and isolate insulin-producing cells from islet preparations. Our study was designed to identify a non-toxic zinc-sensitive fluorescent probe able to selectively label labile zinc in viable beta-cells and to exhibit excitation and emission wavelengths in the visible spectrum, making this technique exploitable by most instruments. We tested Newport Green, a probe excitable at 485 nm with a dissociation constant in the micromolar range corresponding to a low affinity for zinc. The loading of the lipophilic esterified form of Newport Green was easy, rapid, specific, and non-toxic to cells. Confocal microscopy highlighted an intense fluorescence associated with secretory granules. Regression analyses showed a good relationship between zinc fluorescence and islet number (r = 0.98) and between zinc fluorescence and insulin content (r = 0.81). The determination of Zn fluorescence per DNA enabled us to assess the quality of the different islet preparations intended for islet allografting in terms of both purity and viability. Cell sorting of dissociated Newport Green-labeled cells resulted in a clear separation of beta-cells, as judged by insulin content per DNA and immunocytochemical analysis. This zinc probe, the first able to specifically label living cells in the visible spectrum, appears very promising for beta-cell experimentation, both clinically and for basic research.     

The effect of diabetogenic substances on the zinc and calcium content in the pancreatic islet cells of rabbits

The injections of diabetogenic drugs to rabbits induced a decrease in zinc and calcium content in B insulocytes dependent on the degree of these cells damage and hyperglycemia. The content of these metals in A insulocytes was not significantly altered due to a light injury of these cells. A high concentration of calcium in islet A cells is suggested to be a protective factor to the action of alteration agents.     

A two-site mechanism for the inhibition of IAPP amyloidogenesis by zinc

Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic protein co-secreted with insulin in response to glucose levels. The formation of hIAPP amyloid plaques near islet cells has been linked to the death of insulin-secreting β-cells in humans and the progression of type II diabetes. Since both healthy individuals and those with type II diabetes produce and secrete hIAPP, it is reasonable to look for factors involved in storing hIAPP and preventing amyloidosis. We have previously shown that zinc inhibits the formation of insoluble amyloid plaques of hIAPP; however, there remains significant ambiguity in the underlying mechanisms. In this study, we show that zinc binds unaggregated hIAPP at micromolar concentrations similar to those found in the extracellular environment. By contrast, the fibrillar amyloid form of hIAPP has low affinity for zinc. The binding stoichiometry obtained from isothermal titration calorimetry experiments indicates that zinc favors the formation of hIAPP hexamers. High-resolution NMR structures of hIAPP bound to zinc reveal changes in the electron environment along residues that would be located along one face of the amphipathic hIAPP α-helix proposed as an intermediate for amyloid formation. Results from electrospray ionization mass spectroscopy investigations showed that a single zinc atom is predominantly bound to hIAPP and revealed that zinc inhibits the formation of the dimer. At higher concentrations of zinc, a second zinc atom binds to hIAPP, suggesting the presence of a low-affinity secondary binding site. Combined, these results suggest that zinc promotes the formation of oligomers while creating an energetic barrier for the formation of amyloid fibers.     

Dietary zinc restriction promotes degeneration of the endocrine pancreas in mice

Zinc is a key component of several proteins, interacting with the pancreatic hormones insulin and amylin. The role of zinc in insulin oligomerization and crystallinity is well established, although the effects of dietary zinc restriction on both energetic metabolism and β-pancreatic hormonemia and morphology remain unexplored. Here we report the effects of dietary zinc restriction on the endocrine pancreas and metabolic phenotype of mice. Nontransgenic male Swiss mice were fed a low-zinc or control diet for 4 weeks after weanling. Growth, glycemia, insulinemia and amylinemia were lower and pancreatic islets were smaller in the intervention group despite the preserved insulin crystallinity in secretory granules. We found strong immunostaining for insulin, amylin and oligomers in apoptotic pancreatic islet. High production of β-pancreatic hormones in zinc-restricted animals counteracted the reduced islet size caused by apoptosis. These data suggest that zinc deficiency is sufficient to promote islet β-cell hormonal disruption and degeneration.     

Increased level of exogenous zinc induces cytotoxicity and up-regulates the expression of the ZnT-1 zinc transporter gene in pancreatic cancer cells

A balance between zinc uptake by ZIP (SLC39) and efflux of zinc from the cytoplasm into subcellular organelles and out of the cell by ZnT (SLC30) transporters is crucial for zinc homeostasis. It is not clear whether normal and cancerous pancreatic cells respond differently to increased extracellular zinc concentrations. Use of flow cytometry-based methods revealed that treatment with as little as 0.01 mM zinc induced significant cytotoxicity in two human ductal adenocarcinoma cell lines. In contrast, normal human pancreatic islet cells tolerated as high as 0.5 mM zinc. Insulinoma cell lines of mouse and rat origin also succumbed to high concentrations of zinc. Exposure to elevated zinc concentrations enhanced the numbers of carcinoma but not primary islet cells staining with the cell-permeable zinc-specific fluorescent dye, FluoZin-3, indicating increased zinc influx in transformed cells. Mitochondrial membrane depolarization, superoxide generation, decreased antioxidant thiols, intracellular acidosis and activation of intracellular caspases characterized zinc-induced carcinoma cell death. Only the antioxidant glutathione but not inhibitors of enzymes implicated in apoptosis or necrosis prevented zinc-induced cytotoxicity in insulinoma cells. Immunoblotting revealed that zinc treatment increased the ubiquitination of proteins in cancer cells. Importantly, zinc treatment up-regulated the expression of ZnT-1 gene in a rat insulinoma cell line and in two human ductal adenocarcinoma cell lines. These results indicate that the exposure of pancreatic cancer cells to elevated extracellular zinc concentrations can lead to cytotoxic cell death characterized by increased protein ubiquitination and up-regulation of the zinc transporter ZnT-1 gene expression.     

Regulation of RNA metabolism in relation to insulin production and oxidative metabolism in mouse pancreatic islets in vitro

This study was undertaken to investigate the long-term effects of different substrates, in particular glucose, on the regulation of islet RNA metabolism and the relationship of this regulation to the metabolism and insulin production of the islet B-cell. For this purpose collagenase-isolated mouse islets were used either in the fresh state or after culture for 2 or 5 days in RPMI 1640 plus 10% calf serum supplemented with various test compounds. Islets cultured with 16.7 mM glucose contained more RNA than those cultured with 3.3 mM glucose. Culture of islets in glucose at low concentrations inhibited glucose-stimulated RNA synthesis and this inhibitory effect was reversed by prolonged exposure to high glucose concentrations. Culture with 10 mM leucine and 3.3 mM glucose or with 10 mM 2-ketoisocaproate and 3.3 mM glucose increased the total RNA content of islets as compared to that of islets cultured with 3.3 mM glucose alone. Islets cultured with 5 mM theophylline maintained a high RNA content in the presence of 3.3 mM glucose. Theophylline also increased the islet RNA content when added together with 16.7 mM glucose, as compared to 16.7 mM glucose alone. Theophylline probably exerted this effect by decreasing the rate of RNA degradation. Changes in islet RNA metabolism showed a close correlation to changes in islet total protein biosynthesis, whereas islet (pro)insulin biosynthesis and insulin release exhibited different glucose-dependency patterns. The response of islet oxygen uptake to glucose was similar to that of islet RNA and protein biosynthesis. It is concluded that the RNA content of the pancreatic islets is controlled at the levels of both synthesis and degradation. Glucose stimulates the RNA synthesis and inhibits its degradation. Moreover, the results suggest that regulation of RNA synthesis may be mediated through islet metabolic fluxes and the cAMP system.     

Development and characterization of simulant pancreatic islets

Insulin is stored in pancreatic islets as a zinc-insulin complex, and stimulating the islets results in the release of insulin and zinc. Simulant pancreatic islet beads have been developed using agarose beads (50-250 micro m diameter) derivatized with iminodiacetic acid that have been loaded with zinc. A qualitative comparison of the simulant beads with pancreatic islets has been made by staining with dithizone and a zinc-binding fluorescent dye, TSQ. The binding capacity of simulant beads was determined to be 34 micro mol Zn(2+)/g of dried beads using anodic stripping voltammetry. Hydrochloric acid was used to release zinc from beads to mimic the secretion of insulin from pancreatic islets and a release profile was established. The simulant beads can be used to optimize the islet isolation process and reduce the use of real islets in method development.     

Zinc fluxes during acute and chronic exposure of INS-1E cells to increasing glucose levels.

Zinc in beta-cell secretory vesicles is essential for insulin hexamerization, and tight vesicular zinc regulation is mandatory. Little is known about zinc ion fluxes across the secretory vesicle membrane and the influence of changes in the extracellular environment on vesicular zinc. Our study aim was to investigate the effect of acute and chronic exposure to various glucose concentrations on zinc in secretory vesicles, the relation between zinc and insulin, and the presence of two zinc transporters, ZnT1 and ZnT4, in INS-1E cells. Zinc ions were demonstrated and semi-quantified using zinc-sulfide autometallography. Insulin content and secreted insulin were measured. Measurements were made on INS-1E cells after exposure to 2.0, 6.6, 16.7, and 24.6 mmol/l glucose for 1, 24, and 96 hours. 1h: Increasing glucose resulted in no changes in intravesicular zinc ions at 2, and 24.6 mmol/l glucose, but a slight increase at 16.7 mmol/l glucose. 24 and 96 h: Increasing glucose led to decreased vesicular zinc ion content accompanied by a decrease in insulin content. ZnT1 and ZnT4 were present in the cytoplasm. Our results demonstrate that intra-vesicular zinc ions respond to changes in the extra-cellolar glucose concentration, especially during chronic high glucose concentrations, where the content of vesicular zinc ions decreases.     

The Zinc-Myoglobin Relationships in Porcine Muscles

Zinc and myoglobin content in muscles from pigs were studied under various conditions. Zinc concentration was considerably higher in red than in white muscles. In muscles, where the metabolic pattern changes from glycolytic to oxidative during the period from birth to weaning, a simultaneous increase in zinc content was seen. A significant positive correlation exists between myoglobin and zinc content under normal conditions. However, while myoglobin concentration decreases due to iron deficiency anaemia no changes occur in zinc content. It is concluded that no functional link seems to exist between zinc metabolism and myoglobin synthesis in porcine muscles.     

Insulin secretion and islet endocrine cell content at onset and during the early stages of diabetes in the BB rat: effect of the level of glycemic control.

Although it is agreed that autoimmune destruction of pancreatic islets in diabetic BB rats is rapid, reports of endocrine cell content of islets from BB diabetic rats at the time of onset of diabetes vary considerably. Because of the rapid onset of the disease (hours) and the attendant changes in islet morphology and insulin secretion, it was the aim of this study to compare islet beta-cell numbers to other islet endocrine cells as close to the time of onset of hyperglycemia as possible (within 12 h). As it has been reported that hyperglycemia renders the beta cell insensitive to glucose, the early effects of different levels of insulin therapy (well-controlled vs. poorly controlled glycemia) on islet morphology and insulin secretion were examined. When measured within 12 h of onset, insulin content of BB diabetic islets, measured by morphometric analysis or pancreatic extraction, was 60% of insulin content of control islets. Despite significant amounts of insulin remaining in the pancreas, 1-day diabetic rats exhibited fasting hyperglycemia and were glucose intolerant. The insulin response from the isolated perfused pancreas to glucose and the glucose-dependent insulinotropic hormone, gastric inhibitory polypeptide (GIP), was reduced by 95%. Islet content of other endocrine peptides, glucagon, somatostatin, and pancreatic polypeptide, was normal at onset and at 2 weeks post onset. A group of diabetic animals, maintained in a hyperglycemic state for 7 days with low doses of insulin, were compared with a group kept normoglycemic by appropriate insulin therapy. No insulin could be detected in islets of poorly controlled diabetics, while well-controlled animals had 30% of the normal islet insulin content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucocorticoid-induced changes in insulin secretion related to the metabolism and ultrastructure of pancreatic islets

The effect of adrenalectomy and dexamethasone-treatment on insulin secretion was studied and related to the changes observed in the glucose oxidation, calcium uptake, cAMP and insulin content, as well as the ultrastructure of pancreatic rat islets. It was found that adrenalectomy was followed by a decreased glucose-induced insulin secretion, glucose oxidation, calcium uptake, cAMP and insulin content without any remarkable change observed at the ultrastructural level. Conversely, adrenalectomized-rats supplemented with dexamethasone showed an increased glucose-induced insulin secretion, glucose oxidation, calcium uptake and cAMP content but a diminished islet insulin content. At the ultrastructural level, a clear picture of increased secretory activity was found, with diminished number of mature B granules and greater number of pale granules, while rough endoplasmic reticulum and Golgi complex frequently appeared hypertrophic. These changes were only observed in the B cells. On account of our results, we might suggest that insulin secretion is partially controlled by glucocorticoid circulating levels throughout their effect on pancreatic islet metabolism.     

The autoradiographic localization of zinc within the pancreatic islets of the rainbow trout, Salmo gairdneri

This experiment reports on the localization of zinc within the pancreatic islets of Salmo gairdneri. Individual fish were injected with 65Zn and the distribution of the isotope within the islets was determined by autoradiography. Insulin cells were found to accumulate approximately twice as much zinc per unit area as the rest of the islet tissue. It is presumed that this zinc is involved with the crystallization and storage of insulin within the insulin cells. Various histological methods were investigated to ascertain the procedure which best precipitated zinc and at the same time avoided excess leaching.     

Effect of food-deprivation on rat pancreatic islet cells

Somatostatin, insulin and glucagon concentrations in rat pancreas were measured following various intervals of food-deprivation. Tissue concentrations, as measured by radioimmunoassay, were correlated with A-, B-, and D-cell number and size using a scanning integrating image analyzer (Quantimet 720). Alterations in total islet hormone content were not correlated to changes in size or distribution of cells. This implies that changes in tissue content reflect changes in turnover of peptides rather than changes in cell size or number.     

Alterations in pancreatic islet phosphate content during secretory stimulation with glucose.

Isolated rat pancreatic islets were perifused and analyzed for phosphate content immediately following the transient increase in the efflux of orthophosphate which occurs when insulin secretion is stimulated by glucose. In some instances, islets were perifused directly following isolation to minimize preparative delay; in others, islets were prelabeled during incubation with [32P]orthophosphate for 90 min prior to perifusion. In both experimental situations, total islet phosphate content declined 40--50% following exposure to stimulating concentrations of glucose and initiation of enhanced insulin release. In the experiments with prelabeled islets, tissue content of [32P]orthophosphate fell to a similar extent so that the specific radioactivity of islet orthophosphate was unaffected. Inhibited of heightened insulin release with Ni2+ did not modify the decrements in total or radioactive tissue orthophosphate, thus indicating that these responses to islet stimulation reflect events which are proximal to activated exocytosis. Simultaneous analyses for tissue ATP and ADP demonstrated that the efflux in orthophosphate and reduction in tissue orthophosphate content were not mediated via net changes in islet adenine nucleotides. The observations represent the first documentation that a net reduction of tissue inorganic phosphate is one of the early components of stimulus-secretion coupling in isolated pancreatic islets.     

Role of zinc in regulation of arterial blood pressure and in the etiopathogenesis of arterial hypertension

Increased gastrointestinal absorption and urinary excretion of zinc has been confirmed in experimental and clinical studies on primary arterial hypertension as a result from changes of intracellular and extracellular zinc content. In arterial hypertension, the levels of zinc in serum, lymphocyte, and bone decrease while increasing in heart, erythrocytes, kidney, liver, suprarenal glands and spleen. These changes result in the loss of zinc homeostasis that leads to various degrees of deficiency, not entirely compensated by nutritional factors or increased absorption in the gastrointestinal tract. Loss of zinc homeostasis can be both cause and effect of high blood pressure. In the present review, the role of zinc metabolism changes and its mechanisms in arterial hypertension are discussed.     

Time-course of altered islet phospholipids and of calcium binding and ionophoretic properties of islet lipids following glucose stimulation

The time-course of alteration in islet cell phospholipid content following d-glucose exposure in islet cells and in islet cell membranes was related to the ability of lipids extracted from both cultured pancreatic islet cells and from plasma membranes isolated from the islet cells to translocate calcium in two model membrane systems. The first model system (bulk-phase system) detected lipid species with the ability to bind calcium, irrespective of their ability to enhance calcium transport across cell membranes. The second system (multilamellar membrane system) detected lipid species with the ability to both bind calcium and to enhance calcium transport across cell membranes (true ionophores). Pre-exposure to high d-glucose concentration led to a rapid (within 1 min) fall in membrane phosphoinositides. This was partially blocked by mannoheptulose. A concurrent fall in calcium bindig activity of lipids from the plasma membrane was observed. In the whole islet cell fraction, d-glucose induced a marked increase in Ca²⁺ ionophoretic activity. Unlike the fall in membrane polyphosphoinositides and membrane Ca²⁺ binding activity, these changes were dependent on the presence of added extracellular calcium. l-Glucose was without effect on membrane phosphoinositide content. It is concluded that altered membrane and intracellular phospholipids may contribute to the increased availability of intracellular Ca²⁺ following d-glucose stimulation by virtue of theie Ca²⁺ binding and ionophoretic properties.     

Early intervention with liraglutide improves glucose tolerance without affecting islet microcirculation in young Goto-Kakizaki rats

Liraglutide, an analog of glucagon-like peptide-1 (GLP-1), is an effective anti-diabetic agent with few side effects. Since native GLP-1 exerts vascular effects, we investigated changes in pancreatic islet blood flow using a non-radioactive microsphere technique, as well as insulin concentration and glucose tolerance after 17 day treatment with liraglutide in 6-week-old Goto-Kakizaki (GK) rats. Compared to saline-treated control GK rats, liraglutide limited body weight gain, decreased glycemia, improved glucose tolerance and lowered serum insulin concentration. Neither pancreatic or islet blood flow, nor pancreatic insulin content, was affected by liraglutide treatment. We conclude that early intervention with liraglutide decreases glycemia and improves glucose tolerance, thus halting the natural progression towards diabetes, without affecting islet microcirculation or pancreatic insulin content in young female GK rats.     

Autometallography allows ultrastructural monitoring of zinc in the endocrine pancreas

Zinc is intimately involved in insulin metabolism, its major known role being the binding of insulin in osmotically stable hexamers in beta-cell granules. To investigate the anatomical distribution of zinc ions necessary for insulin binding we examined the rat pancreas by autometallography (AMG). AMG demonstrates chelatable zinc and is a sensitive marker for zinc in vesicles and also a surrogate marker for recently described zinc pumps regulating intravesicular zinc metabolism. Zinc ions were found in alpha- and beta-cell granules, primarily in the periphery of the granules. Only occasionally was zinc seen in other islet cell types. AMG allows the study of the microscopic and ultrastructural localisation of free zinc ions in the pancreas. The applicability of the method at the ultrastructural level in particular makes AMG a very sensitive tool in future studies on the role of zinc ions in the pancreas.     

Zinc stress induces changes in apoplasmic protein content and polypeptide composition of barley primary leaves

Excessive zinc in the rooting medium, and consequently increasingzinc content in the shoot of barley (Hordeum vulgare) seedlings,strongly affected the protein content and the polypeptide compositionof the leaf apoplasm. In contrast, only small changes In polypeptide composition (in the IEF range of pH 4–8 and between14 and 80 kDa) were detected In mesophyll and epidermis protoplasts.The zinc stress-induced changes In apoplasmic protein contentwere analysed in detail. The apoplasmic protein content Increasedby more than 3-fold when the zinc concentration in the mediumwas increased from 0.02 to about 200 µmol l^–1; athigher zinc concentrations no further changes occurred. Theincrease in apoplasmic protein content was due both to a generalincrease in abundance of the most dominant apoplasmic polypeptidesand to a pronounced increase in the abundance of specific polypeptidesas monitored in electrophoretic separations or by measuringthe activity of apoplasmic enzymes. The largest induction wasseen for four apoplasmic polypeptides of molecular masses 16,23, 27, and 28 kDa with as yet unknown function. Key words: Apoplasm, barley, leaf, peroxidases, potypeptide, zinc stress     

Accumulation of free tryptophan and tryptairiine in zinc deficient maize seedlings

Maize seedlings were grown in culture solutions containing variouslevels of zinc. The changes in free tryptophan content due to the differentzinc nutritions were estimated by using an amino acid analyzer.Free tryptophan was detected in normal leaves, but the amountswere very small relative to those in zinc deficient plants.The maximum accumulation coincided with the development of severesymptoms associated with advanced zinc deficiency. Changes in tryptamine content due to different zinc nutritionswere estimated by paper chromatography and thin layer chromatography.The content of tryptamine was highest in maize leaves, whichdeveloped symptoms of zinc deficiency. Under conditions of adequatezinc nutrition, tryptamine was also found in maize leaves, butthe amounts were very small in contrast to those in zinc deficientplants. (Received March 25, 1970; )