Beta-cell function in isolated human pancreatic islets in long-term tissue culture

Human pancreatic islets were isolated by collagenase treatment of pancreatic tissue obtained from 27 individuals aged 12 to 69 years. The islets were maintained free floating in tissue culture medium RPMI 1640 supplemented with calf or human serum. In two cases the insulin production was followed up to nearly two years. The insulin production rate of the individual islet preparations varied between 0.2 and 8 ng per islet per day. No significant correlation with donor age or sex was found. The glucose concentration in the medium influenced the insulin release in a dose dependent manner. The acute response of the cultured islets to glucose was evaluated both by batch incubation and by perifusion. Both in the acute and the chronic experiments maximal insulin release was found at 10 mM glucose. In conclusion, these experiments indicate that viable islets of Langerhans can be obtained from adult human pancreatic tissue and that their beta-cell function can be maintained for up to two years. The variation in insulin production rate could not be ascribed to age or sex and may reflect both physiological and methodological factors.     

Enhanced beta-cell mass without increased proliferation following chronic mild glucose infusion

The physiological mechanisms underlying pancreatic beta-cell mass (BCM) homeostasis are complex and not fully resolved. Here we examined the factors contributing to the increased BCM following a mild glucose infusion (GI) whereby normoglycemia was maintained through 96 h. We used morphometric and immunochemical methods to investigate enhanced beta-cell growth and survival in Sprague-Dawley rats. BCM was elevated >2.5-fold over saline-infused control rats by 48 h and increased modestly thereafter. Unexpectedly, increases in beta-cell proliferation were not observed at any time point through 4 days. Instead, enhanced numbers of insulin(+) cell clusters and small islets (400-12,000 microm(2); approximately 23- to 124-microm diameter), mostly scattered among the acini, were observed in the GI rats by 48 h despite no difference in the numbers of medium to large islets. We previously showed that increased beta-cell growth in rodent models of insulin resistance and pancreatic regeneration involves increased activated Akt/PKB, a key beta-cell signaling intermediate, in both islets and endocrine cell clusters. GI in normal rats also leads to increased Akt activation in islet beta-cells, as well as in insulin(+) and insulin(-) cells in the common duct epithelium and endocrine clusters. This correlated with strong Pdx1 expression in these same cells. These results suggest that mechanisms other than proliferation underlie the rapid beta-cell growth response following a mild GI in the normal rat and involve Akt-regulated enhanced beta-cell survival potential and neogenesis from epithelial precursors.     

Glucose-dependent expansion of pancreatic beta-cells by the protein p8 in vitro and in vivo

p8 protein expression is known to be upregulated in the exocrine pancreas during acute pancreatitis. Own previous work revealed glucose-dependent p8 expression also in endocrine pancreatic beta-cells. Here we demonstrate that glucose-induced INS-1 beta-cell expansion is preceded by p8 protein expression. Moreover, isopropylthiogalactoside (IPTG)-induced p8 overexpression in INS-1 beta-cells (p8-INS-1) enhances cell proliferation and expansion in the presence of glucose only. Although beta-cell-related gene expression (PDX-1, proinsulin I, GLUT2, glucokinase, amylin) and function (insulin content and secretion) are slightly reduced during p8 overexpression, removal of IPTG reverses beta-cell function within 24 h to normal levels. In addition, insulin secretion of p8-INS-1 beta-cells in response to 0-25 mM glucose is not altered by preceding p8-induced beta-cell expansion. Adenovirally transduced p8 overexpression in primary human pancreatic islets increases proliferation, expansion, and cumulative insulin secretion in vitro. Transplantation of mock-transduced control islets under the kidney capsule of immunosuppressed streptozotocin-diabetic mice reduces blood glucose and increases human C-peptide serum concentrations to stable levels after 3 days. In contrast, transplantation of equal numbers of p8-transduced islets results in a continuous decrease of blood glucose and increase of human C-peptide beyond 3 days, indicating p8-induced expansion of transplanted human beta-cells in vivo. This is underlined by a doubling of insulin content in kidneys containing p8-transduced islet grafts explanted on day 9. These results establish p8 as a novel molecular mediator of glucose-induced pancreatic beta-cell expansion in vitro and in vivo and support the notion of existing beta-cell replication in the adult organism.     

Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells

We have generated transgenic mice that express green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter (MIP). The MIP-GFP mice develop normally and are indistinguishable from control animals with respect to glucose tolerance and pancreatic insulin content. Histological studies showed that the MIP-GFP mice had normal islet architecture with coexpression of insulin and GFP in the beta-cells of all islets. We observed GFP expression in islets from embryonic day E13.5 through adulthood. Studies of beta-cell function revealed no difference in glucose-induced intracellular calcium mobilization between islets from transgenic and control animals. We prepared single-cell suspensions from both isolated islets and whole pancreas from MIP-GFP-transgenic mice and sorted the beta-cells by fluorescence-activated cell sorting based on their green fluorescence. These studies showed that 2.4 +/- 0.2% (n = 6) of the cells in the pancreas of newborn (P1) and 0.9 +/- 0.1% (n = 5) of 8-wk-old mice were beta-cells. The MIP-GFP-transgenic mouse may be a useful tool for studying beta-cell biology in normal and diabetic animals.     

Modulation of the pancreatic islet beta-cell-delayed rectifier potassium channel Kv2.1 by the polyunsaturated fatty acid arachidonate

Glucose stimulates both insulin secretion and hydrolysis of arachidonic acid (AA) esterified in membrane phospholipids of pancreatic islet beta-cells, and these processes are amplified by muscarinic agonists. Here we demonstrate that nonesterified AA regulates the biophysical activity of the pancreatic islet beta-cell-delayed rectifier channel, Kv2.1. Recordings of Kv2.1 currents from INS-1 insulinoma cells incubated with AA (5 mum) and subjected to graded degrees of depolarization exhibit a significantly shorter time-to-peak current interval than do control cells. AA causes a rapid decay and reduced peak conductance of delayed rectifier currents from INS-1 cells and from primary beta-cells isolated from mouse, rat, and human pancreatic islets. Stimulating mouse islets with AA results in a significant increase in the frequency of glucose-induced [Ca(2+)] oscillations, which is an expected effect of Kv2.1 channel blockade. Stimulation with concentrations of glucose and carbachol that accelerate hydrolysis of endogenous AA from islet phosphoplipids also results in accelerated Kv2.1 inactivation and a shorter time-to-peak current interval. Group VIA phospholipase A(2) (iPLA(2)beta) hydrolyzes beta-cell membrane phospholipids to release nonesterified fatty acids, including AA, and inhibiting iPLA(2)beta prevents the muscarinic agonist-induced accelerated Kv2.1 inactivation. Furthermore, glucose and carbachol do not significantly affect Kv2.1 inactivation in beta-cells from iPLA(2)beta(-/-) mice. Stably transfected INS-1 cells that overexpress iPLA(2)beta hydrolyze phospholipids more rapidly than control INS-1 cells and also exhibit an increase in the inactivation rate of the delayed rectifier currents. These results suggest that Kv2.1 currents could be dynamically modulated in the pancreatic islet beta-cell by phospholipase-catalyzed hydrolysis of membrane phospholipids to yield non-esterified fatty acids, such as AA, that facilitate Ca(2+) entry and insulin secretion.     

Receptors for insulin and insulin-like growth factor-1 and insulin receptor substrate-1 mediate pathways that regulate islet function

The insulin/insulin-like growth factor-1 (IGF-1) signalling pathways are present in most mammalian cells and play important roles in the growth and metabolism of tissues. Most proteins in these pathways have also been identified in the beta-cells of the pancreatic islets. Tissue-specific knockout of the insulin receptor (betaIRKO) or IGF-1 receptor (betaIGFRKO) in pancreatic beta-cells leads to altered glucose-sensing and glucose intolerance in adult mice, and betaIRKO mice show an age-dependent decrease in islet size and beta-cell mass. These data indicate that these receptors are important for differentiated function and are unlikely to play a major role in the early growth and/or development of the pancreatic islets. Conventional insulin receptor substrate-1 (IRS-1) knockouts manifest growth retardation and mild insulin resistance. The IRS-1 knockouts also display islet hyperplasia, defects in insulin secretory responses to multiple stimuli both in vivo and in vitro, reduced islet insulin content and an increased number of autophagic vacuoles in the beta-cells. Re-expression of IRS-1 in cultured beta-cells is able to partially restore the insulin content indicating that IRS-1 is involved in the regulation of insulin synthesis. Taken together, these data provide evidence that insulin and IGF-1 receptors and IRS-1, and potentially other proteins in the insulin/IGF-1 signalling pathway, contribute to the regulation of islet hormone secretion and synthesis and therefore in the maintenance of glucose homeostasis.     

IFN-gamma and tumor necrosis factor-alpha. Cytotoxicity to murine islets of Langerhans

We have previously reported that the cytokines IFN-gamma and TNF-alpha each upregulate the expression of class I MHC proteins and, in combination, induce the expression of class II MHC proteins on pancreatic islet cells. IFN-gamma and TNF-alpha are therefore implicated in the immunologic destruction of beta-cells in insulin-dependent diabetes mellitus. The objective of the present study was to define the effects of IFN-gamma and TNF-alpha on the function and viability of murine pancreatic islet beta-cells in vitro. Exposure of islets for 3 days to 200 U/ml of either IFN-gamma or TNF-alpha did not affect glucose-stimulated insulin release, but at higher concentrations (2000 U/ml) of either cytokine there was significant inhibition of glucose-stimulated insulin release. In combination, IFN-gamma and TNF-alpha each at 200 U/ml caused significant inhibition of glucose-stimulated insulin release; at 2000 U/ml glucose-stimulated insulin release was abolished. In time-course experiments, glucose-stimulated insulin release from islets exposed to IFN-gamma and TNF-alpha each at 1000 U/ml was significantly increased at 4-h (twofold increase compared with control islets), decreased back to control levels at 18 h, significantly inhibited by 24 h (threefold decrease compared with control islets), and completely abolished by 48 h. The progressive impairment of beta-cell function mediated by IFN-gamma plus TNF-alpha was associated with morphologic derangement of the islets that were almost totally disintegrated by day 6 of exposure to the cytokines. At day 6, insulin content of the islets was significantly reduced by exposure to TNF-alpha but not IFN-gamma. The combination of IFN-gamma and TNF-alpha resulted in a further dose-dependent depletion in insulin content compared with TNF-alpha alone. The synergistic functional and cytotoxic effects of IFN-gamma and TNF-alpha are consistent with a direct role for these cytokines in the destruction of beta-cells in insulin-dependent diabetes.     

Evidence for IL-6 production by and effects on the pancreatic beta-cell

IFN-gamma and TNF-alpha injure the pancreatic beta-cell and may be involved in the pathogenesis of autoimmune type 1 diabetes. Because the induction of IL-6 appears to be an important host cell response to injury, we have examined whether IL-6 is produced by murine pancreatic islets or rat insulinoma (RIN-m5F) cells after their exposure to IFN-gamma and TNF-alpha. Islet culture supernatants contained detectable IL-6 activity which was increased 6-fold when islets were exposed to IFN-gamma and 40- and 115-fold when islets were exposed to TNF-alpha and TNF-alpha + IFN-gamma, respectively. A mAb against murine IL-6 abolished (control and IFN-gamma) or significantly reduced (TNF-alpha and TNF-alpha + IFN-gamma) the IL-6 activity in islet supernatants. The magnitude for the effects of IFN-gamma and TNF-alpha on the production of IL-6 from mouse islets was found to be both time and dose dependent. Northern blot hybridization analysis of islet total cytoplasmic RNA with a cDNA probe to murine IL-6 revealed a band at 1.3 kb, the intensity of which increased in islets exposed to IFN-gamma + TNF-alpha. IL-6 activity was also detected in culture supernatants from RIN-m5F cells exposed to TNF-alpha + IFN-gamma. Islets cultured with rIL-6 secreted higher levels of insulin compared with control islets. Pancreatic islet cells, in all probability beta-cells, produce IL-6, the expression of which is up-regulated by IFN-gamma and/or TNF-alpha. In addition to a possible role in regulating pancreatic beta-cell function we propose that IL-6 produced by the pancreatic beta-cell may act as a costimulator for autoreactive B and T lymphocytes in autoimmune diabetes.     

Pancreatic islet immunoreactivity to the Reg protein INGAP.

The Reg-related protein family member INGAP (islet neogenesis-associated protein) is a pleiotropic factor enhancing islet neogenesis, neurite growth, beta-cell protection, and beta-cell function. Using an antibody to the N-termini of INGAP, we have identified that immunoreactivity to INGAP localized to the pancreatic endocrine cells in mouse. INGAP- and insulin-immunoreactive cells are mutually exclusive, with INGAP-immunoreactive cells being preserved after streptozotocin-mediated destruction of beta-cells. Glucagon- and INGAP-immunoreactive cells colocalize, although respective antigen expression occurs in different intracellular locations. These data suggest that INGAP-immunoreactive cells include alpha-cells; however, detection of single INGAP-immunoreactive/glucagon-negative cells indicates that this may not be exclusive. In addition to mouse, detection of islet endocrine cells that were INGAP immunoreactive/glucagon immunoreactive/insulin negative was also observed in islets from human, monkey, and rat. These findings reveal that INGAP and/or related group 3 Reg proteins have a conserved expression in the pancreatic islet.     

Decreased nutrient-stimulated insulin secretion in chronically hypoglycemic late-gestation fetal sheep is due to an intrinsic islet defect

We measured in vivo and in vitro nutrient-stimulated insulin secretion in late gestation fetal sheep to determine whether an intrinsic islet defect is responsible for decreased glucose-stimulated insulin secretion (GSIS) in response to chronic hypoglycemia. Control fetuses responded to both leucine and lysine infusions with increased arterial plasma insulin concentrations (average increase: 0.13 +/- 0.05 ng/ml leucine; 0.99 +/- 0.26 ng/ml lysine). In vivo lysine-stimulated insulin secretion was decreased by chronic (0.37 +/- 0.18 ng/ml) and acute (0.27 +/- 0.19 ng/ml) hypoglycemia. Leucine did not stimulate insulin secretion following acute hypoglycemia but was preserved with chronic hypoglycemia (0.12 +/- 0.09 ng/ml). Isolated pancreatic islets from chronically hypoglycemic fetuses had normal insulin and DNA content but decreased fractional insulin release when stimulated with glucose, leucine, arginine, or lysine. Isolated islets from control fetuses responded to all nutrients. Therefore, chronic late gestation hypoglycemia causes defective in vitro nutrient-regulated insulin secretion that is at least partly responsible for diminished in vivo GSIS. Chronic hypoglycemia is a feature of human intrauterine growth restriction (IUGR) and might lead to an islet defect that is responsible for the decreased insulin secretion patterns seen in human IUGR fetuses and low-birth-weight human infants.     

INGAP-related pentadecapeptide: its modulatory effect upon insulin secretion

We examined the effects of a pentadecapeptide having the 104-118 aminoacid sequence of islet neogenesis-associated protein (INGAP-PP) on insulin secretion, and the morphological characteristics of adult and neonatal pancreatic rat islets cultured in RPMI and 10 mM glucose for 4 days, with or without different INGAP-PP concentrations (0.1-100 mug/ml). A scrambled 15 aminoacid peptide was used as control for the specificity of INGAP-PP effect. Cultured neonatal and adult islets released insulin in response to glucose (2.8-16.7 mM) in a dose-dependent manner, and to leucine and arginine (10 mM). In all cases, the response was greater in adult islets. INGAP-PP added to the culture medium significantly enhanced glucose- and aminoacid-induced insulin release in both adult and newborn rats; however, no changes were observed with the scrambled peptide. Similar results were obtained incubating freshly isolated adult rat islets with INGAP-PP. Whereas INGAP-PP did not induce significant changes in islet survival rate or proportion/number of islet cells, it increased significantly beta-cell size. This first demonstration of the enhancing effect of INGAP-PP on the beta-cell secretory response of adult and newborn islets opens a new avenue to study its production mechanism and potential use to increase the secretory capacity of endogenous islets in intact animals or of islets preserved for future transplants.     

Expression of reg/PSP, a pancreatic exocrine gene: relationship to changes in islet beta-cell mass

A cDNA termed reg was recently isolated by differential screening of a library prepared from regenerating islets isolated from pancreatic remnants of rats subjected to 90% pancreatectomy and nicotinamide treatment. This led to speculation that this gene may be involved in expansion of beta-cell mass. In the current study we have measured reg expression after implantation and resection of a solid insulinoma tumor into rats, maneuvers known, respectively, to reduce and reexpand the volume of beta-cells in the islet. Animals with an implanted insulinoma tumor became profoundly hypoglycemic. Islet beta-cells declined from the normal 75% of total islet volume to less than 30%, in concert with a marked reduction in the reg mRNA level. Removal of the tumor resulted in a sharp increase in beta-cell replication, as measured by [3H]thymidine incorporation and a return to normal beta-cell volume within 4 days of tumor resection. This was associated with a transient induction in reg expression compared to that in tumor-bearing animals, effectively returning the amount of reg mRNA to the levels found in normal animals within 48 h; at later time points after tumor removal (3-7 days) reg expression declined, but then rose toward normal. In situ hybridization analysis localized the initial induction in reg mRNA expression to the exocrine pancreas. Continuous infusion of insulin into normal rats for 4 days, a maneuver that does not significantly reduce beta-cell mass, resulted in dramatically reduced insulin mRNA in islets, but no change in the levels of reg mRNA. We conclude that the diminution in pancreatic beta-cell mass caused by subcutaneous implantation of an insulinoma is associated with reduced reg gene expression and that the increase in beta-cell replication after resection of the tumor is preceded by return of reg gene expression toward normal.     

Effects of c-MYC activation on glucose stimulus-secretion coupling events in mouse pancreatic islets

Alteration of pancreatic beta-cell survival and Preproinsulin gene expression by prolonged hyperglycemia may result from increased c-MYC expression. However, it is unclear whether c-MYC effects on beta-cell function are compatible with its proposed role in glucotoxicity. We therefore tested the effects of short-term c-MYC activation on key beta-cell stimulus-secretion coupling events in islets isolated from mice expressing a tamoxifen-switchable form of c-MYC in beta-cells (MycER) and their wild-type littermates. Tamoxifen treatment of wild-type islets did not affect their cell survival, Preproinsulin gene expression, and glucose stimulus-secretion coupling. In contrast, tamoxifen-mediated c-MYC activation for 2-3 days triggered cell apoptosis and decreased Preproinsulin gene expression in MycER islets. These effects were accompanied by mitochondrial membrane hyperpolarization at all glucose concentrations, a higher resting intracellular calcium concentration ([Ca(2+)](i)), and lower glucose-induced [Ca(2+)](i) rise and islet insulin content, leading to a strong reduction of glucose-induced insulin secretion. Compared with these effects, 1-wk culture in 30 mmol/l glucose increased the islet sensitivity to glucose stimulation without reducing the maximal glucose effectiveness or the insulin content. In contrast, overnight exposure to a low H(2)O(2) concentration increased the islet resting [Ca(2+)](i) and reduced the amplitude of the maximal glucose response as in tamoxifen-treated MycER islets. In conclusion, c-MYC activation rapidly stimulates apoptosis, reduces Preproinsulin gene expression and insulin content, and triggers functional alterations of beta-cells that are better mimicked by overnight exposure to a low H(2)O(2) concentration than by prolonged culture in high glucose.     

Electrospray ionization mass spectrometric analyses of phospholipids from INS-1 insulinoma cells: comparison to pancreatic islets and effects of fatty acid supplementation on phospholipid composition and insulin secretion

Insulin secretion by pancreatic islet beta-cells is impaired in diabetes mellitus, and normal beta-cells are enriched in phospholipids with arachidonate as sn-2 substituent. Such molecules may play structural roles in exocytotic membrane fusion or serve as substrates for phospholipases activated by insulin secretagogues. INS-1 insulinoma cells respond to secretagogues and permit the study of effects of culture with free fatty acids on phospholipid composition and secretion. INS-1 cell glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) lipids are demonstrated here by electrospray ionization mass spectrometry to contain a lower fraction of molecules with arachidonate and a higher fraction with oleate as sn-2 substituent than native islets. Palmitic acid supplementation induces little change in these INS-1 cell lipids, but supplementation with linoleate or arachidonate induces a large rise in the fraction of INS-1 cell GPC species with polyunsaturated sn-2 substituents and a fall in oleate-containing species to yield a GPC profile similar to native islets. The fraction of GPE lipids comprised of plasmenylethanolamine species with polyunsaturated sn-2 substituents in early-passage INS-1 cells is similar to that of islets, but declines on serial passage. Such molecules might participate in exocytotic membrane fusion, and late-passage INS-1 cells have reduced insulin secretory responses. Arachidonate supplementation induces a rise in the fraction of INS-1 cell GPE lipids with polyunsaturated sn-2 substituents and partially restores responses to insulin secretagogues by late-passage INS-1 cells, but does not further amplify secretion by early-passage cells. Effects of extracellular free fatty acids on beta-cell phospholipid composition and secretory responses could be involved in changes in beta-cell function during the period of hyper-free fatty acidemia that precedes diabetes mellitus.     

The hexosamine biosynthesis pathway regulates insulin secretion via protein glycosylation in mouse islets

The hexosamine biosynthesis pathway plays a role in the modification of cellular proteins via the provision of substrate for addition of O-linked N-acetylglucosamine (GlcNAc). The relative importance of the GlcNAc modification of proteins to insulin secretion from pancreatic beta-cells has not been investigated and so remains unclear. In the present study, we show that inhibition of the hexosamine biosynthesis pathway decreases insulin secretion from mouse islets in response to a number of secretagogues, including glucose. This impairment in beta-cell function could not be attributed to reduced islet insulin content, altered ATP levels, or cell death and was restored with the addition of N-acetylglucosamine, a substrate that enters the pathway below the point of inhibition. Western blot analysis revealed that decreased islet protein glycosylation paralleled the decrease in insulin secretion following inhibition of the pathway. In conclusion, the data suggest a role for the hexosamine biosynthesis pathway in regulating the secretion of insulin by altering protein glycosylation. This finding may have implications for the development of type 2 diabetes, as chronic increase in flux through the hexosamine biosynthesis pathway may lead to the deterioration of beta-cell function via abnormal protein glycosylation.     

beta-cell adaptation in 60% pancreatectomy rats that preserves normoinsulinemia and normoglycemia

Islet beta-cells are the regulatory element of the glucose homeostasis system. When functioning normally, they precisely counterbalance changes in insulin sensitivity or beta-cell mass to preserve normoglycemia. This understanding seems counter to the dogma that beta-cells are regulated by glycemia. We studied 60% pancreatectomy rats (Px) 4 wk postsurgery to elucidate the beta-cell adaptive mechanisms. Nonfasting glycemia and insulinemia were identical in Px and sham-operated controls. There was partial regeneration of the excised beta-cells in the Px rats, but it was limited in scope, with the pancreas beta-cell mass reaching 55% of the shams (40% increase from the time of surgery). More consequential was a heightened glucose responsiveness of Px islets so that glucose utilization and insulin secretion per milligram of islet protein were both 80% augmented at normal levels of glycemia. Investigation of the biochemical basis showed a doubled glucokinase maximal velocity in Px islets, with no change in the glucokinase protein concentration after adjustment for the different beta-cell mass in Px and sham islets. Hexokinase activity measured in islet extracts was also minimally increased, but the glucose 6-phosphate concentration and basal glucose usage of Px islets were not different from those in islets from sham-operated rats. The dominant beta-cell adaptive response in the 60% Px rats was an increased catalytic activity of glucokinase. The remaining beta-cells thus sense, and respond to, perceived hyperglycemia despite glycemia actually being normal. beta-Cell mass and insulin secretion are both augmented so that whole pancreas insulin output, and consequently glycemia, are maintained at normal levels.     

Trefoil factor 3 stimulates human and rodent pancreatic islet beta-cell replication with retention of function

Both major forms of diabetes involve a decline in beta-cell mass, mediated by autoimmune destruction of insulin-producing cells in type 1 diabetes and by increased rates of apoptosis secondary to metabolic stress in type 2 diabetes. Methods for controlled expansion of beta-cell mass are currently not available but would have great potential utility for treatment of these diseases. In the current study, we demonstrate that overexpression of trefoil factor 3 (TFF3) in rat pancreatic islets results in a 4- to 5-fold increase in [(3)H]thymidine incorporation, with full retention of glucose-stimulated insulin secretion. This increase was almost exclusively due to stimulation of beta-cell replication, as demonstrated by studies of bromodeoxyuridine incorporation and co-immunofluorescence analysis with anti-bromodeoxyuridine and antiinsulin or antiglucagon antibodies. The proliferative effect of TFF3 required the presence of serum or 0.5 ng/ml epidermal growth factor. The ability of TFF3 overexpression to stimulate proliferation of rat islets in serum was abolished by the addition of epidermal growth factor receptor antagonist AG1478. Furthermore, TFF3-induced increases in [3H]thymidine incorporation in rat islets cultured in serum was blocked by overexpression of a dominant-negative Akt protein or treatment with triciribine, an Akt inhibitor. Finally, overexpression of TFF3 also caused a doubling of [3H]thymidine incorporation in human islets. In summary, our findings reveal a novel TFF3-mediated pathway for stimulation of beta-cell replication that could ultimately be exploited for expansion or preservation of islet beta-cell mass.     

Expression and function of GLUT-1 and GLUT-2 glucose transporter isoforms in cells of cultured rat pancreatic islets.

We have previously investigated glucose induction of glucokinase, glucose usage and insulin release in isolated cultured rat pancreatic islets (Liang, Y., Najafit, H., Smith, R. M., Zimmerman, E. C., Magnuson, M. A., Tal, M., and Mastchinsky, F. M. (1992) Diabetes (1992) 41, 792-806). Here we studied the expression and function of GLUT-1 and GLUT-2 glucose transporter isoforms, using the same system, i.e. isolated pancreatic rat islets immediately after isolation or cultured in the presence of 3 or 30 mM glucose for as long as 10 days. We found by immunofluorescence microscopy and Western and Northern blot analysis of islet extracts that GLUT-1 expression was induced in islet beta-cells in tissue culture both with low or high glucose present. The induction of GLUT-1 was specific to beta-cells but was not present in all beta-cells and was not detected in alpha-cells. GLUT-2 expression was also specific for beta-cells and was not observed in all beta-cells. Some beta-cells in culture coexpressed GLUT-1 and GLUT-2. The expression of the two glucose transporters was regulated in the opposite direction in response to glucose concentration in the culture medium. GLUT-1 was more effectively induced when glucose was low, and GLUT-2 expression was more pronounced when glucose was high in the culture media. Another difference between the two glucose transporters was that GLUT-2 expression was increased while GLUT-1 expression was decreased as culturing continued as long as 7 days. Thus, after 7 days of culture GLUT-2 expression in beta-cells was nearly the same at low and high glucose, whereas GLUT-1 was practically absent no matter what the glucose level was. In attempts to correlate GLUT-1 and GLUT-2 expression to beta-cell function glucose uptake and glucose-stimulated insulin release in fresh and cultured islets were measured. In freshly isolated islet glucose uptake was estimated to be 100-fold in excess of actual glucose use. Glucose uptake was reduced by 7-day culture to about one-third of that observed in freshly isolated islets no matter what the glucose concentration of the culture media. We conclude that in the present experimental system GLUT-1 and GLUT-2 expression and function are not closely associated with glucose usage rates or the secretory function of beta-cells.