Tolbutamide stimulates proliferation of pancreatic beta cells in culture.

To investigate the beta-cell cytotrophic action of tolbutamide, pancreatic cells were dissociated from neonatal rat pancreata and then cultured for 10 days in the presence of various concentrations of tolbutamide. After counting insulin-positive beta cells, dose-response curves were plotted and analyzed. Increasing concentrations of tolbutamide cause an increase in beta-cell numbers until a maximum response is reached at a concentration of about 100 micrograms/ml. At greater concentrations, tolbutamide becomes inhibitory. At concentrations of 100 micrograms/ml of tolbutamide, beta-cell numbers are doubled. We further investigated the effect of glucose on tolbutamide-induced stimulation. Tolbutamide fails to stimulate beta cells after culture in medium containing low glucose concentrations. High concentrations of glucose are required for tolbutamide action. Preexposure for as little as 16 h to low glucose abolishes the stimulatory action of tolbutamide. The experiments indicate that adequate glucose concentrations are necessary for the preservation of tolbutamide-induced beta-cell survival and proliferation. Addition to the culture medium of the calcium channel blocker diltiazem or the calmodulin antagonist chlorpromazine affects a decrease in beta-cell numbers. This decrease depends on the concentration of the drug. Addition of tolbutamide reverses that decrease, which suggests that calcium is required for beta-cell survival and proliferation. Tolbutamide action seems to be specific, since fibroblasts are unaffected at concentrations of tolbutamide that are stimulatory for beta cells. At high concentrations, tolbutamide is cytotoxic for fibroblasts. Comparison with other sulfonylureas supports the superior activity of tolbutamide. The "second generation" sulfonylurea glyburide is cytotoxic.(ABSTRACT TRUNCATED AT 250 WORDS)

Allotransplantation of culture-isolated neonatal rat islet tissue. Absence of MHC class II positive antigen-presenting cells in nonimmunogenic islets.

When highly purified neonatal rat islet tissue, derived after 10 days in vitro, was allografted, it was found to be nonimmunogenic or weakly immunogenic. In contrast, nonislet pancreatic components, derived from the same culture system, transplanted with highly purified islet tissue resulted in rejection in 88% of cases. Extension of the culture period did not result in reduced immunogenicity of the nonislet material. Immunostaining of islet or nonislet tissue from the culture system failed to demonstrate major histocompatibility complex (MHC) class II positive cells in the islet tissue, whereas the presence of MHC class II staining cells in the nonislet components was clearly demonstrable. These results demonstrate that the islet tissue obtained by culture isolation is free of cells capable of stimulating an allogeneic immune response and are consistent with the hypothesis that the absence of MHC class II positive antigen-presenting cells reduces the immunogenicity of the tissue and enhances the survival of allogeneic grafts.

Proliferation of immunocytochemically identified islet beta cells in culture: effect of growth factors, hormones, and nutrients.

Immunocytochemically identified, differentiated, single beta-cells proliferate to form colonies, which then grow into hillocks and islets. Beta-Cell proliferation is most easily quantified during the first week, when colonies are forming. The experimental objective was stimulation of beta-cell proliferation by culture medium supplementation with growth factors, hormones, or nutrients. We found that beta-cell proliferation is stimulated by iron-saturated transferrin, interleukin-1-alpha, fetal calf serum, and glucose. In response to transferrin, proliferation of beta-cells is progressively stimulated, reaching a maximum at 30 micrograms/ml. At greater concentrations the stimulatory effect is progressively lost. Interleukin-1-alpha maximally stimulates beta-cell proliferation at 10 pg/ml, regresses to control levels at 10(3) pg/ml, and inhibits proliferation progressively at greater concentrations. Fetal calf serum maximally stimulates beta-cell proliferation at concentrations of 10%, and glucose stimulates maximally at 15 mM concentrations. The proliferative response to transferrin, interleukin-1-alpha or glucose is serum dependent. Serum and transferrin synergistically stimulate glucose-induced beta-cell proliferation. Interleukin-1-beta, interleukin-2, rat growth hormone, and rat prolactin fail to stimulate beta-cell proliferation.

Clonal growth of immunocytochemically identified islet beta and alpha cells in culture.

The proliferative capacity of individual immunocytochemically identified islet beta cells was investigated in tissue culture. The pancreatic digest was filtered through a 20-micron filter to eliminate partially digested tissue fragments and islets; it was then cultured at low density to allow assessment of single cells. The type of cell was identified immunocytochemically by reaction with antibody to insulin or glucagon, and DNA synthesis was estimated from autoradiographs after incorporation of tritiated thymidine. Single immunocytochemically reactive beta or alpha cells attached to the culture substratum and then proliferated, directly proportional to time in culture. Single beta cells did not incorporate thymidine after 1 day in culture; nonendocrine cells, presumed to be mainly fibroblasts, readily incorporated thymidine. Beyond the first day, about 10% of the beta cells incorporated thymidine. The number of radioactively labeled, immunocytochemically reactive beta cells increased for 5 days in culture and then remained at the elevated level until experiments were terminated at 12 days. DNA synthesis in beta cells occurred in two waves separated by about 3 days, suggesting a generation time of about 3 days for immunocytochemically identified beta cells. Fetal calf serum was found to be an essential culture medium ingredient to sustain thymidine incorporation; horse serum was found to be an unsuitable substitute. Mitotic figures were recorded in differentiated beta and alpha cells. These studies conclusively show that differentiated beta and alpha cells can proliferate in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-reactivity of organs in allograft rejection. Comparison of effect of thyroid allografts on established islet allografts.

The effect of allotransplantation of thyroid or islet allografts into rats with established islet allografts was studied to determine the cross-reactivity of the thyroid and islets in allograft rejection. Islets obtained from cultured neonatal rat (F344) pancreas explants were transplanted bilaterally underneath the kidney capsule of Wistar-Furth rats. After 21 days these allografts did not exhibit signs of rejection. Thyroid (half lobe) from either F344 or Brown Norway rats was transplanted underneath the capsule of the remaining kidney. Transplant of the thyroid from F344 rats resulted in immediate rejection of the islet transplant, whereas transplant of the thyroid from Brown Norway rats was without effect on the islet allograft. This indicates that the thyroid contains immunocompetent cells (cells that present antigen or induce recognition of antigen) that are capable of initiating rejection of established islet allografts. The cytotoxic T-lymphocytes that result are specific for the organ bearing the immunocompetent cells at time of transplantation.

Islet neoformation in tissue culture.

We have devised a tissue culture system that permits de novo formation of islets. Neonatal rat pancreata are enzymatically dissociated into single cells. The cell suspension is filtered through polyester cloth with 20 microns pores to exclude cell aggregates as well as preformed islets and a single cell suspension is then plated into tissue culture dishes at a density precluding reaggregation. Pancreatic cells proliferate forming numerous colonies of epithelioid cells. After a confluent monolayer, cells proliferate into a third dimension, the space occupied by the culture medium. Third dimensional proliferation occurs from basal monolayers of epithelioid colonies. At about 9 days in culture, numerous hillocks are visible that are spaced at about 1 mm from one another. Islets are observed to bud from the hillock surfaces. In 1 micron-thick sections, secretion granules are detected with the light microscope in some islet cells. With the electron microscope three basic cell types are seen. One peripherally located cell type is sparsely granulated and appears to be a precursor cell. The other peripherally located cell type shows a homogeneous population of secretion granules characteristic of A-cells. The third cell type is found in the interior of islets containing granules characteristic of B-cells. Islet cells, but not hillock cells, react immunocytochemically for insulin and glucagon. The cultures secrete 2 to 10-fold the amounts of glucagon present in fresh medium. It is concluded that differentiation of A- and B-cells occurs in neoformed islets.

Specificity of chicken and mammalian transferrins in myogenesis.

Chicken transferrins isolated from eggs, embryo extract, serum or ischiatic-peroneal nerves are able to stimulate incorporation of [3H]thymidine, and promote myogenesis by primary chicken muscle cells in vitro. Mammalian transferrins (bovine, rat, mouse, horse, rabbit, and human) do not promote [3H]thymidine incorporation or myotube development. Comparison of the peptide fragments obtained after chemical or limited proteolytic cleavage demonstrates that the four chicken transferrins are all indistinguishable, but they differ considerably from the mammalian transferrins. The structural differences between chicken and mammalian transferrins probably account for the inability of mammalian transferrins to act as mitogens for, and to support myogenesis of, primary chicken muscle cells.

Regulation of mitotic activity and the cell cycle in primary chick muscle cells by neurotransferrin.

We previously demonstrated that neurotransferrin (NTF), a transferrin extracted from adult chicken peripheral nerves, promotes growth of primary chick muscle cells in the absence of embryo extract. NTF was shown to stimulate DNA synthesis and cell proliferation. In the present study, we demonstrate that NTF is a mitogen using two independent methods; counts of orcein-stained mitotic figures and analysis of cell cycle kinetics with a fluorescence-activated cell sorter. In low-density cultures mitotic activity increases with increasing doses of NTF followed by a plateau at concentrations greater than 6 micrograms/ml. Residual, embryonic mitotic activity progressively declines with time after plating muscle cells in the absence of NTF. Absence of NTF for 2 days causes cells to lose irreversibly their myogenic potential. In the presence of NTF, mitotic activity increases for 2 days followed by a decline concurrent with myoblast fusion and formation of myotubes. Cell cycle analysis showed that NTF addition causes cell populations to shift from G1 to S and G2 + M within 18.5 hr. Muscle cells, plated at high densities in the absence of NTF, show mitotic activities similar to those plated at low densities in the presence of NTF. Addition of NTF to high-density cultures is ineffective in stimulating mitosis. These studies show that at typical cell plating densities, NTF is a required mitogen for primary chick muscle cell cultures.

Peripheral nerve extract promotes long-term survival and neurite outgrowth in cultured spinal cord neurons.

The hypothesis that peripheral, skeletal muscle tissue contains a trophic factor supporting central neurons has recently been investigated in vitro by supplementing the culture medium of spinal cord neurons with muscle extracts and fractions of extract. We extended these studies asking whether or not a trophic factor is present in peripheral nerves, the connecting link between muscle and central neurons via which factors may be translocated from muscle to neurons by the retrograde transport system. Lumbar, 8-day-old chick spinal cords were dissociated into single cells and then cultured in the presence of peripheral nerve extract. Cytosine arabinoside was added to inhibit proliferation of nonneuronal cells. In the presence of nerve extract, spinal cord neurons survived for more than a month, extended numerous neurites, and showed activity of choline acetyltransferase. In the absence of extract, neurons attached and survived for a few days but then died subsequently in less than 10 days. Neurite outgrowth did not occur in the absence of extract. Withdrawal of extract from the medium of established neuronal cultures caused progressive loss of both cells and neurites. Other tissues also contained neuron supporting activity but less than that found in nerve extract. These studies indicate that peripheral nerves contain relatively high levels of spinal cord neuron-directed trophic activity, suggesting translocation of neurotrophic factor from muscle to central target neurons. The neurotrophic factor has long-term (weeks) effects, whereas short-term (days) survival is factor independent.

Appearance of acetylcholinesterase molecular forms in noninnervated cultured primary chick muscle cells.

Asymmetric forms of AChE have generally not been detected in cultured chick skeletal muscle cells in the absence of cocultured neurons. To explore further neurotrophic effects of adult peripheral nerve extracts (NE) on muscle in vitro, we reexamined the appearance of various molecular forms of AChE in cultured chick muscle cells in the presence of NE. The various molecular forms of AChE were distinguished by sucrose gradient sedimentation and radioenzymatic techniques. In the presence of NE, cells proliferated during the first 48 hr of culture, then fused and formed spontaneously contracting myotubes by 6-8 days in culture. Total AChE, 5.4 S, and 11.5 S molecular forms reached activity plateaus by 8 days in culture which persisted until cultures were terminated at day 20. Between 1 and 6 days in culture, 19.5 S AChE (A12) was not detected. The A12 form was first observed at 7 days reaching a maximum of 11.3% of the total AChE at 14 days and then gradually declined to a level of 1% at day 20. Since the A12 form declined in older cultures but comprised 25% in embryonic muscle tissue, we examined the possible requirement of neurons in culture to attain higher levels of A12 AChE. Spinal cord neurons were plated onto 6-day muscle cultures and AChE activities were measured between 8 and 20 days. The results showed that 19.5 S AChE activity in the presence of both spinal cord neurons and NE was no greater than that found in the presence of NE alone. To suppress spontaneous contraction, 0.6 microM tetrodotoxin (TTX) or 15 microM d-tubocurarine (dTC) were added to 5-day-old muscle cultures at a time when myotubes were differentiated but contractile activity had not begun. TTX had cytotoxic effects and inhibited further development of myotubes. In contrast, dTC had no deleterious effect on morphological development, eliminated contraction, but did not interfere with the appearance of any forms of AChE including the A12 form. These studies show that primary chick muscle cells are capable of producing the A12 form of AChE if cultured in NE-supplemented medium. In this culture system, production of the A12 form does not require activity or innervation.

The identification of neurotrophic factor as a transferrin.

Partially purified neurotrophic factor (NTF) from chicken nerves comigrated with transferrin and a component in several preparations known to have neurotrophic effects on cultured skeletal muscle cells. One-dimensional gel electrophoretograms of proteolytic fragments of NTF and fragments obtained from transferrins purified from chicken eggs, serum and embryos were indistinguishable. These purified transferrins, like NTF, all stimulated the incorporation of [3H]thymidine and supported myotube formation to a similar degree as NTF. These studies suggest that NTF is a transferrin-like protein and that both transferrins and NTF act by initially promoting myoblast proliferation and subsequently supporting myogenesis in chick muscle cultures.

Dose-dependent initiation of myogenesis by neurotrophic factor.

The kinetics of primary chick muscle cells in response to partially purified neurotrophic factor (NTF) were investigated. Chick muscle cells are routinely plated in the absence of NTF but in the presence of horse serum to allow cell attachment to the substratum; a maximum number of cells attaches 5-8 hr after plating, but cells do not proliferate in the absence of NTF, or do so extremely slowly. Subsequent to a routine attachment period of 6 hr, the medium is aspirated and replaced with medium containing 2-20 micrograms/ml of NTF as well as horse serum. Dose responsiveness at elevated NTF concentrations is observed only if F12 medium, supplemented with serum, insulin, transferrin, and selenium is used. In the presence of commercial F10, although supplemented with serum, dose responsiveness is not apparent at more than 2 micrograms/ml of NTF. Upon exposure to NTF, in the presence of supplemented F12 and serum, an 8-hr lag ensues; then, thymidine is incorporated at NTF dose-dependent rates by growing cell populations. The rates of incorporation into cell populations depend on the initial number of cells plated and the amount of NTF supplied. Cell counting indicates that dose-dependent proliferation of muscle cells has occurred during the first 48 hr of exposure to NTF. The specific activity of NTF is repeatedly shown to be at least 10-fold greater than unfractionated nerve extract. Shortly after 48 hr of exposure to NTF, myoblasts characteristically begin to fuse with one another, and myogenesis is visible. These studies show that the initiation of in vitro myogenesis in primary chick muscle cells depends on the amount of NTF supplied. The work also indicates a requirement for optimum medium conditions in order to detect dose-responses to NTF.

Muscle satellite cells in urodele amphibians: faciliatated identification of satellite cells using ruthenium red staining.

The ruthenium red (RR) stained forelimb musculature of three species of urodeles Triturus (Notophthalmus) viridescens, Amblystoma maculatum, Amblystoma opacum in various stages of growth were examined with the electron microscope for the presence of satellite cells. It was found that RR staining facilitated greatly the identification of satellite cells. In young larvae of all three species satellite cells were detected with a frequency of 29% to 48% per total number of nuclei. In adult Triturus and Amblystoma maculatum satellite cells were no longer detected; instead "pericytes" as described by Hay ('74) were seen with a frequency of 12% and 3% respectively. During metamorphosis of Triturus satellite cells, with part of their myofiber-satellite cell intercellular space filled with basement membrane material, occurred at a peak frequency. The cells presumably are intermediate in the formation of "pericytes." At ten days after metamorphosis satellite cells and intermediate cells were no longer detected and the limb musculature contained only "pericytes" similar to the ones observed in adult newts. The significance of the presence of satellite cells in relation to limb regeneration and muscle regeneration is discussed.