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M Asfari

Publications and source records attributed to M Asfari.

12 recordsLinked to original sources
PubMed2005

Short-term treatment with mycophenolic acid and tacrolimus is tolerogenic for INS-1 cell clone transplantation and the deleterious effects of the drugs are limited: in vivo and in vitro studies.

One of the major requirements for a successful and life-lasting organ transplant is the access to safe, least toxic and permanent tolerance-inducing drugs. In this study we wished to evaluate the effects of tolerogenic doses of the immunosuppressive drugs mycophenolic acid (MPA) and tacrolimus (Tac) on clonal beta-cell lines, both in vivo and in vitro. Here we demonstrate that combined administration of low-dose MPA and Tac for 23 days induced permanent tolerance in an allogeneic beta-cell line transplant in Wistar rat liver through the portal vein. This short-term treatment of tolerogenic doses of the two drugs was deleterious to the survival of the transplanted cells but a small percentage of the cells could resist the effect and become fully active when the drugs were removed. The surviving cells, retrieved from growth in vivo, did not exhibit increased resistance in comparison to the original cells when tested in vitro at two glucose concentrations, 10 and 20 mM. The presence of a small percentage of resistant cells at the two glucose concentrations was also detected in the in vitro study after a continuous 8-day treatment demonstrating that the in vivo resistance was not related to micro-environmental protection but possibly to a phenotypic cell state that is yet to be determined.

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PubMed1997

Induction by glucose of genes coding for glycolytic enzymes in a pancreatic beta-cell line (INS-1).

Chronic elevation in glucose has pleiotropic effects on the pancreatic beta-cell including a high rate of insulin secretion at low glucose, beta-cell hypertrophy, and hyperplasia. These actions of glucose are expected to be associated with the modulation of the expression of a number of glucose-regulated genes that need to be identified. To further investigate the molecular mechanisms implicated in these adaptation processes to hyperglycemia, we have studied the regulation of genes encoding key glycolytic enzymes in the glucose-responsive beta-cell line INS-1. Glucose (from 5 to 25 mM) induced phosphofructokinase-1 (PFK-1) isoform C, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4-fold), and L-pyruvate kinase (L-PK) (7-fold) mRNAs. In contrast the expression level of the glucokinase (Gk) and 6-phosphofructo-2-kinase transcripts remained unchanged. Following a 3-day exposure to elevated glucose, a similar induction was observed at the protein level for PFK-1 (isoforms C, M, and L), GAPDH, and L-PK, whereas M-PK expression only increased slightly. The study of the mechanism of GAPDH induction indicated that glucose increased the transcriptional rate of the GAPDH gene but that both transcriptional and post transcriptional effects contributed to GAPDH mRNA accumulation. 2-Deoxyglucose did not mimic the inductive effect of glucose, suggesting that increased glucose metabolism is involved in GAPDH gene induction. These changes in glycolytic enzyme expression were associated with a 2-3-fold increase in insulin secretion at low (2-5 mM) glucose. The metabolic activity of the cells was also elevated, as indicated by the reduction of the artificial electron acceptor 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium. A marked deposition of glycogen, which was readily mobilized upon lowering of the ambient glucose, and increased DNA replication were also observed in cells exposed to elevated glucose. The results suggest that a coordinated induction of key glycolytic enzymes as well as massive glycogen deposition are implicated in the adaptation process of the beta-cell to hyperglycemia to allow for chronically elevated glucose metabolism, which, in this particular fuel-sensitive cell, is linked to metabolic coupling factor production and cell activation.

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PubMed1996

Discordant Regulation of insulin-like growth factor-II and insulin-like growth factor binding protein-2 gene expression in a rat beta-cell line.

Expression and regulation of IGFBP-2 and IGF-II by GH and glucose in the rat insulin producing beta-cell line, INS-1, were determined. These cells express high levels of the insulin-like growth factor II (IGF-II) gene which is regulated by glucose. GH has no regulatory activities on IGF-II or IGFBP-2 gene expression. The levels of IGF-II and IGFBP-2 mRNA at increasing glucose concentrations are inversely regulated and exogenously added insulin inhibits IGFBP-2 gene expression at low glucose. These observations suggest that the expression of IGFBP-2 gene in this cell line is under the negative control of the endogenously produced insulin (and/or IGF-II) which accumulates to effective levels in the medium when the cells are cultured at high glucose concentrations.

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PubMed1995

Growth hormone (GH) and prolactin (PRL) regulate IGFBP-3 gene expression in rat beta-cells.

We examined the effects of growth hormone (GH), prolactin (PRL) and insulin-like growth factor-I (IGF-I) on DNA synthesis, and insulin-like growth factor binding protein 3 (IGFBP-3) gene expression, in a rat beta-cell line INS-1. GH and PRL were equally effective in increasing DNA synthesis and IGFBP-3 gene expression. IGF-I also stimulated DNA synthesis, but its effect on IGFBP-3 mRNA accumulation was moderate. The GH activities in this cell line were not mediated by IGF-I production: (1) no IGF-I mRNA could be detected in poly-A RNA preparation, and IGF-I level was very low; (2) neither IGF-I mRNA nor the protein, were increased upon GH treatment; (3) DNA synthesis stimulatory activities of GH and IGF-I were additive; (4) the GH-induced IGFBP-3 mRNA increase was not inhibited by cycloheximide treatment. Thus, the activities of GH in this cell line are not mediated by a protein synthesis.

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PubMed1995

Insulin-like growth factor-II gene expression in a rat insulin-producing beta-cell line (INS-1) is regulated by glucose.

A highly differentiated rat glucose-responsive insulin producing cell line INS-1 expresses high levels of insulin-like growth factor-II (IGF-II). Basal levels of IGF-II gene mRNA were expressed in cells cultured at 1-6 mmol/l glucose. At glucose concentrations of 10-20 mmol/l, IGF-II mRNA was increased more than threefold after 44 h of incubation. Levels of IGF-II mRNA in INS-1 cells incubated at 5.6 and 20 mmol/l glucose in the presence of 4 micrograms/ml actinomycin D are comparable and are not reduced during 20 h of treatment, indicating the high stability of IGF-II mRNA in this cell line. From the three rat IGF-II promoters, promoter 3 is by far the most active in INS-1 cells. The IGF-II promoter 3 activity and IGF-II mRNA production at high glucose concentrations increased threefold over their respective levels at low glucose concentration, suggesting that the glucose-induced IGF-II gene expression in this beta-cell line might be transcriptionally controlled. The up-regulation of IGF-II mRNA by glucose was not due to the increased intracellular cyclic AMP levels or protein kinase C activation. A protein kinase C activator had no effect on IGF-II gene expression, and an adenylate cyclase activator (forskolin), suppressed the stimulatory effects of glucose on the IGF-II mRNA. Under all the experimental conditions examined, the IGF-II and insulin genes were differentially regulated in INS-1 cells. The IGF-II gene expression and DNA synthesis, however, were regulated in parallel, suggesting that these two cellular activities are closely associated.

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PubMed1995

Expression and regulation of growth hormone (GH) and prolactin (PRL) receptors in a rat insulin producing cell line (INS-1).

We have examined the expression of growth hormone (GH) and prolactin (PRL) receptors, and the binding parameters of human GH (hGH), on the rat insulin producing cell line INS-1. We found that, like normal insulin producing beta-cells, INS-1 cells express both GH and PRL receptors, and the majority of human GH (hGH) binding sites on this cell line are of lactogenic specificity. As calculated from Scatchard plots, about 6600 hGH binding sites with a Ka of 6.2 x 10(8) M-1 are present per cell. Northern blot analysis showed two mRNA species of 4 and 1.6 kb for the GH receptor and, one major species of 10.5 kb for the PRL receptor. The PRL receptor mRNA was up-regulated by bovine GH (bGH), rat PRL (rPRL), in a time- and dose-dependent manner. On the contrary, bGH and rPRL, down-regulated the expression of GH receptor gene. The importance of this differential regulation in in vivo and in vitro studies are discussed.

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PubMed1992

Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines.

New insulin-secreting cell lines (INS-1 and INS-2) were established from cells isolated from an x-ray-induced rat transplantable insulinoma. The continuous growth of these cells was found to be dependent on the reducing agent 2-mercaptoethanol. Removal of this thiol compound caused a 15-fold drop in total cellular glutathione levels. These cells proliferated slowly (population doubling time about 100 h) and, in general, showed morphological characteristics typical of native beta-cells. Most cells stained positive for insulin and did not react with antibodies against the other islet hormones. The content of immunoreactive insulin was about 8 micrograms/10(6) cells, corresponding to 20% of the native beta-cell content. These cells synthesized both proinsulin I and II and displayed conversion rates of the two precursor hormones similar to those observed in rat islets. However, glucose failed to stimulate the rate of proinsulin biosynthesis. In static incubations, glucose stimulated insulin secretion from floating cell clusters or from attached cells. Under perifusion conditions, 10 mM but not 1 mM glucose enhanced secretion 2.2-fold. In the presence of forskolin and 3-isobutyl-1-methylxanthine, increase of glucose concentration from 2.8-20 mM caused a 4-fold enhancement of the rate of secretion. Glucose also depolarized INS-1 cells and raised the concentration of cytosolic Ca2+. This suggests that glucose is still capable of eliciting part of the ionic events at the plasma membrane, which leads to insulin secretion. The structural and functional characteristics of INS-1 cells remained unchanged over a period of 2 yr (about 80 passages). Although INS-2 cells have not been fully characterized, their insulin content was similar to that of INS-1 cells and they also remain partially sensitive to glucose as a secretagogue. INS-1 cells retain beta-cell surface antigens, as revealed by reactivity with the antigangloside monoclonal antibodies R2D6 and A2B5. These findings indicate that INS-1 cells have remained stable and retain a high degree of differentiation which should make them a suitable model for studying various aspects of beta-cell function.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Effects of cytokines on rat insulinoma INS-1 cells.

To investigate further the role of cytokines in the pathogenesis of type I insulin-dependent diabetes mellitus, the effects of interleukin-1 beta (IL-1), tumour necrosis factor-alpha (TNF) and gamma-interferon (IFN) were tested on rat insulinoma INS-1 cells. Whereas TNF and IFN had, respectively, a minor or no effect on insulin production, IL-1 caused a time- and dose-dependent decrease in insulin release and lowered the insulin content as well as the preproinsulin mRNA content of INS-1 cells. Both IL-1 and TNF exerted a cytostatic effect, estimated by a decrease in [3H]thymidine incorporation, while only IL-1 decreased cell viability as measured by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The glutathione content of INS-1 cells was shown to be modulated by the presence of 2-mercaptoethanol in the culture medium, but was not affected by IL-1 or TNF. In conclusion, INS-1 cell culture is considered to be a useful model for studying the effect of cytokines on insulin-producing cells. The differentiated features of these cells will permit several questions to be addressed regarding the mechanism of action of IL-1 and eventually other cytokines, both at the level of gene expression and of intracellular signalling.

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PubMed1989

Transcriptional induction of glucokinase gene by insulin in cultured liver cells and its repression by the glucagon-cAMP system.

Primary cultures of rat hepatocytes were used to investigate the regulation of glucokinase gene expression by insulin and glucagon. Insulin added in physiological concentrations to the culture medium causes de novo induction of glucokinase mRNA. The induced plateau is reached 4 to 8 h after insulin addition, and the mRNA level remains high as long as insulin is present. Comparison of the potencies of insulin, proinsulin, and insulin-like growth factor I in this system indicates that induction by insulin is mediated via the insulin receptor. The magnitude of the insulin effect is independent of the extracellular glucose concentration. Run-on transcription assays with isolated nuclei show that the mRNA build up depends primarily on a specific stimulation of glucokinase gene transcription. Glucagon added to hepatocytes together with a supramaximal concentration of insulin prevents induction of glucokinase mRNA in a dose-dependent manner. The inhibitory effect of glucagon is mimicked by 8-(4-chlorophenylthio)-cAMP. The effect of this agent has also been tested in hepatocytes first induced for maximal glucokinase gene transcription by culture with insulin alone for 12 h. The transcriptional activity of the gene as measured by run-on assay was completely turned off within 30 min after addition of the cyclic nucleotide. Under these conditions, glucokinase mRNA decays rapidly, with an apparent half-life of 45 min. The mRNA degradation rate was similarly rapid after insulin withdrawal from induced cells. Thus, a cAMP-mediated repression mechanism is a key aspect in the regulation of glucokinase gene transcription in the hepatocyte. Insulin may act by relieving the gene from repression.

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PubMed1988

Transplantation studies of a putative lymphosarcoma of Xenopus.

The fate of the putative transplantable Xenopus lymphosarcoma (M. Balls, Cancer Res., 22: 1142-1154, 1962) was studied under three experimental conditions: (a) xenotransplantation, i.e., transplantation of live "tumor" tissue between adults of Xenopus borealis and X. laevis; (b) inoculation of live "tumor" cells from X. borealis into the blastocoele of X. laevis embryos; and (c) transplantation of "tumor" tissue into recipient adults immunologically unresponsive to the donor tissue antigens. This condition is fulfilled by using X. laevis-X. gilli (LG) hybrids [H.R. Kobel and L. Du Pasquier. In: J.B. Solomon and J.D. Horton (eds.), Developmental Immunology, pp. 229-306. The Netherlands: Elsevier/North-Holland Biomedical Press, 1977] as donors, and triploid X. laevis-X. gilli and X. borealis (LGB) hybrids [C.H. Thiébaud, Dev. Biol., 98: 245-249, 1983] as recipients. In all transplantation experiments, donor and recipient cells could be unambiguously distinguished upon quinacrine staining that yields typical nuclear patterns, for instance bright patchiness in X. borealis, visible also in LGB cells. The results of xenotransplantation between X. borealis and X. laevis indicated that all developing "tumors" were composed of the recipient cell phenotype. The inoculation of live "tumor" cells from X. borealis "tumor" into the blastocoele of X. laevis embryos resulted in "tumor" formation in the recipient tadpoles and in metamorphosed animals. The cell constituting these "tumors" all were of recipient, X. laevis cell phenotype. Finally, "tumor" tissues from LG clones transplanted into LGB hosts were replaced by "tumors" formed of cells with recipient, LGB phenotype. These experiments indicate that this Xenopus tumor-like growth is a transmissible and not a transplantable disorder.

Animals↗
PubMed1988

Mycobacterium-induced infectious granuloma in Xenopus: histopathology and transmissibility.

The acid-fast bacteria present in the transmissible putative Xenopus lymphosarcoma (px1) (M. Balls, Cancer Res., 22: 1142-1154, 1962) were isolated and identified as Mycobacterium marinum. M. marinum induces an infectious granuloma in Xenopus which is histopathologically indistinguishable from Xenopus "lymphosarcoma" (px1). It had been reported that the filtrate (0.22-micron pore size) and the supernate of ultracentrifugation (20,000 x g) of the pxl tissue homogenate were bacteria free and could transmit the disease (R.H. Clothier and M. Balls, Oncology, 28: 445-457, 1973; R.H. Clothier and M. Balls, Oncology, 28: 458-480, 1973). Both features, however, also correspond to the transmission of M. marinum-induced granuloma. The granuloma induced by the ultrafiltrate of granulomatous tissue homogenate may be due to the granulomatogenic component of the bacteria-containing tissue, since no bacteria were detected in such filtrate. The 100,000 x g centrifugation supernate of the same homogenate, however, contains bacteria which induce granuloma. Since no other feature of pxl corresponds to neoplasia, it is concluded that as diagnosed by Dawe [C.J. Dawe, Natl. Cancer Inst. Monogr., 32: 7-28, 1969; C.J. Dawe. In: M. Mizell (ed.), Recent Results in Cancer Research, pp. 429-440. New York: Springer-Verlag, 1969; C.J. Dawe. In: R.M. Dutcher (ed.), Comparative Leukemia Research. Bibliotheca Haematologica pp. 634-637. New York: Karger, 1970], this spontaneous abnormal growth of Xenopus actually is an infectious granuloma.

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