Serum-deprived Conditions Research Articles

Reduction of apoptosis through the mitochondrial pathway by the administration of acetyl- l-carnitine to mouse fibroblasts in culture

It is shown in literature that stress, such as deprivation of trophic factors and hypoxia, induces apoptosis in cultured cells and in tissues. In light of these results, we explored the possibility of protecting cells from programmed death by improving the metabolism of the mitochondrion. To this end, acetyl- l-carnitine was administered at various concentrations under conditions of serum deprivation. The choice of this drug was based on the accepted notion that acetyl- l-carnitine is able to stabilize mitochondrial membranes and to increase the supply of energy to the organelle. The results presented here indicate that the drug protects cells from apoptotic death: this is demonstrated by a lower positivity to the TUNEL reaction and by a strong reduction of the apoptotic DNA ladder in serum-deprived cells. The involvement of the mitochondrial apoptotic pathway was assessed by cytochrome C release and immunoreactivity to caspase 3. Moreover, acetyl- l-carnitine stimulates cell proliferation.

Ex vivo amplification of T cells from human cord blood

The current status of immunotherapy for cancer is here summarized with particular attention to the new methodologies developed for ex vivo expansion of T cells from neonatal cord blood. Umbilical cord blood (UCB) mononuclear cells (MNC) generate CD45RA naïve T lymphocytes when cultured under serum-deprived conditions with appropriate combinations of growth factors. These ex vivo generated T cells resemble precursors for the lymphoid lineage present in adult bone marrow in terms of active transcription of RAG-2 and pTα.

Mediators of endothelial cell injury in low perfusion conditions

Abstract Introduction: During free tissue transfer operations, endothelial cells (EC) are exposed to a period of ischemia that can affect outcome. We compared gene expression profiles of ECs in normal culture conditions to serum-deprived conditions as an in vitro model of tissue stress. Methods: ECs from multiple body sites were grown in EGM-2MV with 5% fetal bovine serum (FCS). For serum-deprived samples, ECs were switched to EGM-2MV with 0.25% FCS. After 24h, ECs were harvested and mRNA was extracted. Gene expression patterns were compared between the normal and serum-deprived samples using cDNA microarray. Specific gene expression was then confirmed by quantitative PCR. Results: There are 1083 genes that show more than a four fold difference in expression when comparing normal versus serum-deprived growth conditions. ECs exposed to serum deprivation up regulate inflammatory and coagulation factor genes while cell cycle genes are down regulated. Up regulated inflammatory mediators included MCP-1, interferon-alpha, TGF-alpha, colony stimulating factor 2, TNFC, and TNF receptor-associated factor 5. Coagulation factors included thrombin and the thrombin receptor. The down regulated cell cycle genes included cyclin B2 and c-myc binding protein. Conclusions: Our data suggest that serum deprivation induces gene expression of inflammatory mediators and coagulation factors that can lead to poor outcome during tissue transfer. Understanding the mechanism of EC injury may lead to the development of rationally designed therapies aimed at minimizing complications and improving outcomes.

Increased expression and activation of CD30 induce apoptosis in human blood eosinophils.

Eosinophils are one of the major effector cells in asthma, and controlling the number and survival of eosinophils might attenuate the severity of asthma. This result could be achieved by inducing eosinophil apoptosis. Apoptosis allows the removal of cells without inducing an inflammatory response. Our knowledge of the factors involved in regulating eosinophil apoptosis remains limited. CD30 molecule has been associated with T cell-negative selection and in TCR-mediated apoptosis. In this study we examined the expression and role of CD30 in apoptosis of human blood eosinophils. Percentage of apoptotic eosinophils was determined by annexin V-propidium iodide labeling, and CD30 expression was examined by flow cytometry. Spontaneous apoptosis was induced by serum deprivation, and survival was conferred by incubating cells with 10% FBS and IL-5. CD30 surface expression was up-regulated in eosinophils incubated for 24 h as compared with freshly isolated eosinophils, and both CD30 expression and eosinophil apoptosis increased in a time-dependent manner. We also measured CD30 mRNA expression by quantitative real-time RT-PCR and determined that CD30 transcripts increased in eosinophils undergoing apoptosis only under serum deprivation conditions. The agonistic CD30 Abs, Ber-H8 and HeFi-1, significantly enhanced eosinophil apoptosis. FBS and IL-5 failed to inhibit or suppress the CD30 agonistic-induced apoptosis. These data support the role of CD30 activation in eosinophil apoptosis. This research will help in furthering our understanding of eosinophil apoptosis and therefore might contribute to the development of better therapeutic modalities in the treatment and/or cure of allergic inflammation in bronchial asthma.

The cellular prion protein (PrP C) prevents apoptotic neuronal cell death and mitochondrial dysfunction induced by serum deprivation

Prion diseases are transmissible neurodegenerative disorders that are invariably fatal in humans and animals. Although the nature of the infectious agent and pathogenic mechanisms of prion diseases are not clear, it has been reported that prion diseases may be associated with aberrant metabolism of cellular prion protein (PrP C). In various reports, it has been postulated that PrP C may be involved in one or more of the following: neurotransmitter metabolism, cell adhesion, signal transduction, copper metabolism, antioxidant activity or programmed cell death. Despite suggestive results supporting each of these mechanisms, the physiological function(s) of PrP C is not known. To investigate whether PrP C can prevent apoptotic cell death in prion diseases, we established the cell lines stably expressing PrP C from PrP knockout (PrP −/−) neuronal cells and examined the role of PrP C under apoptosis and/or serum-deprived condition. We found that PrP −/− cells were vulnerable to apoptotic cell death and that this vulnerability was rescued by the expression of PrP C. The expression levels of apoptosis-related proteins including p53, Bax, caspase-3, poly(ADP-ribose) polymerase (PARP) and cytochrome c were significantly increased in PrP −/− cells. In addition, Ca 2+ levels of mitochondria were increased, whereas mitochondrial membrane potentials were decreased in PrP −/− cells. These results strongly suggest that PrP C may play a central role as an effective anti-apoptotic protein through caspase-dependent apoptotic pathways in mitochondria, supporting the concept that disruption of PrP C and consequent reduction of anti-apoptotic capacity of PrP C may be one of the pathogenic mechanisms of prion diseases.

Dietary nucleotides and intestinal cell lines: I. modulation of growth

Emerging evidence suggests that nucleotide-free diets induce immune suppression and may seriously influence intestinal growth, maturation, and function. Thus, DNA-Na+ salt from fish soft roe, used as a nucleotide source, was investigated in regard to its effect on intestinal growth under optimal and nutrition–depleted conditions. The effect of another dietary additive, RNA from bakers yeast ( S. cerevisiae) and various deoxy-mononucleotides have also been clarified. DNA-Na+, RNA, dAMP, dCMP, dGMP, and TMP were added to a human malignant intestinal cell line, CaCo-2 and a rat intestinal cell line of normal origin, IEC-6. Growth effects were ascertained by measuring the amount of tritium-labeled thymidine incorporated into the cell DNA. Our results show that DNA-Na+, RNA, and deoxy-mononucleotides have beneficial effects on growth, trying to compensate for the glutamine- and partially serum-deprived culture condition, depending on the intestinal cell line investigated. Especially the “normal” IEC-6 cell line seemed to benefit from the exogenous nucleotide supply. dAMP increased mostly CaCo-2 growth under optimal conditions. Both dCMP and dGMP supported growth of both cell lines under most culture conditions. In contrast, TMP acted as a general growth inhibitor. Of interest also was the growth-suppressing ability of DNA-Na+ in particular, but also RNA, on vigorously proliferating CaCo-2 cells cultured under optimal nutrition supply. This activity should be investigated in regard to other hyperproliferating and malignant cell types.

The Akt-regulated Forkhead Transcription Factor FOXO3a Controls Endothelial Cell Viability through Modulation of the Caspase-8 Inhibitor FLIP

FLICE-inhibitory protein (FLIP) is a homolog of caspase-8 that lacks catalytic activity and has been shown to be important in protecting endothelial cells from apoptosis. The serine/threonine kinase Akt/PKB was recently reported to promote FLIP expression in endothelial and tumor cells. Here we examined the role of the forkhead transcription factor FOXO3a, a downstream target of Akt, in controlling FLIP regulation in endothelial cells. FOXO3a nuclear translocation was regulated by Akt in human umbilical vein endothelial cells. Transduction of a nonphosphorylatable, constitutively active mutant of FOXO3a (TM-FOXO3a) led to the down-regulation of FLIP levels. Transduction with TM-FOXO3a also increased caspase-8 activity and promoted apoptosis in endothelial cells. Conversely, transduction of a dominant-negative mutant of FOXO3a up-regulated FLIP levels and protected endothelial cells from apoptosis under serum deprivation conditions. Restoration of intracellular FLIP blocked caspase-8 activation and inhibited apoptosis in TM-FOXO3a-transduced cells. These data suggest that FOXO3a is a downstream target of Akt in endothelial cells that can promote apoptosis via FLIP down-regulation and activation of the extrinsic apoptotic pathway.

Novel Angiogenic Compounds for Targeted Drug Delivery

ABSTRACTInduction of angiogenesis is necessary for the success of engineered implantable tissues in order to meet oxygen and nutrient requirements of cells during tissue repair. Insufficient vascularization in bone graft reconstruction may impede healing and initiate hypoxic cell death at the interior of the implant. As a result, endogenous growth factors have been studied to enhance angiogenesis during wound repair. However, these peptide-based molecules are highly sensitive to processing that occurs during scaffold biomaterial fabrication and treatment for tissue engineering purposes. We report here the development of new small molecule regulators of angiogenesis that may circumvent the impediments associated with protein-based growth factor delivery. In this study, we report the design and evaluation of SC–3–143 as a regulator of endothelial function. We show that the compound significantly increases the formation of microvascular networks in vitro, and selectively enhances endothelial survivability by reducing endothelial cell death under serum deprived culture conditions.

Transferrin ensures survival of ovarian carcinoma cells when apoptosis is induced by TNFalpha, FasL, TRAIL, or Myc.

The activation of Myc induces apoptosis of human ovarian adenocarcinoma N.1 cells when serum factors are limited. However, the downstream mechanism that is triggered by Myc is unknown. Myc-activation and treatment with the proapoptotic ligands TNFalpha, FasL, and TRAIL induced H-ferritin expression under serum-deprived conditions. H-ferritin chelates intracellular iron and also intracellular iron sequestration by deferoxamine-induced apoptosis of N.1 cells. Supplementation of serum-free medium with holo-transferrin blocked apoptosis of N.1 cells that was induced by Myc-activation or by treatment with TNFalpha, FasL, and TRAIL, whereas apotransferrin did not prevent apoptosis. This suggests that intracellular iron depletion was a trigger for apoptosis and that transferrin-bound iron rescued N.1 cells. Furthermore, apoptosis of primary human ovarian carcinoma cells, which was induced by TNFalpha, FasL, and TRAIL, was also inhibited by holo-transferrin. The data suggest that Myc-activation, FasL, TNFalpha, and TRAIL disturbed cellular iron homeostasis, which triggered apoptosis of ovarian carcinoma cells and that transferrin iron ensured survival by re-establishing this homeostasis.

PDTC, metal chelating compound, induces G1 phase cell cycle arrest in vascular smooth muscle cells through inducing p21Cip1 expression: involvement of p38 mitogen activated protein kinase.

Pyrrolidine dithiocarbamate (PDTC), a metal chelating compound, is known to induce cell death in vascular smooth muscle cells (VSMC). However, the molecular mechanism for PDTC-induced VSMC death is not well understood. Addition of PDTC reduced cell growth and DNA synthesis on VSMC in low density conditions. However, in serum depleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. Several metal chelators prevented the cell death induced by PDTC. In a serum-deprived condition, addition of exogenous metals, copper, iron, and zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as copper, iron, and zinc in serum may mediate the cytotoxic effect of PDTC. At low VSMC density in 10% FBS, treatment of PDTC, which induced a cell-cycle block in G1-phase, induced down-regulation of cyclins and CDKs and up-regulation of the CDK inhibitor p21 expression, whereas up-regulation of p27 or p53 by PDTC was not observed. Finally, we determined PDTC-mediated signaling pathway involved in VSMC death. Among relevant pathways, PDTC induced marked activation of p38MAPK and JNK. Expression of dominant negative p38MAPK and SB203580, a p38MAPK specific inhibitor, blocked PDTC-dependent p38MAPK, growth inhibition, and p21 expression. These data demonstrate that the p38MAPK pathway participates in p21 induction, which consequently leads to decrease of cyclin D1/cdk4 and cyclin E/cdk2 complexes and PDTC-dependent VSMC growth inhibition. In conclusion, an understanding of the molecular mechanisms of PDTC in VSMC provides a theoretical basis for clinical approaches using antioxidant therapies in atherosclerosis.

Identifying the factors and signal pathways necessary for anchorage-independent growth of Ha-ras oncogene-transformed NIH/3T3 cells

Ha-ras Val 12 overexpression was positively correlated with colony formation by NIH/3T3 derivative “2–12” cells harboring an inducible Ha-ras Val 12 transgene. The ras-farnesylation inhibitor, Lovastatin, completely suppressed colony formation at higher dosages. However, Ha-ras oncogene overexpression alone could not stimulate colony formation under serum-deprived conditions, suggesting that ras is required but not sufficient for supporting colony formation. Substituting cow colostrum (AC-2®) for serum did not result in colony formation from 2–12 cells in soft agar, suggesting the colostrum lacked or contained insufficient amounts of factors that stimulate colony formation. Supplementation of AC-2®-containing medium with growth factors, such as insulin-like growth factor-1 (IGF-1), partially restored the capability of anchorage-independent cell growth induced by Ha-ras overexpression. Consistently, antibodies specific for IGF-1 receptors only partially blocked colony formation from 2–12 cells. The data indicate that multiple factors, including IGF-1, are required for Ha-ras-dependent colony formation. Signal transduction studies revealed that, under Ha-ras overexpression conditions, IGF-1 utilizes phosphatidyl inositol 3-kinase and NF-κB to transduce colony formation-related signaling.

Amplification of T cells from human cord blood in serum-deprived culture stimulated with stem cell factor, interleukin-7 and interleukin-2.

We report the effects exerted by cytokine combinations, including stem cell factor (SCF), interleukin-7, interleukin-4 and interleukin-2, on the amplification of T cells from cord blood (CB) mononuclear cells cultured for 10-11 days under serum-deprived conditions. Of all the combinations investigated, SCF+interleukin-7 sustained the best fold increase (FI) of total nucleated cells (FI=6.4+/-1.17), amplifying preferentially CD4(+) over CD8(+) T-cell subsets (FI=4.72+/-0.79 vs 2.73+/-1.2, respectively, P<0.05). The addition of interleukin-2 to this combination did not significantly increase the total number of cells generated (FI=7.4+/-2.27), but allowed preferential amplification of CD8(+) over CD4(+) T cells (FI=6.04+/-0.14 vs 1.67+/-0.6, respectively, P<0.05). Single-strand conformation polymorphism analysis of the T-cell receptor V(beta)-chain rearrangements expressed by the expanded T cells indicated that the complexity of the T-cell repertoire had increased after 10 days of culture in the presence of SCF and IL-7. Interestingly, a modest expansion (FI=8.67+/-1.5) of myeloid progenitor cells was also observed in these cultures. These results indicate that it is possible to expand specific T-cell subsets for adoptive immunotherapy without losing myeloid progenitor cells necessary for neutrophil recovery after CB transplantation, by modulating the cytokines added to the cultures.

Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes.

Humanin (HN) has been reported to be an endogenous peptide that exerts highly selective neuroprotection against cell death induced by various types of Alzheimer's disease-related insults. We previously proposed the much broader cytoprotective potential of HN from the result that HN suppressed serum-deprivation-induced death of rat pheochromocytoma cells. In this study, we showed that HN also suppressed death of human lymphocytes cultured under serum-deprived condition. Further, we revealed, by assaying metabolic activity and survival rate, that HN was a potent factor capable of increasing the metabolic activity of individual serum-deprived lymphocytes. To our knowledge, there is no report described about a rescue factor that increases the metabolic activity of individual serum-deprived cells and prolongs their survival. This novel feature of HN may enable us to apply this peptide for the management of diseases involving poor metabolic activity, such as mitochondria-related disorders and brain ischemia.

Regulation of Angiogenesis by Glycogen Synthase Kinase-3β

Glycogen synthase kinase-3beta (GSK3beta) plays important roles in metabolism, embryonic development, and tumorigenesis. Here we investigated the role of GSK3beta signaling in vascular biology by examining its function in endothelial cells (ECs). In EC, the regulatory phosphorylation of GSK3beta was found to be under the control of phosphoinositide 3-kinase-, MAPK-, and protein kinase A-dependent signaling pathways. The transduction of a nonphosphorylatable constitutively active mutant of GSKbeta promoted apoptosis under the conditions of prolonged serum deprivation or the disruption of cell-matrix attachments. Conversely, the transduction of catalytically inactive GSK3beta promoted EC survival under the conditions of cellular stress. Under normal cell culture conditions, the activation of GSK3beta signaling inhibited the migration of EC to vascular endothelial growth factor or basic fibroblast growth factor. Angiogenesis was inhibited by GSK3beta activation in an in vivo Matrigel plug assay, whereas the inhibition of GSK3beta signaling enhanced capillary formation. These data suggest that GSK3beta functions at the nodal point of converging signaling pathways in EC to regulate vessel growth through its control of vascular cell migration and survival.

High glutamate decreases S100B secretion stimulated by serum deprivation in astrocytes.

S100B is a calcium-binding protein expressed and secreted by astrocytes, playing a neurotrophic role in neighboring cells. A protective role of the S100B against glutamate-induced excitotoxicity has recently been proposed. We investigated S100B secretion in rat hippocampal astrocytes exposed to high concentrations of glutamate during serum deprivation (stimulated condition) or not (basal condition), for 30 min. Glutamate at 1 mM had no effect on basal secretion of S100B, but it decreased S100B secretion in serum-deprived astrocytes after 1 h. Secretion was inhibited by Rp-cAMPS or H89. In addition, serum deprivation was accompanied by a transitory increase of intracellular content of cAMP. Our results suggest that high levels of glutamate in a serum-deprived condition could impair S100B secretion from hippocampal astrocytes.

Localization and functional role of hepatocyte growth factor (HGF) and its receptor c-met in the rat developing cerebral cortex

Development of the cerebral cortex is a series of precisely timed proliferative, migratory, and maturational processes. Hepatocyte growth factor (HGF) is a pleiotrophic cytokine, which plays important roles in the organogenesis and regeneration of various tissues, both during development and in the adult, due to its mitogenic, motogenic and morphogenic activities. In the present study, we examined expression and functional roles of HGF and c-Met during development of the rat cerebral cortex. Quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) revealed that expression levels of c- met and HGF mRNAs were increased in the cerebral cortex during late embryonic development and peaked at E18. Immunohistochemical analyses revealed that c-Met-immunoreactivity (IR) was localized to the preplate (PP), with weaker-IR in neuroepithelial layer (NE) at embryonic day 14 (E14). At E16, c-Met-IR was present in the cortical plate (CP) and the intermediate zone (IZ), with a weak presence in the ventricular zone (VZ). On the other hand, HGF-IR was present in NE and VZ at E14 and E16, respectively. HGF-IR appeared in cortical plate tissue from E16 onward. Double labeling immunofluorescent cytochemical studies revealed that c-Met-IR was localized both in TuJ-1-IR- and non-TuJ-1-IR-cells, purified from E18 cerebral cortex in vitro, suggesting the presence of c-Met-IR in postmitotic neurons as well as in neuroepithelial cells. c-Met-IR was strong in cell bodies and neurites shortly after in vitro culture, while at 7DIV c-Met-IR decreased in neurites and was evident in growth cones. HGF dose dependently supported neuronal survival in vitro under serum-deprived conditions. In a transwell culture chamber, HGF increased neuronal migration, and co-incubation with functional blocking antibody against HGF abrogated this motogenic effect of HGF. These lines of evidence suggest that HGF is involved in the development and maintenance of cortical neurons during differentiation, motogenesis, neuritogenesis and neuronal survival.

The Soluble Notch Ligand, Jagged-1, Inhibits Proliferation of CD34+ Macrophage Progenitors

The Notch/Notch ligand system controls diverse cellular processes. The proteolytic cleavage generates transmembrane and soluble forms of Notch ligands. We examined the effect of a soluble Notch ligand, human Jagged-1, on human cord blood (CB) CD34+ cells, under serum-deprived conditions, using soluble human Jagged-1-immunoglobulin G1 chimera protein (hJagged-1). Soluble hJagged-1 inhibited myeloid colony formation but not erythroid-mix or erythroid colony formation, in the presence of stem cell factor (SCF), interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, thrombopoietin, and erythropoietin. Cytological analysis revealed that the decrease in myeloid colonies resulted mainly from the inhibition of macrophage colony formation. Furthermore, soluble hJagged-1 led to the inhibition of macrophage colony formation supported by M-CSF plus SCF and GM-CSF plus SCF. Delayed-addition experiments and the analysis of colony sizes demonstrated that soluble hJagged-l inhibited the growth of macrophage progenitors by acting in the early stage of macrophage development. The direct action of hJagged-1 was confirmed by the enhanced expression of the HES-1 (hairy enhancer of the split-1) gene. These results suggest that soluble hJagged-1 may regulate human hematopoiesis in the monocyte/macrophage lineage.

ZnCl 2 prevents c- myc repression and apoptosis in serum-deprived Syrian hamster embryo cells

In order to understand the c- myc implication in the apoptotic process better, we investigated the influence of ZnCl 2 on its expression in normal and transformed Syrian hamster embryo (SHE) cells in relation to apoptosis induced by serum withdrawal. Normal primary SHE cells exposed to a serum-free medium undergo rapid apoptosis characterised by a dramatic down-regulation of c- myc transcription. In these normal cells treated with ZnCl 2, c- myc expression is maintained in serum-starved conditions while apoptosis is inhibited. The results shed light on the involvement of c- myc expression in the survival of normal cells in the absence of growth factors. The regulation of c- myc expression appears to be influenced by zinc treatment as an inhibitor of apoptosis, but mechanisms sustaining the level of c- myc transcription remain to be demonstrated. The hypothesis that maintenance of c- myc expression allows cells to escape apoptosis is in accordance with results in transformed SHE cells that underwent low apoptosis and poor down-regulation of c- myc in serum-deprived conditions.

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor

It is shown that neokyotorphin (the α-globin fragment 137–141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.

Leptin increases the viability of isolated rat pancreatic islets by suppressing apoptosis.

To test the hypothesis that leptin secreted from adipose tissue is a mediator linking obesity and pancreatic islet hypertrophy, we examined the effects of leptin on proliferative and apoptotic responses in rat islet cells. Rat pancreatic islets were isolated and incubated with 0, 1, 5, or 75 nM leptin for 24 h under serum-deprived conditions. Cell viability was assessed with 2,5-diphenyltetrazolium bromide and trypan blue dye exclusion tests. Cell proliferation and apoptosis were evaluated with 5-bromo-2'-deoxyuridine incorporation into DNA and DNA ladder formation, respectively. Incubation for 24 h with 1 and 5 nM leptin, the concentrations observed in obese subjects, increased the viability of isolated pancreatic islet cells. Five nanomolar concentrations of leptin did not stimulate 5-bromo-2'-deoxyuridine incorporation into incubated islet cells, indicating no influence on cell proliferation, but did inhibit DNA ladder formation, a hallmark of cell apoptosis. Moreover, 5 nM leptin reduced the triglyceride content and suppressed inducible nitric oxide synthase mRNA expression in incubated islets. These results suggest that leptin increased viable cell numbers via suppression of apoptosis in isolated pancreatic islet cells under these experimental conditions. This mechanism might account at least in part for an obesity-induced increase in pancreatic beta-cell mass.