RESUMEN
The mechanism by which estradiol, testosterone, 1,25(OH)2 vitamin D3, and triiodothyronine promote tissue growth is unknown, although, in some tissues, a role for local IGF-I has been suggested. We previously showed that HTLV-II-transformed T-cell lines from healthy adults augmented basal colony formation in response to peptide (growth hormone, parathormone, and adrenocorticotrophin) and glycoprotein (thyroid-stimulating hormone) hormones through stimulation of local IGF-I. T-cell lines from African Efe Pygmies, however, were resistant to the direct growth-promoting action of IGF-I, as well as to the growth-promoting action of growth hormone, parathormone, adrenocorticotrophin, and thyroid-stimulating hormone. We, therefore, used these cell lines to determine the mechanism of T-cell growth in response to steroid and thyroid hormones. We quantified colony formation of American control T-cell lines in the presence and absence of alpha IR-3 antibody against the type 1 IGF receptor and Pygmy T-cell lines in response to estradiol (36.7-1835 pmol/ liter), testosterone (34.7-17,350 pmol/liter), 1,25(OH)2 vitamin D3 (2.4-24,000 pmol/liter), and triiodothyronine (1536-192,000 pmol/liter). There were no statistically significant differences by ANOVA in overall response curves for any of the four hormones comparing control clonal responses in the presence or absence of alpha IR-3 and no statistically significant difference in overall responsiveness between control and Pygmy T-cell lines. From these data, we conclude that (i) normal T-cell lines grow in response to estradiol, testosterone, 1,25(OH)2 vitamin D3, and triiodothyronine; (ii) these responses are not mediated through local IGF-I since they are not blocked by pretreatment with antibody to the type 1 IGF receptor; and (iii) Pygmy T-cell lines, which are genetically resistant to IGF-I, grow equivalently to control T-cell lines in response to estradiol, testosterone, 1,25(OH)2 vitamin D3, and triiodothyronine, further underscoring the IGF-I independence of this stimulation in our system.
Asunto(s)
Población Negra , Calcitriol/farmacología , Estradiol/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Testosterona/farmacología , Triyodotironina/farmacología , Análisis de Varianza , Línea Celular , Virus Linfotrópico T Tipo 2 Humano , Humanos , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Efe Pygmies of northeast Zaire have the shortest mean adult stature of any population on earth. Although various alterations in the GH/insulin-like growth factor I (IGF-I) axis have been suggested, the basis for short stature in the Pygmy is unknown. We previously described IGF-I unresponsiveness in a T lymphoblast cell line derived from an Efe Pygmy, and studies in five additional lines have confirmed severe IGF-I resistance in these cells. We have now performed experiments to determine the molecular basis for the IGF-I resistance in these cells. We found markedly decreased cell surface expression of IGF-I receptors with normal ligand binding affinity. The Pygmy IGF-I receptors were not autophosphorylated and did not transmit a signal in response to physiological concentrations of IGF-I. There was a substantially decreased level of IGF-I receptor messenger ribonucleic acid in the Pygmy cells with a normal messenger ribonucleic acid half-life. The nucleotide sequence of the full-length IGF receptor complementary DNA in Pygmy 1 showed no significant variation. These results indicate decreased IGF-I receptor gene transcription and IGF-I receptor signaling as the primary variation in the Pygmy cell lines. The findings point to the IGF-I receptor as the locus governing short stature in the African Pygmy and suggest that human stature may be genetically controlled by expression of the IGF-I receptor.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Nativos de Hawái y Otras Islas del Pacífico , Receptores de Somatomedina/metabolismo , Linfocitos T/metabolismo , Secuencia de Bases , Línea Celular Transformada , ADN Complementario/genética , República Democrática del Congo , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Membranas Intracelulares/metabolismo , Sondas Moleculares/genética , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Grupos Raciales , Receptores de Somatomedina/genética , Transducción de SeñalRESUMEN
To investigate IGF-I resistance in African Efe Pygmies, we examined clonal responsiveness to IGF-I in Epstein-Barr virus-transformed B-lymphocytes from three Efe Pygmies and three American control subjects. The Efe B-lymphoblasts did not increase clonal responsiveness when incubated with IGF-I (as high as 250 micrograms/liter) in contrast to the control B-lymphoblasts which showed a bimodal dose-response with a maximal stimulation of 50% above baseline. The proliferative response of Efe B-lymphoblasts was similar to that of control B-lymphoblasts when incubated with another growth factor, phorbol 12-myristate 13-acetate, which does not activate the IGF-I receptor. These findings indicate that Efe Pygmy B-lymphoblasts are resistant to IGF-I as measured by in vitro clonal proliferation assays. Coupled with our previous report of IGF-I unresponsiveness in Efe Pygmy HTLV-II-transformed T-lymphocytes, these data suggest that IGF-I resistance is generalized and may play a central role in the etiology of short stature in this population.
Asunto(s)
Linfocitos B/virología , Población Negra , Factor I del Crecimiento Similar a la Insulina/metabolismo , Receptor IGF Tipo 1/metabolismo , Adulto , Linfocitos B/metabolismo , Estatura , Peso Corporal , República Democrática del Congo/etnología , Hormona de Crecimiento Humana/metabolismo , Humanos , Técnicas In Vitro , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Masculino , Persona de Mediana Edad , Tirotropina/metabolismoRESUMEN
Tissue inhibitor of metalloproteinases-1 (TIMP-1), the major physiological matrix metalloproteinase inhibitor and a potent antimetastatic factor, also stimulates the growth of erythroid progenitors (erythroid-potentiating activity). We analyzed the relationship between the growth factor activity and protease inhibition by preparing purified TIMP-1 "knockout" proteins lacking in vitro antiproteolytic activity. The growth-stimulatory effect of these N-terminal TIMP-1 point mutants, as tested in an in vitro assay using erythroid precursors (erythroid burst-forming units) was equal to that of unmutated TIMP-1. A fully antiproteolytic C-terminal TIMP-1 truncation also stimulated growth in the erythroid burst-forming unit assay. The results indicate that the influence of TIMP-1 on erythroid precursor growth is independent of its ability to inhibit metalloproteinases. TIMP-1 is analogous to proteins that have both proteolytic and growth factor activity, such as plasmin, thrombin, and urokinase. However, TIMP-1 is novel in this regard because it is a metalloproteinase inhibitor. We show that the antiproteolytic and growth factor activities of the TIMP-1 molecule are physically and functionally distinct.
Asunto(s)
Eritropoyesis/fisiología , Glicoproteínas/fisiología , Metaloendopeptidasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Transformada , Células Cultivadas , Chlorocebus aethiops , ADN Complementario/genética , Células Precursoras Eritroides/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Genes myc , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/farmacología , Humanos , Metaloproteinasa 3 de la Matriz , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Relación Estructura-Actividad , Inhibidores Tisulares de MetaloproteinasasRESUMEN
Previous investigations suggested that resistance to GH was the cause of short stature of African Pygmies. Because many of the actions of GH are mediated by insulin-like growth factor I (IGF-I), we sought to determine whether Pygmy tissue was responsive to IGF-I. An initial effort to obtain HTLV-II-transformed T lymphoblast cell lines resulted in a single cell line that showed complete resistance to both IGF-I and GH in a clonal proliferation assay as well as decreased IGF-I binding. In the current study, we examined T cell lines from seven Efe Pygmy subjects, three neighboring Lese farmers, and six American controls and quantified clonal responses to IGF-I, GH, and insulin. The T cell lines from the Efe Pygmies were all completely resistant to the growth-promoting actions of IGF-I concentrations less than 250 micrograms/L and GH concentrations less than 500 micrograms/L. The Lese population, with whom there is admixture with the Efe population, showed heights and clonal responses to IGF-I and GH intermediate between those of Pygmies and American controls. The Pygmy T cell lines showed reduced clonal proliferation in response to high insulin concentrations known to act through the IGF-I receptor. These findings indicate that genetic IGF-I resistance is present in the T cell lines of Efe Pygmies and suggest that unresponsiveness to IGF-I may be responsible for their short stature.
Asunto(s)
Estatura , Factor I del Crecimiento Similar a la Insulina/farmacología , Nativos de Hawái y Otras Islas del Pacífico , Linfocitos T/efectos de los fármacos , Adulto , Línea Celular Transformada , República Democrática del Congo/etnología , Resistencia a Medicamentos , Hormona del Crecimiento/farmacología , Humanos , Resistencia a la Insulina , Masculino , Persona de Mediana Edad , Grupos Raciales , Valores de Referencia , Estados Unidos/etnologíaRESUMEN
We used an in vitro T-lymphoblast clonal proliferation assay to quantify human IGF-I (hIGF-I)-, human PTH (hPTH)-, human ACTH (hACTH)-, and human TSH (hTSH)-stimulated growth of human T-cell leukemia virus-II-transformed T-lymphoblast cell lines from normal individuals and to elucidate the role of IGF-I as the mediator of hPTH-, hACTH-, and hTSH-induced T-cell growth. Normal T-lymphoblast cell lines respond to hIGF-I in a bimodal fashion. The mean first peak response was 143 +/- 9.8% above baseline (defined as 100%) occurring at 8 micrograms/L, and the mean second peak response was 154 +/- 14.4% occurring at 100 micrograms/L. Both responses were completely blocked after incubation with alpha IR-3, an MAb to the IGF-I receptor (by analysis of variance, p = 0.015 between full response curves). After stimulation with hPTH, the mean peak clonal response of normal T-lymphoblast cell lines was 189 +/- 7.0%; after incubation with alpha IR-3, the mean peak clonal response was 108 +/- 7.9% (p = 0.0015 between full response curves). The mean peak clonal response of normal T-lymphoblast cell lines after hACTH stimulation was 192 +/- 8.6%; preincubation with alpha IR-3 reduced the mean peak clonal response to 94 +/- 1.2% (p < 0.0001 between full response curves). With hTSH stimulation, the mean peak clonal response of normal T-lymphoblast cell lines was 167 +/- 7.0%; after incubation with alpha IR-3, the mean peak clonal response was 94 +/- 8.2% (p = 0.003 between full response curves).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hormona Adrenocorticotrópica/farmacología , Factor I del Crecimiento Similar a la Insulina/fisiología , Hormona Paratiroidea/farmacología , Linfocitos T/efectos de los fármacos , Tirotropina/farmacología , Adulto , Población Negra , Estatura/fisiología , División Celular/fisiología , Línea Celular Transformada , Transformación Celular Viral , Virus Linfotrópico T Tipo 2 Humano , Humanos , Valores de Referencia , Reproducibilidad de los Resultados , Estimulación QuímicaRESUMEN
Growth hormone (GH) and insulin have both mitogenic and metabolic actions. The growth-promoting effects of GH in vivo are thought to be mediated by insulin-like growth factor-I (IGF-I), whereas the metabolic effects of GH are thought to be either direct or mediated by factors other than IGF-I. In previous studies using HTLV-II-transformed T-lymphoblast cell lines established from normal individuals, we have shown that GH preincubation induces resistance to the growth-promoting (mitogenic) action of insulin. In this study, using T-cell lines from 3 American control subjects, 1 African control subject, and 1 African Pygmy (the latter previously shown to be resistant to the growth-promoting actions of both IGF-I and GH), we examined the role of local IGF-I in the mediation of GH-induced resistance to the mitogenic action of insulin. In these studies, we quantified the stimulation of T-cell colony formation in response to insulin in the presence and absence of either GH or IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Población Negra , Etnicidad , Hormona del Crecimiento/farmacología , Resistencia a la Insulina/inmunología , Factor I del Crecimiento Similar a la Insulina/farmacología , Insulina/farmacología , Activación de Linfocitos , Receptor IGF Tipo 1/fisiología , Linfocitos T/inmunología , Adulto , África/etnología , Anticuerpos Monoclonales/farmacología , Línea Celular , Línea Celular Transformada , Relación Dosis-Respuesta a Droga , Virus Linfotrópico T Tipo 2 Humano/inmunología , Humanos , Inmunoglobulina G/farmacología , Cinética , Masculino , Receptor IGF Tipo 1/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
The cause of short stature in African Pygmies is unknown, but some evidence suggests that they are GH resistant. Since IGF-I mediates many actions of GH, we sought to determine if Pygmy tissue is responsive to IGF-I. We established HTLV-II-transformed cell lines from 1 Efe Pygmy, 1 African control, and 3 American controls, and quantified in vitro colony formation in response to IGF-I, GH, and insulin, and assessed IGF-I receptor binding. The Pygmy T-cell line showed no clonal responsiveness following stimulation with physiologic concentrations of IGF-I or any concentration of GH, but responded normally to insulin. IGF-I binding studies showed no binding to the Pygmy T-cell line with normal binding to control cells. The primary abnormality in this Pygmy T-cell line is IGF-I resistance at the receptor level with secondary GH resistance.
Asunto(s)
Hormona del Crecimiento/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Linfocitos T/efectos de los fármacos , Adulto , Población Negra , Línea Celular Transformada , Transformación Celular Viral , República Democrática del Congo , Etnicidad , Virus Linfotrópico T Tipo 2 Humano/genética , Humanos , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Cinética , Masculino , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Linfocitos T/metabolismoRESUMEN
GH is the hormone primarily responsible for regulating body size within the genetic program. While GH has pleiotropic actions on cellular growth and metabolism, most of its effects are believed to be mediated by a single GH receptor. This receptor is not functional in tissues from patients with Laron dwarfism. We used human T-cell leukemia virus-immortalized T-lymphoblast cell lines from Laron dwarfs and normal individuals to examine the mechanism of GH-induced insulin resistance at the cellular level. GH (5-500 micrograms/L) caused a profound decrease in the sensitivity of normal T-lymphoblasts in response to all insulin concentrations (P < 0.0001 vs. insulin alone); pretreatment with GH and GH receptor antibody significantly improved sensitivity to all concentrations of insulin (P = NS vs. insulin alone). Preincubation with GH and PRL receptor antibody was associated with partial improvement in insulin sensitivity (P = 0.004 vs. insulin alone). Thus, in normal T-cell lines, the major pathway of GH-induced insulin resistance appears to be directed by the GH receptor, with a smaller effect mediated through the PRL receptor. While T-cell lines from Laron dwarfs do not respond to GH in clonal proliferation assays, GH (50 and 100 micrograms/L) caused profound insulin resistance in these cells (P = 0.008 and P < 0.0001, respectively, vs. insulin alone). GH receptor antibody did not abrogate this effect at any insulin concentration (P = NS vs. insulin alone), but there was partial restoration of insulin sensitivity when GH and PRL receptor antibody were coincubated (P = 0.0069 vs. insulin alone). Thus, in Laron T-cell lines, PRL and perhaps other lactogenic receptors appear to mediate GH-induced insulin resistance. The kinetics of GH-induced insulin resistance in Laron T-cells were also distinct from the pattern seen in normal T-cells, and unlike in normal cells, GH had no effect on insulin-like growth factor-I-induced clonal expansion of Laron T-cell lines (P = NS vs. insulin-like growth factor-I alone). These results provide evidence for an alternative pathway of GH action revealed in cells lacking classical growth responses to GH.
Asunto(s)
Enanismo/fisiopatología , Hormona del Crecimiento/farmacología , Resistencia a la Insulina , Receptores de Prolactina/fisiología , Receptores de Somatotropina/fisiología , Linfocitos T/efectos de los fármacos , Adolescente , Adulto , Anticuerpos/inmunología , Línea Celular Transformada , Enanismo/patología , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Masculino , Prolactina/farmacología , Receptores de Prolactina/inmunología , Receptores de Somatotropina/inmunologíaRESUMEN
We used normal and HTLV-II-transformed T-lymphocytes as target cells to study clonal proliferative responses to physiologic and supraphysiologic concentrations of insulin and IGF-I. Responses of both growth factors were measured in the presence and absence of alpha IR-3, and IGF-I receptor-blocking antibody. A biphasic response to insulin was noted in all cell lines with the first peak [78 +/- 6.6% (mean +/- SE) above control] occurring at 1.4 or 1.6 nmol/liter and a second peak (84 +/- 4.9% above control) occurring at 18.0 nmol/liter. Following preincubation with alpha IR-3, the overall clonal profile in response to insulin was significantly reduced [F(7,56) = (10.4, p < .0001] as a result of blunting at high physiologic and supraphysiologic insulin concentrations, i.e., > or = 1.6 nmol/liter. As expected, the overall clonal profile in response to IGF-I was blocked by alpha IR-3 [F(4,32) = 11.6, p < .0001]. These data show that insulin at both physiologic and supraphysiologic concentrations, as well as IGF-I, stimulate virally transformed T-lymphoblast growth. The significant inhibition of growth responses to high concentrations of insulin and to IGF-I by alpha IR-3 suggests mediation of these effects through the IGF-I receptor. Similar studies were performed using freshly isolated, phytohemagglutinin (PHA)-stimulated T-lymphocytes. The overall response to insulin was significantly reduced compared to the profile of transformed T-lymphoblasts [F(7,70) = 4.9, p = .0002] as a result of blunting at physiologic insulin concentrations < 1.8 nmol/liter. In response to IGF-I, the clonal profile of PHA-stimulated T-lymphocytes was slightly reduced compared to that of virally transformed T-lymphoblasts [F(4,40) = 3.4, p = .0174]. Thus, both insulin and IGF-I receptor-effector mechanisms are involved in the growth of virally transformed T-lymphoblasts, whereas the IGF-I receptor-effector mechanism appears to play a more significant role in the growth of normal, mitogen-activated T-lymphocytes.
Asunto(s)
Transformación Celular Viral , Virus Linfotrópico T Tipo 2 Humano/fisiología , Factor I del Crecimiento Similar a la Insulina/farmacología , Insulina/farmacología , Linfocitos T/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Transformada , Células Cultivadas , Células Clonales/efectos de los fármacos , Humanos , Receptor IGF Tipo 1/antagonistas & inhibidores , Estimulación QuímicaRESUMEN
OBJECTIVE: To assess insulin and insulin-like growth factor I (IGF-I) action in women with polycystic ovarian syndrome (PCOS). DESIGN: Hyperinsulinemia was determined by measuring the insulin responses during a 2-hour oral glucose tolerance test (OGTT). Quantification of in vivo insulin action was determined by a frequently sampled intravenous (IV) OGTT with minimal modeling analysis. In vitro sensitivity to insulin at physiological and supraphysiological concentrations and to IGF-I was assessed by examining colony formation of two hematopoietic cell populations, burst-forming units of the erythroid line (BFU-E) and human leukemia virus immortalized T-cell lines. (The proliferative responses of BFU-E, a primary tissue explant, are presumably conditioned by factors in the immediate blood-borne environment, whereas proliferative responses of T-cell lines are presumed to reflect intrinsic target-cell hormone sensitivity.) SETTING: Tertiary care research institution. PATIENTS: Eight patients (4 obese and 4 nonobese) with PCOS and three healthy women for reference controls. RESULTS: Nonobese (P less than 0.04) and obese patients with PCOS (P less than 0.01) both demonstrated significant hyperinsulinemia after OGTT. In vivo insulin resistance was observed in both nonobese (P less than 0.03) and obese PCOS subjects (P less than 0.01) using frequently sampled IV OGTT. Both nonobese (P less than 0.03) and obese patients with PCOS (P less than 0.01) had blunted in vitro clonal responses of BFU-E, with normal T-cell line clonal responsiveness to physiological levels of insulin and normal BFU-E and T-cell line clonal responses to IGF-I. CONCLUSIONS: These findings demonstrate the following in both nonobese and obese patients with PCOS: (1) there is in vivo hyperinsulinemia and resistance to insulin action on glucose disposal; (2) with BFU-E, there is in vitro resistance to the mitogenic action of insulin but normal responsiveness to IGF-I; and (3) there is normal in vitro mitogenic responsiveness of T-cell lines to both insulin and IGF-I. The intrinsically normal mitogenic responsiveness to insulin and, especially to IGF-I, whether or not under the influence of the bloodborne milieu, provides a mechanism whereby hyperinsulinemia could directly contribute to the ovarian abnormalities that characterize PCOS.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/farmacología , Insulina/farmacología , Síndrome del Ovario Poliquístico/fisiopatología , Adulto , Análisis de Varianza , Andrógenos/sangre , Línea Celular Transformada , Ensayo de Unidades Formadoras de Colonias , Estradiol/sangre , Estrona/sangre , Femenino , Hormona Folículo Estimulante/sangre , Prueba de Tolerancia a la Glucosa , Humanos , Hidrocortisona/sangre , Hiperinsulinismo , Insulina/sangre , Resistencia a la Insulina , Hormona Luteinizante/sangre , Activación de Linfocitos , Obesidad/complicaciones , Obesidad/fisiopatología , Síndrome del Ovario Poliquístico/sangre , Síndrome del Ovario Poliquístico/complicaciones , Valores de Referencia , Linfocitos TRESUMEN
Tissue inhibitor of metalloproteinase (TIMP) was purified and molecularly cloned on the basis of its erythroid-potentiating activity (EPA). TIMP/EPA appears to be a bifunctional molecule with both growth factor and anti-enzymatic activity. Recently, a second TIMP-related molecule was identified and we have investigated its possible erythroid-potentiating activity. Native, purified human TIMP-2 was assayed for erythroid-potentiating activity using an in vitro erythroid burst formation assay and was compared with that of previously characterized recombinant EPA/TIMP-1. The results demonstrate that both members of the tissue inhibitor of metalloproteinase family, TIMP-1 and TIMP-2, possessed erythroid potentiating activity which was inhibited by antibodies developed to neutralize EPA. These results suggest that TIMP-2 shares a common structural domain with EPA/TIMP-1 that is responsible for the erythroid-potentiating activity of these inhibitors. Therefore, TIMP-1 and TIMP-2, with both anti-protease activity and growth factor activity, join a family of bifunctional molecules such as fibroblast growth factor and thrombin which have both enzymatic and growth factor activity.
Asunto(s)
División Celular/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Sustancias de Crecimiento/farmacología , Proteínas de Neoplasias/farmacología , Proteínas Portadoras/farmacología , Epítopos , Humanos , Proteínas de Neoplasias/inmunología , Proteínas Recombinantes/farmacología , Relación Estructura-Actividad , Inhibidor Tisular de Metaloproteinasa-2RESUMEN
Insulin resistance may be due directly to genetically programmed disorders of insulin action or acquired defects in which environmental factors influence insulin action. To address the issue of this distinction, we studied the ability of insulin to stimulate colony formation in primary cultures of erythroid progenitors (assumed to retain environmental influences) and immortalized T lymphocytes (presumed to reflect only genetic influences). Four patients with hyperinsulinemia and disturbed glucose metabolism were studied (2 patients with acanthosis nigricans, 1 of whom had circulating anti-insulin-receptor antibodies, 1 with partial lipodystrophy, and 1 with Cushing's syndrome). The mean colony-forming ability of their erythroid progenitor cells in response to insulin stimulation (less than or equal to 1.6 pM) was significantly blunted compared with control cells (P less than 0.05). The mean responsiveness of their immortalized T-lymphoblast cell lines to similar insulin concentrations was no different than that of control T-lymphocyte lines, consistent with an acquired cause for the observed insulin resistance in each case. A T-lymphocyte line from a patient with leprechaunism, however, showed no stimulation in response to physiological concentrations of insulin. With these same in vitro methodologies, there was normal T-lymphocyte line responsiveness to insulinlike growth factor I (IGF-I) or insulin concentrations greater than 8.6 pM; both of these responses could be completely blocked by preincubation with an antibody to the IGF-I receptor. These findings suggest that, despite resistance to physiological levels of insulin, the high circulating insulin concentrations present in the serum of these patients could mediate unwanted tissue-specific growth through an intact IGF-I receptor-effector mechanism.
Asunto(s)
Hiperinsulinismo/fisiopatología , Resistencia a la Insulina/fisiología , Acantosis Nigricans/fisiopatología , Adolescente , Células Cultivadas , Niño , Ensayo de Unidades Formadoras de Colonias , Femenino , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Hiperinsulinismo/genética , Insulina/farmacología , Resistencia a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , Cinética , Receptores de Superficie Celular/análisis , Receptores de Somatomedina , Linfocitos T/citología , Linfocitos T/efectos de los fármacosRESUMEN
It has become evident that locally produced insulin-like growth factors-I and -II (IGF-I and IGF-II) play an important role in the medication of GH action upon tissues. To explore this concept with respect to immunocompetent cells, we analyzed IGF production and clonogenic responsiveness of immortalized human T-cell lines established from seven normal controls and four Laron dwarfs. While the normal T-cell lines showed significant augmentation of basal colony formation in response to both IGF-I and GH, little increase in clonogenesis in response to GH was seen with the Laron T-cell lines. Assay of basal and GH-stimulated conditioned media demonstrated low, but measurable, levels of IGF-I and IGF-II from both normal and Laron T-cells. Under serum-free incubation conditions, GH stimulation of normal T-cell lines failed to generate significant increases in mean IGF-I or IGF-II concentration and no increase in the mean IGF-II concentration in conditioned medium were observed after GH stimulation of Laron T-cell lines. Nevertheless, the increased cloning efficiency of the normal T-cell lines in response to either GH or IGF-I was nearly completely abrogated by preincubation of cells with antibodies to either IGF-I or the type I IGF receptor. These studies, thus, support a role for locally generated IGF-I in the mediation of GH action on T-lymphocytes and indicate that this effect is mediated via the type I IGF receptor.
Asunto(s)
Transformación Celular Viral , Hormona del Crecimiento/farmacología , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 2 Humano/genética , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Somatomedinas/biosíntesis , Linfocitos T/citología , División Celular/efectos de los fármacos , Línea Celular , Células Clonales , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Interleucina-2/farmacología , Cinética , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
The clinical entity of Laron dwarfism is characterized by resistance to both endogenous and exogenous GH and may be due to a deficiency or absence of functional GH receptors. We previously showed that two types of hematopoietic cells derived from these patients are resistant to the in vitro growth-promoting action of GH at concentrations below 500 micrograms/L. In the current study we found that Laron T-cell lines had a mean peak augmentation of basal colony formation of 22 +/- 3.4% above baseline in response to a GH concentration of 10,000 micrograms/L. Since cloned cDNAs for human and rabbit GH receptors and rat PRL receptors show a high degree of sequence homology, we undertook studies of PRL action in cells from patients with Laron dwarfism to determine if the Laron defect was also associated with PRL unresponsiveness. Quantitating the augmentation of colony formation by T-lymphoblast cell lines established from three Laron dwarfs, we found normal responsiveness to PRL at concentrations of 25-10,000 micrograms/L. It is, thus, possible that the responsiveness of Laron T-cell lines to very high concentrations of GH could be mediated through an intact PRL (or other lactogenic) receptor based on the known affinity of GH for these receptors in other systems. These data suggest that cells from patients with Laron dwarfism have normal in vitro responsiveness to PRL and that the defect in Laron dwarfism appears to be specific to the GH receptor-effector pathway. It remains to be determined whether intact alternative lactogenic receptor mechanisms subserve any clinical effects of GH in patients with Laron dwarfism.
Asunto(s)
Enanismo Hipofisario/metabolismo , Hormona del Crecimiento/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Línea Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Humanos , Prolactina/farmacología , Receptores de Somatotropina/deficiencia , Receptores de Somatotropina/efectos de los fármacosRESUMEN
We recently identified a female leprechaun infant with marked hyperinsulinemia [as high as 10,975 microU/ml (78,746 pmol/liter)], presumably secondary to insulin resistance. She had two physical findings suggestive of possible insulin action: cystic ovarian enlargement with gonadotropin-independent steroid secretion and persistent, severe myocardial hypertrophy. To examine the pathophysiology of this disorder we measured the in vitro sensitivity to insulin and other growth factors of erythroid progenitors and a T-lymphoblast cell line derived from her peripheral blood. Resistance to insulin was demonstrated by failure of her circulating erythroid progenitor cells to augment proliferation in response to physiologic concentrations of insulin (1-10 ng/ml). An immortalized T lymphoblast cell line was established by transforming the cells with the human retrovirus human T cell leukemia virus II. This cell line showed little or no response to physiologic concentrations of insulin contrary to consistently observed stimulation of colony formation by cell lines similarly derived from normals. The patient's T lymphoblasts, however, showed normal sensitivity to insulin-like growth factor I. In response to supraphysiologic insulin concentrations (25-1000 ng/ml), leprechaun T lymphoblasts showed significant augmentation of colony formation (peak 189% above baseline at 50 ng/ml); normal T lymphoblasts also showed responsiveness at these high insulin concentrations. Preincubation with a monoclonal antibody against the insulin-like growth factor I receptor (alpha IR-3 at 5000 ng/ml) blocked the in vitro effect of physiologic concentrations of insulin-like growth factor and supraphysiologic concentrations of insulin on leprechaun and control T lymphoblast colony formation, but had no clear effect upon the response to physiologic insulin concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hiperinsulinismo/genética , Resistencia a la Insulina/genética , Insulina/farmacología , Adulto , Ensayo de Unidades Formadoras de Colonias , Enanismo/sangre , Enanismo/genética , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Hiperinsulinismo/sangre , Técnicas In Vitro , Lactante , Recién Nacido , Activación de Linfocitos/efectos de los fármacos , Masculino , Pubertad Precoz/sangre , Pubertad Precoz/genética , SíndromeRESUMEN
We report here the development of a rapid and quantitative method for measuring in vitro T cell transformation by human T cell leukemia viruses type I (HTLV-I) and type II (HTLV-II). This method is based on our finding that cocultivation of lethally irradiated HTLV-producing cells with peripheral blood lymphocytes (PBLs) preactivated for 24 hours with phytohemagglutinin and interleukin-2 (IL-2) induces colony formation in methylcellulose-containing medium. Colonies of about 200 cells can be clearly distinguished from background aggregates within four to six days after cocultivation. These colonies gradually increase in size and reach 300 to 1,000 cells within 14 days after cocultivation. Cells of these colonies were infected, as evidenced by expression of viral p19 antigen and the presence of HTLV proviral sequences. These cells proved to be transformed in terms of IL-2-independent continuous growth in liquid medium. Colony formation was found to depend in a linear fashion upon the percentage of the infected cells present in the irradiated cell population and is sufficiently sensitive to detect as few as 1% of virus-producing cells.
Asunto(s)
Transformación Celular Viral , Ensayo de Unidades Formadoras de Colonias , Ensayo de Tumor de Célula Madre , Ensayo de Unidades Formadoras de Colonias/normas , Deltaretrovirus , Infecciones por Deltaretrovirus/patología , Estudios de Evaluación como Asunto , Femenino , Humanos , Linfocitos/patología , Células Madre Neoplásicas/patología , Ensayo de Tumor de Célula Madre/normasRESUMEN
Tissues from patients with Laron dwarfism are resistant to the actions of endogenous or exogenous GH. As a result, insulin-like growth factor I (IGF-I) levels are low, possibly contributing to the severe growth deficiency that occurs in patients with this syndrome. In this study, we found that erythroid progenitor cells and permanently transformed T-cell lines from two patients with Laron dwarfism responded in vitro to added IGF-I in concentrations ranging between 1-10 ng/mL despite no stimulatory response to added GH in concentrations of up to 500 ng/mL. Normal or near-normal responsiveness to insulin was also demonstrated. The persistence of GH resistance in the cultured T-cell lines confirms the primary genetic nature of the defect in Laron dwarfism. The preservation of in vitro growth responsiveness to IGF-I in hematopoietic tissue from the Laron dwarfs suggests that affected individuals are sensitive to this factor and may respond to it in vivo.
Asunto(s)
Enanismo/genética , Eritrocitos/patología , Hormona del Crecimiento/farmacología , Células Madre Hematopoyéticas/patología , Factor I del Crecimiento Similar a la Insulina/farmacología , Somatomedinas/farmacología , Linfocitos T/patología , Adolescente , Adulto , División Celular/efectos de los fármacos , Transformación Celular Viral , Células Cultivadas , Deltaretrovirus , Enanismo/patología , Humanos , MasculinoRESUMEN
Migration-inhibitory factor (MIF) is a lymphokine that acts to localize mononuclear phagocytes (monocytes and macrophages) and perhaps to activate them. Mo cells are a human T cell leukemia virus II-infected T cell line previously shown to secrete large quantities of MIF upon stimulation with phytohemagglutinin and phorbol myristate acetate. MIF was purified from Mo cell-conditioned medium by gel filtration, phenyl-Sepharose affinity chromatography, isoelectrofocusing, and reverse-phase high-performance liquid chromatography (RP-HPLC). Overall purification was 6,000-fold. The purified MIF fraction was found to display potent colony-stimulating factor (CSF) activity when assayed on human bone marrow cells. The double peak of MIF activity as shown by C 18-RP-HPLC coincided with the double peak of CSF activity. A monoclonal antibody selected for its anti-MIF activity absorbed both the CSF and the MIF activity. These findings indicate that MIF and CSF are either identical molecules or closely related molecules with common structural elements.
Asunto(s)
Factores Estimulantes de Colonias/análisis , Linfocinas/análisis , Línea Celular , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Deltaretrovirus , Fluorometría , Humanos , Focalización Isoeléctrica , Fitohemaglutininas/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Six nonobese women with polycystic ovarian disease (PCOD) showed significant hyperinsulinemia, compared with controls after oral glucose (P less than 0.05). As an indicator of insulin sensitivity, in vitro proliferation of erythrocyte progenitor cells of PCOD subjects exposed to physiologic concentrations of insulin was significantly blunted (P less than 0.001). Monocyte insulin receptor binding was not impaired in the PCOD subjects. Three of the PCOD patients were treated with a long-acting gonadotropin-releasing hormone agonist for 6 months, which resulted in marked suppression of ovarian androgen secretion but no demonstrable changes in in vivo or in vitro indicators of insulin resistance. Thus insulin resistance in PCOD subjects appears to be unrelated to ovarian hyperandrogenism (or acanthosis or obesity). Although certain tissues are insulin-resistant in PCOD patients, the ovary may remain sensitive and overproduce androgens in response to high circulating insulin levels.