RESUMEN
AIM: The aim of this randomized placebo-controlled study was to evaluate the safety and efficacy of pioglitazone administered alone or in combination with metformin in reducing insulin dosage requirements for improved glycaemic control in patients with type 2 diabetes previously poorly controlled with combination therapy. METHODS: In this multicentre, double-blind study, 222 patients with haemoglobin A1c (HbA(1c))>8.0% at screening treated with combination therapy initially were given titrated insulin therapy (to fasting plasma glucose <140 mg/dl) and then were randomly assigned to 20-week treatment with pioglitazone or placebo in combination with insulin, with or without concurrent metformin therapy. More than 98% of patients were taking metformin prior to and during the study. RESULTS: Pioglitazone significantly reduced (p < 0.05) insulin dose requirements 2 weeks after treatment initiation. At study end relative to baseline, pioglitazone reduced daily insulin dosages by 12.0 units (p < 0.001), a 21.5% (12.0/55.8 units at baseline) group mean average reduction. Relative to placebo, pioglitazone reduced daily insulin dosages by 12.7 units [95% confidence interval [CI]: -17.5, -8.0], while improving mean HbA(1c) levels [adjusted mean HbA(1c) change: pioglitazone, -1.6% vs. placebo, -1.4% (not statistically different)]. Pioglitazone also significantly increased high-density lipoprotein cholesterol levels [adjusted mean difference: +4.5 (95% CI: 2.6-6.5) mg/dl], decreased triglyceride levels [-43.9 (-69.2, -18.6) mg/dl], shifted low-density lipoprotein (LDL) particle concentrations from small [pattern B, -13.6% (-17.7%, -9.5%)] to large [pattern A, +15.1% (10.8%, 19.5%)] and increased mean LDL particle size [+3.8 (2.6, 4.9) A]. More pioglitazone-treated patients experienced oedema (9.0 vs. 4.5%) and weight gain (9.1 vs. 2.7%) than placebo patients. CONCLUSIONS: Pioglitazone in combination with insulin therapy improved glycaemic control, reduced insulin dose requirements and improved lipid profiles in patients with type 2 diabetes previously poorly controlled with combination therapy.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Insulina/uso terapéutico , Lípidos/sangre , Tiazolidinedionas/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Colesterol/sangre , Diabetes Mellitus Tipo 2/sangre , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Femenino , Hemoglobina Glucada/metabolismo , Humanos , Hipoglucemiantes/uso terapéutico , Insulina/administración & dosificación , Masculino , Persona de Mediana Edad , Pioglitazona , Triglicéridos/sangreRESUMEN
The mechanism by which insulin decreases protein degradation is unknown. We examined insulin binding and degradation (125I[A14]insulin) and protein degradation (3H-leucine labeling) in Chinese hamster ovary (CHO) cells transfected with wild-type (WI) and mutant human insulin receptors. The deltaExon-16 mutant is missing the juxtamembrane domain that mediates endocytosis. The delta343 mutant receptor lacks the tyrosine kinase structural domain but retains the juxtamembrane internalization domain. The mutant deltaNPEY lacks the single NPEY sequence located 16 residues after the end of the transmembrane domain. Null transfected cells (NEO) not expressing human receptors were studied as controls. The WT and deltaNPEY cells equivalently internalized and degraded insulin; delta343 cells internalized and degraded insulin, but at a reduced rate; deltaExon-16 cells internalized and degraded significantly less insulin than the other mutants; NEO cells showed essentially no internalization and degradation. In contrast, all cell types showed the same efficacy at inhibition of protein degradation, albeit at different potencies. These results suggest insulin actions are mediated by multiple and redundant effector systems, but that receptor tyrosine kinase activity is not required for inhibition of protein degradation.
Asunto(s)
Acetilcisteína/análogos & derivados , Insulina/metabolismo , Insulina/farmacología , Proteínas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor de Insulina/metabolismo , Acetilcisteína/farmacología , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Inhibidores Enzimáticos/farmacología , Humanos , Leucina/metabolismo , Datos de Secuencia Molecular , Mutación , Receptor de Insulina/genética , Transfección , Tirosina/metabolismoRESUMEN
A total of 302 diabetic patients were selected from regular attendants of the Tikur Anbessa Hospital (TAH) diabetic clinic to determine the prevalence of retinopathy from December 1994 to March 1995. The mean age was 41.4 +/- 14.4 years (range 14-85). There were 160 males (53%) and 142 females (47%). One hundred forty (46%) were type 1 and 162 (53.6%) were type 2. The mean duration of diabetes was 9.4 +/- 5.4 years and the mean Hemoglobin Alc (HbAlc) was 10.4 + 2.2%. On the day of the examination the mean fasting blood glucose (FBG) and random blood glucose (RBG) were 195.5 +/- 79.9 mg/dl and 273.1 +/- 114.5 mg/dl respectively. The mean serum total cholesterol, triglycerides, LDL, VLDL and GDL were 166.5 +/- 45.5 mg/dl, 129.9 +/- 92.4 mg/dl, 94.5 +/- 36.4 mg/dl, 24.4 +/- 15.1 mg/dl and 44.3 +/- 11.5 mg/dl respectively. The overall prevalence of retinopathy was 37.8% out of which 108 patients (36.1%) had background retinopathy and 5 patients (1.7%) had proliferative retinopathy. The retina could not be visualized in three patients because of dense cataract. Retinopathy correlated positively with age, duration of diabetes and blood pressure respectively, however no significant correlation was seen with mean total HgAlc and serum lipids. Prevalence of retinopathy was comparable in type 1 and type 2 (p > 0.05). The prevalence of retinopathy in our patients relative to the duration of diabetes mellitus is high. Therefore, improving facilities for the diagnosis and treatment of retinopathy is recommended.
Asunto(s)
Retinopatía Diabética/epidemiología , Adolescente , Adulto , Distribución por Edad , Anciano , Anciano de 80 o más Años , Glucemia/análisis , Estudios Transversales , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Retinopatía Diabética/sangre , Retinopatía Diabética/diagnóstico , Retinopatía Diabética/etiología , Etiopía/epidemiología , Femenino , Hospitales de Enseñanza/estadística & datos numéricos , Humanos , Masculino , Persona de Mediana Edad , Evaluación de Necesidades , Servicio Ambulatorio en Hospital/estadística & datos numéricos , Vigilancia de la Población , Prevalencia , Distribución por SexoRESUMEN
We assessed the roles of insulin receptor substrate-1 (IRS-1) and Shc in insulin action on farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) using Chinese hamster ovary (CHO) cells that overexpress wild-type human insulin receptors (CHO-hIR-WT) or mutant insulin receptors lacking the NPEY domain (CHO-DeltaNPEY) or 3T3-L1 fibroblasts transfected with adenoviruses that express the PTB or SAIN domain of IRS-1 and Shc, the pleckstrin homology (PH) domain of IRS-1, or the Src homology 2 (SH2) domain of Shc. Insulin promoted phosphorylation of the alpha-subunit of FTase and GGTase I in CHO-hIR-WT cells, but was without effect in CHO-DeltaNPEY cells. Insulin increased FTase and GGTase I activities and the amounts of prenylated Ras and RhoA proteins in CHO-hIR-WT (but not CHO-DeltaNPEY) cells. Overexpression of the PTB or SAIN domain of IRS-1 (which blocked both IRS-1 and Shc signaling) prevented insulin-stimulated phosphorylation of the FTase and GGTase I alpha-subunit activation of FTase and GGTase I and subsequent increases in prenylated Ras and RhoA proteins. In contrast, overexpression of the IRS-1 PH domain, which impairs IRS-1 (but not Shc) signaling, did not alter insulin action on the prenyltransferases, but completely inhibited the insulin effect on the phosphorylation of IRS-1 and on the activation of phosphatidylinositol 3-kinase and Akt. Finally, overexpression of the Shc SH2 domain completely blocked the insulin effect on FTase and GGTase I activities without interfering with insulin signaling to MAPK. These data suggest that insulin signaling from its receptor to the prenyltransferases FTase and GGTase I is mediated by the Shc pathway, but not the IRS-1/phosphatidylinositol 3-kinase pathway. Shc-mediated insulin signaling to MAPK may be necessary (but not sufficient) for activation of prenyltransferase activity. An additional pathway involving the Shc SH2 domain may be necessary to mediate the insulin effect on FTase and GGTase I.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Transferasas Alquil y Aril/metabolismo , Insulina/farmacología , Proteínas/metabolismo , Receptor de Insulina/fisiología , Células 3T3 , Adenoviridae , Animales , Células CHO , Cricetinae , Farnesiltransferasa , Proteína Adaptadora GRB2 , Humanos , Proteínas Sustrato del Receptor de Insulina , Cinética , Ratones , Fosfoproteínas/metabolismo , Fosforilación , Prenilación de Proteína , Subunidades de Proteína , Receptor de Insulina/genética , Proteínas Recombinantes/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Transfección , Dominios Homologos srcRESUMEN
OBJECTIVES: To assess the coronary artery risk factors in Ethiopian diabetic patients. DESIGN: A cross sectional study done on a representative sample of diabetic patients to evaluate the coronary risk factors. SETTING: Hospital-based study. SUBJECTS: A total of 302 diabetic patients randomly selected from the 3000 regularly attending diabetic patients in the Tikur Anbessa Hospital Diabetes Centre of whom 161(53.3%) were males, 141(46.7%) were females, 140(46.4%) were Type 1 and 162(53.6%) were Type 2. OUTCOME MEASURES: Assessment of the coronary risk factors in diabetic patients. RESULTS: There were 20(6.6%) smokers and no ex-smokers. All smokers were males of whom five (25%) were Type 1 and 15(75%) were Type 2. Hypertension and obesity were found in 64(21.2%) and 69(22.8%) patients respectively. Sixty one (20.2%) patients and 43(14.2%) patients had hypercholesterolaemia and hypertriglyceridaemia respectively. CONCLUSION: Our diabetic patients share the risk factors for developing coronary artery disease like any other diabetic patient in the developed countries but at a lower level.
Asunto(s)
Enfermedad Coronaria/epidemiología , Diabetes Mellitus/epidemiología , Angiopatías Diabéticas/epidemiología , Adulto , Comorbilidad , Estudios Transversales , Etiopía/epidemiología , Femenino , Humanos , Hiperlipidemias/epidemiología , Masculino , Persona de Mediana Edad , Factores de Riesgo , FumarRESUMEN
The cytoplasmic juxtamembrane domain of the human insulin receptor (hIR) contains a single copy of the tetrameric amino acid sequence Asn-Pro-Glu-Tyr (NPEY) (residues 969-972 in the exon 11-containing B-isoform), which exists in the context of NPXY. In this study, we examined the role of NPEY972 in mediating insulin signal transduction and cellular biological effects. Transfected Chinese hamster ovary cell lines expressing either the wild-type hIR-B isoform (hIR.WT) or a mutant receptor lacking the NPEY972 sequence (hIRDeltaNPEY) and control Chinese hamster ovary.Neo cells were used to comparatively analyze the following insulin effects: in vivo receptor tyrosine autophosphorylation and kinase activity, signal transduction to downstream signaling molecules, and stimulation of glycogen and DNA synthesis. The results showed that in comparison to hIR.WT, the hIRDeltaNPEY mutant demonstrated the following: (a) normal insulin-mediated receptor tyrosine phosphorylation, but approximately 50% reduction in phosphorylation of p185-(insulin receptor substrate-1) and binding of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase), (b) an enhanced stimulation of PI 3-kinase enzymatic activity, (c) a complete inability to phosphorylate Shc, (d) minimal impairment of insulin sensitivity for glycogen synthesis, and (e) an augmented response to insulin-stimulated DNA synthesis via a high capacity, low sensitivity pathway. These results demonstrate the following: 1) the NPEY972 sequence is important but not absolutely essential for coupling of hIR kinase to insulin receptor substrate-1 and p85 or for mediating insulin's metabolic and mitogenic effects, 2) the NPEY972 sequence is necessary for Shc phosphorylation, and 3) the absence of Shc phosphorylation releases the constraints on maximal insulin-stimulated mitogenic response, thus indicating that alternate signaling pathway(s) exist for this insulin action. This alternate pathway appears to be associated with enhanced activation of PI 3-kinase and is of high capacity and low sensitivity.
Asunto(s)
Insulina/metabolismo , Mitógenos/metabolismo , Oligopéptidos/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal , Animales , Células CHO , Cricetinae , ADN/metabolismo , Glucógeno/biosíntesis , Humanos , Proteínas Sustrato del Receptor de Insulina , Oligopéptidos/genética , Fosfatidilinositol 3-Quinasas , Fosfoproteínas/metabolismo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptor de Insulina/genética , Timidina/metabolismoRESUMEN
Protein tyrosine phosphatase 1B (PTP1B) is a protein tyrosine phosphatase of unknown function, although increasing evidence supports a role for this phosphatase in insulin action. We have investigated the interaction of PTP1B with the insulin receptor using a PTP1B glutathione S-transferase (GST) fusion protein with a point mutation in the enzyme's catalytic domain. This fusion protein is catalytically inactive, but the phosphatase's phosphotyrosine binding site is maintained. The activated insulin receptor was precipitated from purified receptor preparations and whole-cell lysates by the inactive PTP1B-GST, demonstrating a direct association between the insulin receptor and PTP1B. A p120 of unknown identity was also precipitated from whole-cell lysates by the PTP1B fusion protein, but IRS-1 (pp185) was not. A catalytically inactive [35S]PTP1B-fusion protein bound directly to immobilized insulin receptor kinase domains and was displaced in a concentration-dependent manner. Finally, tyrosine-phosphorylated PTP1B was precipitated from whole-cell lysates by an anti-insulin receptor antibody after insulin stimulation. The site of interaction between PTP1B and the insulin receptor was studied using phosphopeptides modeled after the receptor's kinase domain, the NPXY domain, and the COOH-terminal. Each phosphopeptide inhibited the PTP1B-GST:insulin receptor interaction. Study of mutant insulin receptors demonstrated that activation of the kinase domain is necessary for the PTP1B:insulin receptor interaction, but receptors with deletion of the NPXY domain or of the COOH-terminal can still bind to the PTP1B-GST. We conclude that PTP1B can associate directly with the activated insulin receptor at multiple different phosphotyrosine sites and that dephosphorylation by PTP1B may play a significant role in insulin receptor signal transduction.
Asunto(s)
Proteínas Tirosina Fosfatasas/metabolismo , Receptor de Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos , Sitios de Unión , Línea Celular , Clonación Molecular , Glutatión Transferasa , Humanos , Immunoblotting , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fosfopéptidos/química , Fosfopéptidos/aislamiento & purificación , Mutación Puntual , Proteínas Tirosina Fosfatasas/química , Ratas , Receptor de Insulina/química , Receptor de Insulina/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
The intracellular portion of the human insulin receptor (hIR) beta-subunit contains distinct functional domains including the exon 16-encoded juxtamembrane (JM) domain that mediates endocytosis, and the tyrosine kinase (TK) domain that mediates insulin's metabolic and mitogenic actions. To explore the functional relationship between these domains and to determine the role of insulin internalization in insulin action, we constructed and studied the endocytic and signaling properties of an hIR mutant truncated at Glu-1012. This truncation removes the carboxyl-terminal 343 amino acids containing essentially all of the TK domain but leaves behind the exon 16-encoded JM domain that is necessary for endocytosis, plus an additional 35 amino acids downstream. The wild-type (hIR-WT) and mutant (hIR delta 343) receptors were stably expressed in CHO cells and their abilities to mediate various insulin-stimulated functions were comparatively analyzed. In cells expressing hIR-WT, insulin markedly enhanced tyrosine phosphorylation of the beta-subunit and of the endogenous 185 kDa substrate whereas these effects were completely absent in cells expressing hIR delta 343. The hIR delta 343 receptors retained the ability to internalize a significant amount of surface-bound insulin at 37 degrees C. However, they were unable to mediate either the short or long-term biological effects of insulin as determined by assaying insulin-stimulated glucose uptake (assessed by 2-deoxyglucose uptake), protooncogene expression (measured by Northern blot analysis of c-fos mRNA) and DNA synthesis (measured by 3H-thymidine incorporation). These results indicate that the hIR beta-subunit JM and TK domains can be functionally uncoupled, and that insulin internalization in the absence of hIR TK domain and kinase activity is insufficient for mediating intracellular insulin action.
Asunto(s)
Insulina/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Eliminación de Secuencia , Animales , Transporte Biológico , Células CHO , División Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cricetinae , Medio de Cultivo Libre de Suero , ADN/biosíntesis , Endocitosis , Exones , Ácido Glutámico , Humanos , Insulina/farmacología , Cinética , Sustancias Macromoleculares , Receptor de Insulina/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Timidina/metabolismo , TransfecciónRESUMEN
The human insulin receptor (hIR) cytoplasmic juxtamembrane domain contains two tyrosine (Y) residues which exist in GPLY and NPEY motifs that have been implicated in endocytic function. We have previously shown that the NPEY motif is not necessary for endocytosis of the B isoform (exon 11+) of hIR. To examine the role of the GPLY sequence in transmembrane insulin signaling and endocytic functions of hIR-B, we constructed a mutant receptor, hIR delta GPLY, that lacks the GPLY sequence (residues 962-965), and stably expressed it in CHO cells. When compared to wild type hIR-B (hIR-WT) similarly expressed in CHO cells, the hIR delta GPLY mutant exhibited higher insulin binding affinity (EC50 of 1.0 vs 3.5 nM) and normal insulin-stimulated receptor tyrosine autophosphorylation and kinase activity towards the endogenous 185 kDa insulin receptor substrate. The hIR delta GPLY receptor also exhibited normal endocytic functions as hIR-WT in that: a) the internalization of surface photoaffinity labeled hIR delta GPLY was similar to that of hIR-WT, and b) the rate and extent of 125I-insulin internalization and degradation at 37 degrees C were also unimpaired. Therefore, these results demonstrate that the GPLY sequence is not necessary for transmembrane insulin signaling and endocytic functions of the hIR-B isoform.
Asunto(s)
Receptor de Insulina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN/química , Endocitosis , Humanos , Insulina/farmacología , Datos de Secuencia Molecular , Fosforilación , Fosfotirosina , Receptor de Insulina/química , Eliminación de Secuencia , Transducción de Señal , Relación Estructura-Actividad , Tirosina/análogos & derivados , Tirosina/fisiologíaRESUMEN
The insulin receptor is known to interact with the SH2 domain proteins p85 (the regulatory subunit of phosphatidylinositol 3-kinase), Syp (a tyrosine phosphatase), and GAP (GTPase-activating protein). In this study, we mapped the insulin receptor binding sites for each of these proteins by examining the ability of phosphopeptides, corresponding to insulin receptor phosphorylation sites, and mutant insulin receptors to inhibit an insulin receptor-SH2 domain interaction. Precipitation of partially purified insulin receptors by glutathione S-transferase fusion proteins containing the N-terminal SH2 domains of p85 and GAP and both SH2 domains of Syp was demonstrated. The effect of the addition of each phosphopeptide on insulin receptor precipitation was tested. pY1322, the C-terminal insulin receptor peptide, inhibited insulin receptor precipitation by both p85- and Syp-GST. The NPXY internalization domain peptide inhibited insulin receptor precipitation by GAP-GST. These data were confirmed by mutant insulin receptor experiments. The insulin receptor C-terminal mutants, delta CT and Y/F2, were not precipitated by p85- or Syp-GST and the NPXY mutant insulin receptors, delta Ex16 and HI delta NPEY, were not precipitated by GAP-GST. Therefore, we conclude that p85 and Syp bind to the insulin receptor C terminus at tyrosine 1322 and GAP binds to the insulin receptor NPXY domain at tyrosine 960.
Asunto(s)
Fosfopéptidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas/metabolismo , Receptor de Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Activadoras de GTPasa , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas , Fosfotirosina , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Ratas , Proteínas Tirosina Fosfatasas con Dominio SH2 , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
The tetrameric amino acid sequence AsnProXTyr (NPXY), where X represents any amino acid, is conserved in the intracytoplasmic domains of several membrane proteins and has been postulated to play a role in receptor-mediated endocytosis. The human insulin receptor (hIR) contains a single copy of the sequence AsnProGluTyr (NPEY) in its intracytoplasmic domain. To determine if this putative consensus sequence is necessary for endocytic functions of hIR, we constructed a mutant receptor, hIR delta NPEY, that lacks NPEY sequence, stably expressed this mutant receptor in Chinese hamster ovary cells, and then studied its endocytic functions. When compared to wild type hIR similarly expressed in Chinese hamster ovary cells, the hIR delta NPEY mutant exhibited: 1) normal subunit organization and insulin binding affinity; 2) essentially normal internalization of covalent photoaffinity labeled insulin-receptor complexes; and 3) normal internalization of receptor-bound [125I]insulin as well as normal degradation and release of the internalized insulin. Therefore, we conclude that the NPEY sequence in the juxtamembrane domain of hIR is not necessary for its endocytic function.
Asunto(s)
Endocitosis/fisiología , Señales de Clasificación de Proteína/fisiología , Receptor de Insulina/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Cricetinae , Cricetulus , ADN/genética , Femenino , Amplificación de Genes/genética , Insulina/metabolismo , Radioisótopos de Yodo , Datos de Secuencia Molecular , Mutación/genética , Ovario/citología , Ovario/metabolismo , Ovario/ultraestructura , Reacción en Cadena de la Polimerasa , Señales de Clasificación de Proteína/análisis , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , TransfecciónRESUMEN
The effects of insulin on the steady-state level of human insulin receptor (hIR) mRNA were examined in the HepG2 human liver cell line using Northern blot analysis of either total cellular or poly(A)+ RNA. In control cells, up to six (4.5, 5.2, 7.4, 8.5, 9.4 and 10.8 kb) hybridizable species of hIR mRNA were identified, with the 8.5 and 10.8 kb species being most prominent. Incubation for 18 hrs with 1 microM insulin resulted in a similar decrease (to approximately 35% of control) of all the hIR mRNA species. The insulin effect was dose-dependent and was half-maximal by 2-3 hrs and maximal by 4-6 hrs of incubation at 37 degrees C. The hIR mRNA levels remained maximally insulin suppressed for up to 18 hrs but thereafter the effect became attenuated. These results indicate that insulin downregulates the level of hIR mRNA with a biphasic time-course and that this process is most likely part of the general mechanism by which insulin maintains the homeostatic control of its cellular receptor levels.
Asunto(s)
Insulina/fisiología , Receptor de Insulina/genética , Northern Blotting , Regulación hacia Abajo , Regulación de la Expresión Génica , Humanos , ARN Mensajero/genética , Receptor de Insulina/metabolismo , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Internalization, degradation, and insulin-induced down-regulation of insulin receptors were studied comparatively in transformed Chinese hamster ovary (CHO) cell lines, CHO.T and CHO.IR.ros, respectively expressing either the wild-type human insulin receptor (hIR) or a mutated hybrid receptor in which the transmembrane and cytoplasmic domains of hIR were replaced by corresponding domains of the transforming protein p68gag-ros (v-ros) of avian sarcoma virus UR2. At 37 degrees C, degradation of insulin receptors photoaffinity labeled on the cell surface (440 kDa) was most rapid for the hybrid hIR.ros (t1/2 1.0 +/- 0.1 h), intermediate for the wild-type hIR (t1/2 2.7 +/- 0.5 h), and slowest for the endogenous CHO insulin receptors (t1/2 3.7 +/- 0.7 h). Initial intracellular accumulation of the hIR.ros hybrid was also most rapid, reaching maximal amounts in 20 min following which the receptors disappeared rapidly from the intracellular compartment. In contrast, intracellular accumulation of the receptors in the CHO.T and CHO cells was slower, reaching maximal amounts in 60 min, and rapid disappearance of the receptors from the intracellular compartment did not occur. Chloroquine, a lysosomotropic agent, inhibited degradation of both the wild-type hIR and the chimeric hIR.ros and increased their intracellular accumulation. However, the chloroquine effect was much more marked for the hIR.ros receptors whose intracellular accumulation was increased by greater than 300% (in comparison with approximately 60% increase for the wild-type hIR), demonstrating marked intracellular degradation of the hybrid hIR.ros at chloroquine-sensitive sites. Insulin-induced down-regulation of the cell surface hIR.ros (52% loss in 3 h) was also more marked than the wild-type hIR (approximately 30% loss in 3 h). Thus, in the hybrid hIR.ros receptor, which was shown previously to exhibit insulin-stimulated autophosphorylation and kinase activity but not insulin-stimulated metabolic function, the capacity for internalization and down-regulation is not only preserved but is also markedly accelerated. These findings suggest that 1) the postreceptor coupling mechanisms mediating insulin-induced receptor internalization, degradation, and down-regulation are different from those mediating metabolic functions; and 2) v-ros may contain the structural information directing accelerated receptor catabolism.
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Proteínas Oncogénicas Virales , Proteínas Tirosina Quinasas Receptoras , Receptor de Insulina/metabolismo , Marcadores de Afinidad , Animales , Línea Celular , Membrana Celular/metabolismo , Cloroquina/farmacología , Cricetinae , Citoplasma/metabolismo , Regulación hacia Abajo , Humanos , Insulina/farmacología , Cinética , Mutación , Proteínas Oncogénicas Virales/metabolismo , Fotoquímica , Receptor de Insulina/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Insulin receptors on the surface of isolated rat adipocytes were photoaffinity labeled at 12 degrees C with the iodinated photoreactive insulin analogue, 125I-B2 (2-nitro-4-azidophenylacetyl)-des-PheB1-insulin, and the pathways in the intracellular processing of the labeled receptors were studied at 37 degrees C. During 37 degrees C incubations, the labeled 440-kDa insulin receptors were continuously internalized (as assessed by trypsin inaccessibility) and degraded such that up to 50% of the initially labeled receptors were lost by 120 min. Metabolic poisons (0.125-0.75 mM 2,4-dinitrophenol (DNP) and 1-10 mM NaF), which led to dose-dependent depletion of adipocyte ATP pools, inhibited receptor loss, and caused up to 3-fold increase in intracellular receptor accumulation. This effect was due to inhibition of intracellular receptor degradation, and there was no apparent effect of the metabolic poisons on initial internalization of the receptors. Following maximal intracellular accumulation of labeled insulin receptors in the presence of NaF or DNP, removal of these agents resulted in a subsequent, time-dependent degradation of the accumulated receptors. However, when the lysosomotropic agent, chloroquine (0.2 mM), was added immediately following removal of the metabolic poisons, further degradation of the intracellularly accumulated receptors was prevented, suggesting that the chloroquine-sensitive degradation of insulin receptors occurs distal to the site of inhibition by NaF or DNP. To confirm this, maximal intracellular accumulation of labeled receptors was first allowed to occur in the presence of chloroquine and the cells were then washed and reincubated in chloroquine-free media in the absence or presence of NaF or DNP. Under these conditions, degradation of the intracellularly accumulated receptors continued to occur, and NaF or DNP failed to block the degradation. In summary, these results indicate that the loss of cell surface insulin receptors in adipocytes involves: 1) initial internalization of the receptors to a nondegradative intracellular compartment by a process that is relatively insensitive to ATP depletion, followed by 2) a highly energy-dependent unidirectional translocation of the receptors from this compartment to chloroquine-sensitive site(s) of degradation.
Asunto(s)
Tejido Adiposo/metabolismo , Cloroquina/farmacología , Metabolismo Energético , Receptor de Insulina/metabolismo , 2,4-Dinitrofenol , Adenosina Trifosfato/metabolismo , Tejido Adiposo/efectos de los fármacos , Marcadores de Afinidad , Animales , Azidas/metabolismo , Transporte Biológico Activo , Dinitrofenoles/farmacología , Metabolismo Energético/efectos de los fármacos , Insulina/análogos & derivados , Insulina/metabolismo , Radioisótopos de Yodo , Cinética , Masculino , Fragmentos de Péptidos/metabolismo , Fotoquímica , Ratas , Ratas Endogámicas , Fluoruro de Sodio/farmacología , Tripsina/metabolismoRESUMEN
We recently demonstrated that chymotrypsin substrate analogues inhibit receptor-mediated insulin internalization in isolated rat adipocytes. In this study, the effect on glucose transport of inhibiting insulin internalization with these agents was examined. Glucose transport was assayed by measuring [3H]-2-deoxyglucose uptake, and internalized insulin was measured after rapidly dissociating surface-bound insulin with an acidic buffer. The chymotrypsin substrate analogue N-acetyl-Tyr ethyl ester inhibited insulin internalization by 85% while increasing surface-bound insulin by 80-110%. Under these conditions, ATP levels were minimally altered, and basal glucose transport was unchanged; however, insulin-stimulated glucose transport was decreased by 86%. The inhibition of insulin-stimulated glucose transport was not overcome by supramaximal concentrations (400 ng/ml) of insulin. When insulin internalization and insulin-stimulated glucose transport were measured in the presence of increasing concentrations of N-acetyl-Tyr ethyl ester (0.1-1 mM), a strong and highly significant correlation (r = .97, P less than .001) was found between inhibition of insulin internalization and inhibition of insulin-stimulated glucose uptake. Fragments of N-acetyl-Tyr ethyl ester that do not inhibit insulin internalization were also without effect on insulin-stimulated glucose transport. In addition to N-acetyl-Tyr ethyl ester, four other chymotrypsin substrate analogues that are effective inhibitors of insulin internalization also markedly inhibited insulin-stimulated glucose transport. These results indicate that insulin internalization and insulin-stimulated glucose transport share a common postbinding step in adipocytes and that this step is inhibitable by chymotrypsin substrate analogues.
Asunto(s)
Tejido Adiposo/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Adenosina Trifosfato/análisis , Tejido Adiposo/análisis , Tejido Adiposo/efectos de los fármacos , Animales , Quimotripsina/metabolismo , Desoxiglucosa/metabolismo , Relación Dosis-Respuesta a Droga , Insulina/farmacología , Antagonistas de Insulina/farmacología , Masculino , Ratas , Ratas Endogámicas , Tirosina/análogos & derivados , Tirosina/farmacologíaRESUMEN
The dynamics of the internalization of photoaffinity-labelled insulin-receptor complexes was investigated in isolated rat adipocytes by using tryptic proteolysis to probe both the orientation and cellular location of the labelled complexes. In cells that were labelled at 16 degrees C and not prewarmed, 150 micrograms of trypsin/ml rapidly degraded the labelled 125 kDa insulin-receptor subunit into a major proteolytic fragment of 70 kDa and minor amounts of 90- and 50-kDa fragments. With milder trypsin treatment conditions (100 micrograms of trypsin/ml, 15 s at 37 degrees C), the 90 kDa peptide (different from the 90 kDa beta-subunit of the insulin receptor) appeared as a major intermediate proteolytic product, but this species was rapidly and completely converted into the 70- and 50-kDa fragments with continued exposure to trypsin, such that it did not accumulate to appreciable amounts in cells that were not prewarmed before trypsin exposure. By contrast, trypsin treatment of cells prewarmed to 37 degrees C for various times showed that: first, a proportion of the labelled 125 kDa receptors was internalized (became trypsin-insensitive); secondly, the 90 kDa tryptic peptide was formed in large amounts, with proportionate decreases occurring in the amounts of the 70- and 50-kDa tryptic peptides. The increased accumulation of the 90 kDa tryptic peptide from cells preincubated at 37 degrees C, but not at 16 degrees C, indicated that trypsin cleavage sites within the 90 kDa segment of the insulin-receptor alpha-subunit that were exposed at 16 degrees C were made inaccessible by incubation at 37 degrees C, a finding that is consistent with generation of a cryptic domain of the receptor subunit. The tryptic generation of the 90 kDa peptide at 37 degrees C was rapid, becoming half-maximal in 4.4 +/- 0.6 min and maximal in 15-20 min, preceded the intracellular accumulation of labelled receptors (half-maximal in 12.6 +/- 0.7 min and maximal in 30-40 min), was highly correlated with receptor internalization, and was not observed in cultured IM-9 lymphocytes, a cell line in which photolabelled insulin receptors are primarily lost by shedding into the incubation media. These results show that, in adipocytes incubated at 37 degrees C, rapid masking of a previously (at 16 degrees C) accessible domain of the insulin-receptor alpha-subunit occurs and that this dynamic process happens at an early stage in the internalization of insulin-receptor complexes.
Asunto(s)
Tejido Adiposo/metabolismo , Receptor de Insulina/metabolismo , Animales , Cloroquina/farmacología , Electroforesis en Gel de Poliacrilamida , Técnicas In Vitro , Insulina/metabolismo , Linfocitos/metabolismo , Sustancias Macromoleculares , Masculino , Fragmentos de Péptidos/análisis , Ratas , Ratas Endogámicas , Receptor de Insulina/efectos de los fármacos , Temperatura , Tripsina/farmacologíaRESUMEN
The effects of metalloendoprotease inhibitors on insulin binding, internalization, and processing were studied in isolated rat adipocytes. The metalloendoprotease inhibitor phosphoramidon caused a marked (threefold) increase in intracellular insulin accumulation without affecting surface binding. The dipeptide metalloendoprotease substrate analogues benzyloxycarbonyl-Gly-Phe-NH2 and benzyloxycarbonyl-Gly-Leu-NH2 caused similar large increases in intracellular insulin but also caused a doubling of cell surface bound insulin. The effect on surface binding was due to increased insulin receptor affinity as demonstrated by Scatchard analysis and the benzyloxycarbonyl-Gly-Phe NH2 induced inhibition of the dissociation of prebound insulin from the cell surface. These results suggest a role for endogenous metalloendoprotease-like enzymes in insulin processing by rat adipocytes.
Asunto(s)
Tejido Adiposo/metabolismo , Insulina/metabolismo , Inhibidores de Proteasas/farmacología , Animales , Células Cultivadas , Cloroquina/farmacología , Dipéptidos/farmacología , Endocitosis/efectos de los fármacos , Endopeptidasas , Glicopéptidos/farmacología , Cinética , Metaloendopeptidasas , Metaloproteínas , Ratas , Receptor de Insulina/metabolismoRESUMEN
To explore the possible role of proteolytic step(s) in receptor-mediated endocytosis of insulin, the effects of inhibitors of various classes of proteases on the internalization process were studied in isolated rat adipocytes. Intracellular accumulation of receptor-bound 125I-insulin at 37 degrees C was quantitated after rapidly dissociating surface-bound insulin with an acidic buffer (pH 3.0). Of the 23 protease inhibitors tested, only chymotrypsin substrate analogues inhibited insulin internalization. Internalization was decreased 62-90% by five different chymotrypsin substrate analogues: N-acetyl-Tyr ethyl ester, N-acetyl-Phe ethyl ester, N-acetyl-Trp ethyl ester, benzoyl-Tyr ethyl ester, and benzoyl-Tyr amide. The effect of the substrate analogues in inhibiting insulin internalization was dose-dependent, reversible, and required the full structural complement of a chymotrypsin substrate analogue. Cell surface receptor number was unaltered at 12 degrees C. However, concomitant with their inhibition of insulin internalization at 37 degrees C, the chymotrypsin substrate analogues caused a marked increase (160-380%) in surface-bound insulin, indicating trapping of insulin-receptor complexes on the cell surface. Additionally, 1 mM N-acetyl-Tyr ethyl ester decreased overall insulin degradation by 15-20% and also prevented the chloroquine-mediated increase in intracellular insulin, further indicating that surface-bound insulin was prevented from reaching intracellular chloroquine-sensitive degradation sites. The internalization of insulin receptors that were photoaffinity labeled on the cell surface with B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin was also inhibited 70-90% by the five chymotrypsin substrate analogues, as determined by the effects of the analogues on the accumulation of trypsin-insensitive (intracellular) 440-kD intact labeled receptors. In summary, these results show that chymotrypsin substrate analogues efficiently inhibit the internalization of insulin and insulin receptors in adipocytes and implicate a possible role for endogenous chymotrypsin-like enzyme(s) or related substances in receptor-mediated endocytosis of insulin.
Asunto(s)
Tejido Adiposo/metabolismo , Quimotripsina/antagonistas & inhibidores , Endocitosis/efectos de los fármacos , Insulina/metabolismo , Inhibidores de Proteasas/farmacología , Receptor de Insulina/metabolismo , Marcadores de Afinidad , Aminopeptidasas/antagonistas & inhibidores , Animales , Carboxipeptidasas/antagonistas & inhibidores , Cloroquina/farmacología , Cinética , Masculino , Ratas , Relación Estructura-Actividad , Transglutaminasas/antagonistas & inhibidores , Tirosina/análogos & derivados , Tirosina/farmacologíaRESUMEN
We studied the ability of isolated adipocytes from normal and type II diabetic subjects to internalize and process [125I]insulin. Adipocytes were incubated with [125I]insulin at 16 or 37 C, and at various times total cell-associated, surface-bound, and intracellular insulin were quantitated using an acid-barbital extraction technique which quickly removes cell surface insulin, leaving behind the intracellular insulin. Insulin internalization was slow in normal adipocytes at 16 C, such that only 13% of total cell-associated insulin was intracellular after 2 h of incubation. In contrast, internalization was rapid at 37 C, such that the intracellular pool of insulin was near maximal by 30 min and accounted for approximately 40% of the total cell-associated insulin. Sephadex G-50 column chromatography of the intracellular insulin demonstrated that more than 95% of this pool coeluted with native insulin. In adipocytes from the diabetic subjects, approximately 45% of total cell-associated insulin was intracellular after 30 min of incubation at 37 C. After 60 min of incubation at 37 C, the percentages of total cell-associated and surface-bound insulin were significantly lower in adipocytes from diabetic compared to normal subjects [1.81 +/- 0.31% (+/- SEM) vs. 2.92 +/- 0.24% (P less than 0.05) and 0.97 +/- 0.14% vs. 1.72 +/- 0.15% (P less than 0.01), respectively]. The percentage of insulin in the intracellular compartment was also slightly lower in adipocytes from diabetic compared to normal subjects (0.84 +/- 0.19% vs. 1.20 +/- 0.16%; P greater than 0.05). The lysosomotropic agent chloroquine increased total cell-associated insulin, and this was due entirely to an increase in intracellular insulin. In adipocytes from normal subjects, chloroquine increased intracellular insulin by 32% at 30 min, by 89% at 60 min, by 140% at 90 min, and by 178% at 120 min. In comparison to the normal adipocytes, the chloroquine-mediated increase in intracellular insulin was lower in adipocytes from the diabetic subjects (-8.1% at 30 min, 37% at 60 min, 58% at 90 min, and 63% at 120 min; P less than 0.05 at all time points). These results indicate that insulin is rapidly internalized in human adipocytes at 37 C such that approximately half of total cell-associated insulin is intracellular the intracellular insulin is largely intact; and intracellular processing of insulin by a chloroquine-sensitive pathway(s) is impaired in adipocytes from type II diabetic subjects.
Asunto(s)
Tejido Adiposo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Adulto , Cloroquina/farmacología , Cromatografía en Gel , Humanos , Técnicas In Vitro , Persona de Mediana Edad , Receptor de Insulina/metabolismo , SolubilidadRESUMEN
We have studied the functional and structural characteristics of insulin receptors on cultured rat hypothalamic cells. The receptors on these cells are specific for insulin, but have a lower binding affinity than that measured in nonneuronal tissues. Neither acute (2-h) nor long term (24-h) exposure of the hypothalamic cells to high insulin concentrations resulted in receptor down-regulation. However, insulin is internalized in these cells and accumulated in the presence of the lysomotropic agent chloroquine. Acute exposure to insulin does not alter initial rate of 2-deoxyglucose transport in hypothalamic cells, but does cause a stimulation of aminoisobutyric acid uptake. Photoaffinity labeling of the receptors of the hypothalamic cells with a biologically active photosensitive insulin revealed a major specifically labeled band of 115K mol wt and a minor band of 40K mol wt under disulfide-reducing conditions compared to bands of 125K and 90K mol wt seen after labeling of the insulin receptors of adipocytes. The receptor proteins in hypothalamic cells under nonreducing conditions (420K, 370K, and 310K mol wt) were also smaller than those in adipocytes. Thus, the insulin receptors of cultured hypothalamic cells differ from insulin receptors on peripheral target tissues in both functional and structural aspects.