RESUMO
We aimed to determine the importance of leptin in the regulation of luteinizing hormone (LH) and growth hormone (GH) secretion in ovariectomized (OVX) ewes. Lean and fat sheep were produced by dietary manipulation over 8 months and were then fasted for 32 h. Plasma concentrations of glucose, insulin and leptin were higher in the fat group. Fasting decreased plasma concentrations of glucose and insulin and increased concentrations of nonesterified fatty acids (NEFA) in fat and lean ewes, but leptin concentrations were reduced in the fat group only. Plasma GH concentrations were higher in the lean group and LH concentrations were lower; there was no effect of fasting. These data suggested that long-term changes in plasma leptin concentrations might affect LH and GH secretion, but acute changes with fasting had no effect. OVX ewes of normal body weight were fasted for 72 h with or without intracerebroventricular (i.c.v.) infusion of leptin (4 microg/h), achieving similar metabolic effects to the 32 h fast. The 72-h fast increased LH pulse amplitude, mean GH and cortisol concentrations, but these changes were corrected towards normal by leptin treatment. Thus, leptin could attenuate fasting-induced alterations in the secretion of LH, GH and cortisol. Finally, we food-restricted OVX ewes for 4 months (lean), leading to a 20-kg reduction in body weight. Plasma concentrations of leptin and insulin were decreased, and plasma GH concentrations increased, but there was no effect on plasma concentrations of LH, glucose or NEFA. Icv infusion of leptin did not affect any endocrine or metabolic parameter in these ewes. In summary, maintenance of a lean or fat condition for a prolonged period (8 months) or an extended fasting (72 h) can affect LH and GH secretion, but short-term food restriction (4 months) affected only GH secretion and short-term fasting (32 h) had no effect on either LH or GH secretion. This is in spite of altered plasma leptin concentrations in all circumstances studied. Although leptin treatment can restore plasma concentrations of LH, GH and cortisol towards normal in sheep fasted for 72 h, some other factor(s) must signal to the brain to cause shifts in neuroendocrine function in other conditions where nutritional/metabolic status is altered.
Assuntos
Jejum/sangue , Hormônio do Crescimento/sangue , Leptina/fisiologia , Hormônio Luteinizante/sangue , Desnutrição/sangue , Animais , Glicemia/metabolismo , Composição Corporal/fisiologia , Ácidos Graxos não Esterificados/sangue , Feminino , Hormônio do Crescimento/metabolismo , Hidrocortisona/sangue , Injeções Intraventriculares , Insulina/sangue , Leptina/administração & dosagem , Hormônio Luteinizante/metabolismo , Ovariectomia , Ovinos , Fatores de TempoRESUMO
Ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) have been implicated in bone calcification, type II diabetes, control of purinergic signalling and tumour invasion. The gene for the plasma cell membrane glycoprotein PC-1 in the mouse (Enpp1) has been known since 1970 to exist in two allelic forms, but their structural basis was heretofore unknown. We show that the Enpp1a and Enpp1b alleles differ by only two amino acids, at positions 650 and 679 in the C-terminal nuclease-like domain. Histidine 650 but not arginine 679 forms an essential part of the Enpp1a epitope recognized by monoclonal antibody IR-518. Sequences of LEW and LOU rats and the rat glioma cell line C6 differ from that of the mouse by about 60 amino acids. The LOU and C6 cell line sequences differ by only three amino acids, but differ from the LEW sequence by 10 amino acids. All three rat strains possess the mouse Enpp1b allele at positions 650 and 679. Despite numerous other differences from the mouse, rats immunized with Enpp1a mouse cells have generated monoclonal antibodies specific for the Enpp1a allele, suggesting that amino acids 650 and 679 may be particularly immunogenic. The cytoplasmic tails of the mouse and rat are highly conserved, but are significantly different from human cytoplasmic tails.
Assuntos
Anticorpos Heterófilos/imunologia , Alótipos de Imunoglobulina/imunologia , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/imunologia , Pirofosfatases/genética , Pirofosfatases/imunologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos LewRESUMO
We investigated the signals involved in the apical targeting of dipeptidyl peptidase IV (DPP IV/CD26), an archetypal type II transmembrane glycoprotein. A secretory construct, corresponding to the DPP IV ectodomain, was first stably expressed in both the enterocytic-like cell line Caco-2 and the epithelial kidney MDCK cells. Most of the secretory form of the protein was delivered apically in MDCK cells, whereas secretion was 60% basolateral in Caco-2 cells, indicating that DPP IV ectodomain targeting is cell-type-dependent. A chimera (CTM-GFP) containing only the cytoplasmic and transmembrane domains of mouse DPP IV plus the green fluorescent protein was then studied. In both cell lines, this chimera was preferentially expressed at the apical membrane. By contrast, a secretory form of GFP was randomly secreted, indicating that GFP by itself does not contain cryptic targeting information. Comparison of the sequence of the transmembrane domain of DPP IV and several other apically targeted proteins does not show any consensus, suggesting that the apical targeting signal may be conformational. Neither the DPP IV nor the CTM-GFP chimera was enriched in lipid rafts. Together these results indicate that, besides the well-known raft-dependent apical targeting pathway, the fate of the CTM domain of DPP IV may reveal a new raft-independent apical pathway.
Assuntos
Dipeptidil Peptidase 4/metabolismo , Glicoproteínas de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Sinais Direcionadores de Proteínas , Animais , Sítios de Ligação , Células CACO-2 , Linhagem Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Dimerização , Cães , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Enzymes of the nucleotide pyrophosphatase/phosphodiesterase (NPPase) family are expressed at opposite surfaces in polarized epithelial cells. We investigated the targeting signal of NPP1, which is exclusively expressed at the basolateral surface. Full-length NPP1 and different constructs and mutants were transfected into the polarized MDCK cell line. Expression of the proteins was analyzed by confocal microscopy and surface biotinylation. The basolateral signal of NPP1 was identified as a di-leucine motif located in the cytoplasmic tail. Mutation of either or both leucines largely redirected NPP1 to the apical surface. Furthermore, addition of the conserved sequence AAASLLAP redirected the apical nucleotide pyrophosphatase/phosphodiesterase NPP3 to the basolateral surface. Full-length NPP1 was not significantly internalized. However, when the cytoplasmic tail was deleted upstream the di-leucine motif or when the six upstream flanking amino acids were deleted, the protein was mainly found intracellularly. Endocytosis experiments indicated that these mutants were endocytosed from the basolateral surface. These results identify the basolateral signal of NPP1 as a short sequence including a di-leucine motif that is dominant over apical determinants and point to the importance of surrounding amino acids in determining whether the signal will function as a basolateral signal only or as an endocytotic signal as well.
Assuntos
Endocitose/fisiologia , Leucina/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Pirofosfatases/metabolismo , Transdução de Sinais/fisiologia , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos/fisiologia , Animais , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Polaridade Celular/fisiologia , Citoplasma/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação/fisiologia , Ratos , Propriedades de Superfície , TransfecçãoRESUMO
Whilst there have been many studies in various species examining the effects of leptin on food intake, there is a paucity of data comparing responsiveness in the two sexes. We have, therefore, addressed this issue in sheep. Because this species shows seasonal variation in voluntary food intake (VFI), we also considered the possibility that there might be seasonal variation in the responsivity to leptin. Centrally administered leptin was relatively ineffective as a satiety factor in either sex during AUTUMN: In Spring, leptin had a profound inhibitory effect on VFI in the females, but only a slight effect in males. These data indicate that responsiveness to leptin depends on sex and also on season in animals that are substantially affected by photoperiod.
Assuntos
Ingestão de Alimentos/efeitos dos fármacos , Leptina/farmacologia , Estações do Ano , Caracteres Sexuais , Animais , Peso Corporal , Feminino , Leptina/administração & dosagem , Masculino , Orquiectomia , Ovariectomia , Fotoperíodo , Saciação/efeitos dos fármacos , OvinosRESUMO
Although existing data suggest an influence of leptin on circulating levels of growth hormone (GH), the action site and properties of leptin are still controversial. Using primary cultured ovine pituitary cells, we studied the direct effect of leptin on the secretion of GH. Pituitary cells were dissociated by collagenase and subjected to Percoll gradient centrifugation to enrich the somatotroph population to 60-80% of cells. Treatment of primary cultured ovine somatotrophs with leptin (10(-9)-10(-7) M) for 30 min did not affect basal, GH-releasing hormone (GHRH) (10(-7) M)- or GH-releasing peptide-2 (GHRP-2)(10(-7) M)-stimulated GH secretion. Following treatment of cells for 1-3 d with leptin, GHRH-stimulated GH secretion was reduced and GHRP-2-stimulated GH secretion increased. The combined effect of GHRH and GHRP-2 on GH secretion was not altered by the treatment of cells with leptin for 3 d. GHRH receptor mRNA levels in cultured somatotrophs were decreased but GHRP receptor mRNA levels were increased by 3-d leptin treatment. These results suggest that leptin has a long-term effect on somatotrophs to reduce GHRH receptor synthesis leading to a decrease in GHRH-stimulated GH secretion. Leptin appears, however, to have an opposite effect on GHRP receptor synthesis leading to an increase in GHRP-stimulated GH secretion.
Assuntos
Hormônio do Crescimento/metabolismo , Leptina/farmacologia , Hipófise/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo , Hormônio do Crescimento/análise , Hormônio do Crescimento/genética , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Técnicas In Vitro , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Hipófise/metabolismo , RNA Mensageiro/análise , Receptores de Neuropeptídeos/análise , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/análise , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Proteínas Recombinantes/farmacologia , Ovinos , Fatores de TempoRESUMO
Leptin can act as a satiety factor and exert neuroendocrine effects, but most studies have been performed in fasted animals. We aimed to determine the effect of chronic under-nutrition on the response to a 3-day intracerebroventricular infusion of leptin with regard to food intake and the secretion of pituitary hormones. Ovariectomised ewes (n=6) had a mean (+/-s.e.m. ) bodyweight of 56+/-0.8 kg on a diet available ad libitum (ad lib) or 33.4+/-1 kg on a restricted diet. The differential bodyweight was achieved by dietary means over a period of 6 months prior to the commencement of the study. Leptin (4 microg/h) or vehicle (artificial cerebrospinal fluid (aCSF)) was infused into the third cerebral ventricle for 3 days. Blood samples were taken prior to commencement and on day 3 of infusion for the assay of plasma hormone levels. The experiment was repeated one week later in a cross-over design. Food intake and metabolic status were monitored daily. The luteinising hormone (LH) pulse amplitude was lower (P<0.05) but plasma growth hormone (GH) levels were higher (P<0.05) in the food-restricted animals. Plasma levels of glucose, lactate, insulin, urea and triglycerides were similar in the two groups but non-esterified fatty acid levels were higher (P<0.01) in the animals on an ad lib diet. Leptin reduced (P<0.05) food intake only in the animals fed an ad lib diet. Leptin increased (P<0.05) the secretion of LH in the food-restricted group only and increased (P<0.05) GH irrespective of bodyweight. In conclusion, leptin does not alter food intake in animals on a restricted diet but can increase the secretion of LH in the same animals. The treatment of leptin was not sufficient to reduce plasma GH levels in the food-restricted animals, suggesting that other factors or mechanisms must be involved in the regulation of this axis.
Assuntos
Hormônio do Crescimento/metabolismo , Leptina/administração & dosagem , Hormônio Luteinizante/metabolismo , Distúrbios Nutricionais/fisiopatologia , Resposta de Saciedade/efeitos dos fármacos , Análise de Variância , Animais , Doença Crônica , Estudos Cross-Over , Ácidos Graxos não Esterificados/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Hormônio Foliculoestimulante/metabolismo , Hormônio do Crescimento/sangue , Injeções Intraventriculares , Leptina/farmacologia , Hormônio Luteinizante/sangue , Ovariectomia , OvinosRESUMO
1. Leptin is secreted by fat and acts on the brain. 2. Central infusion of leptin reduces food intake but does not alter endocrine secretions in normally fed sheep. 3. Leptin treatment can correct for altered hormonal secretion in fasted animals. 4. Alterations in bodyweight (leptin status) affect the expression of a number of genes in the hypothalamus that are involved in the regulation of food intake and neuroendocrine function. 5. Leptin receptors are found in both the hypothalamus and pituitary and direct action of leptin can be demonstrated on the somatotrophs in the pituitary.
Assuntos
Peso Corporal/fisiologia , Proteínas de Transporte/metabolismo , Ingestão de Alimentos/fisiologia , Hipotálamo/metabolismo , Leptina/metabolismo , Receptores de Superfície Celular , Animais , Peso Corporal/efeitos dos fármacos , Proteínas de Transporte/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Hormônio do Crescimento/sangue , Humanos , Hipotálamo/efeitos dos fármacos , Leptina/farmacologia , Neuropeptídeo Y/metabolismo , Receptores para Leptina , OvinosRESUMO
Tissue-nonspecific alkaline phosphatase (TNAP) is essential for bone matrix mineralization, but the central mechanism for TNAP action remains undefined. We observed that ATP-dependent (45)Ca precipitation was decreased in calvarial osteoblast matrix vesicle (MV) fractions from TNAP-/- mice, a model of infantile hypophosphatasia. Because TNAP hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP(i)), we assessed phosphodiesterase nucleotide pyrophosphatase (PDNP/NTPPPH) activity, which hydrolyzes ATP to generate PP(i). Plasma cell membrane glycoprotein-1 (PC-1), but not the isozyme B10 (also called PDNP3) colocalized with TNAP in osteoblast MV fractions and pericellular matrix. PC-1 but not B10 increased MV fraction PP(i) and inhibited (45)Ca precipitation by MVs. TNAP directly antagonized inhibition by PC-1 of MV-mediated (45)Ca precipitation. Furthermore, the PP(i) content of MV fractions was greater in cultured TNAP-/- than TNAP+/+ calvarial osteoblasts. Paradoxically, transfection with wild-type TNAP significantly increased osteoblast MV fraction NTPPPH. Specific activity of NTPPPH also was twofold greater in MV fractions of osteoblasts from TNAP+/+ mice relative to TNAP-/- mice. Thus TNAP attenuates PC-1/NTPPPH-induced PP(i) generation that would otherwise inhibit MV-mediated mineralization. TNAP also paradoxically regulates PC-1 expression and NTPPPH activity in osteoblasts.
Assuntos
Fosfatase Alcalina/metabolismo , Calcificação Fisiológica/fisiologia , Glicoproteínas de Membrana/genética , Osteoblastos/fisiologia , Diester Fosfórico Hidrolases , Células 3T3 , Fosfatase Alcalina/deficiência , Fosfatase Alcalina/genética , Animais , Cálcio/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados , Modelos Animais de Doenças , Matriz Extracelular/fisiologia , Matriz Extracelular/ultraestrutura , Regulação Enzimológica da Expressão Gênica , Genótipo , Hipofosfatasia/enzimologia , Hipofosfatasia/genética , Isoenzimas/deficiência , Isoenzimas/genética , Isoenzimas/metabolismo , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Pirofosfatases/deficiência , Pirofosfatases/genética , Pirofosfatases/metabolismo , Crânio/fisiologia , Transcrição Gênica , TransfecçãoRESUMO
Ecto-enzymes are catalytic membrane proteins with their active sites outside the cell. They include cholinesterase, which inactivates acetylcholine, and angiotensin-converting enzyme, which converts angiotensin I to biologically active angiotensin II, and numerous other peptidases, transpeptidases, nucleotidases, phosphodiesterases, and phosphatases. Many CD antigens of leukocytes are ecto-enzymes; some CD antigens for which no function is currently known are probably ecto-enzymes. Expression is highly regulated and correlated with differentiation and activation. Some are highly restricted in distribution; others are ubiquitous. Many are shared between leukocytes and non-hematogenous cells. Biological functions appear to depend on the type and location of the cell in which expression occurs, and include recycling of nutrients, local control of response to cytokines and hormones, bone formation, cell mobility, invasion, and metastasis. Many novel regulatory functions of ecto-enzymes remain to be discovered, and may reveal new mechanisms of local extracellular control of cellular function.
Assuntos
Antígenos de Diferenciação/metabolismo , Membrana Celular/enzimologia , Hidrolases/metabolismo , Nucleotídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Transferases/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação/química , Antígenos de Diferenciação/genética , Humanos , Hidrolases/química , Hidrolases/genética , Dados de Sequência Molecular , Nucleosídeos/metabolismo , Especificidade por Substrato , Transferases/química , Transferases/genéticaRESUMO
The transferrin receptor contains a highly conserved Arg-Gly-Asp (RGD) sequence in the C-terminal region where transferrin is thought to bind. RGD sequences are commonly involved in cell adhesion. This sequence is crucial for transferrin binding, suggesting possible evolutionary links between molecules mediating iron uptake and cell adhesion.
Assuntos
Sequência de Aminoácidos , Sequência Conservada , Oligopeptídeos , Receptores da Transferrina/metabolismo , Transferrina/metabolismo , Fosfatase Alcalina/genética , Humanos , Dados de Sequência Molecular , Ligação Proteica , Receptores da Transferrina/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
We have studied the effect of leptin on food intake and neuroendocrine function in ovariectomized ewes. Groups (n = 5) received intracerebroventricular infusions of either vehicle or leptin (20 microg/h) for 3 days and were blood sampled over 6 h on days -1, 2, and for 3 h on day 3 relative to the onset of the infusion. The animals were then killed to measure hypothalamic neuropeptide Y expression by in situ hybridization. Plasma samples were assayed for metabolic parameters and pituitary hormones. Food intake was reduced by leptin, but did not change in controls. Leptin treatment elevated plasma lactate and nonesterified fatty acids, but did not affect glucose or insulin levels, indicating a state of negative energy balance that was met by the mobilization of body stores. Pulse analysis showed that the secretion of LH and GH was not affected by leptin treatment, nor were the mean plasma concentrations of FSH, PRL, or cortisol. Expression of messenger RNA for neuropeptide Y in the arcuate nucleus was reduced by the infusion of leptin, primarily due to reduced expression per cell rather than a reduction in the number of cells observed. Thus, the action of leptin to inhibit food intake is dissociated from neuroendocrine function. These results suggest that the metabolic effects of leptin are mediated via neuronal systems that possess leptin receptors rather than via endocrine effects.
Assuntos
Comportamento Alimentar/efeitos dos fármacos , Sistemas Neurossecretores/fisiologia , Obesidade , Ovário/fisiologia , Hormônios Hipofisários/metabolismo , Proteínas/farmacologia , Animais , Apetite/efeitos dos fármacos , Feminino , Humanos , Hidrocortisona/sangue , Hibridização In Situ , Leptina , Neuropeptídeo Y/análise , Ovariectomia , OvinosRESUMO
Many developmentally regulated membrane proteins of lymphocytes are ecto-enzymes, with their active sites on the external surface of the cell. These enzymes commonly have peptidase, phosphodiesterase or nucleotidase activity. Their biological roles are just beginning to be discovered. Although their expression is usually associated with particular stages of lymphoid differentiation, the same gene products are often expressed on the surface of certain non-lymphoid cell types outside the immune system, indicating that their functions cannot be unique to lymphocytes, nor can they be ubiquitous. The plasma cell membrane protein PC-1 (phosphodiesterase I; EC 3.1.4.1/nucleotide pyrophosphatase; EC 3.6.1.9), which was one of the first serological markers for lymphocyte subsets to be discovered, is a typical example. Within the immune system, PC-1 is confined to plasma cells, which represent about 0.1% of lymphocytes. However, PC-1 is also expressed on cells of the distal convoluted tubule of the kidney, chondrocytes, osteoblasts, epididymis and hepatocytes. Recent work has shown that PC-1 is a member of a multigene family of ecto-phosphodiesterases that currently has two other members, PD-1 alpha (autotaxin) and PD-1 beta (B10). Within this family, the extracellular domains are highly conserved, especially around the active site. In contrast, the transmembrane and cytoplasmic domains are highly divergent. Individual members of the eco-phosphodiesterase family have distinct patterns of distribution in different cell types, and even within the same cell. For example, PC-1 is present only on the basolateral surface of hepatocytes, while B10 (PD-1 beta) is confined to the apical surface. Analysis of conservation and differences in the sequence of their cytoplasmic tails may illuminate intracellular targetting signals. Ecto-phosphodiesterases may play a part in diverse activities in different tissues, including recycling of nucleotides. They may also regulate the concentration of pharmacologically active extracellular compounds such as adenosine or its derivatives and cell motility. Some members may modulate local concentrations of pyrophosphate, and hence influence calcification in bone and cartilage.
Assuntos
Linfócitos/enzimologia , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/química , Pirofosfatases/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
We have studied the direct effect of leptin on the secretion of GH from the anterior pituitary gland of the sheep. Using primary cultures of ovine pituitary cells, leptin (10(-9)-10(-7) M) treatment for 30 min did not affect basal or GHRH (10(-7) M)-stimulated GH secretion. Following treatment for 24 h, a dose of 10(-7) M leptin stimulated basal GH secretion. In contrast, doses of 10(-7) and 10(-8) M leptin inhibited GHRH-stimulated GH secretion after 24 h of treatment. These results suggest that leptin can have long-term effects on the somatotropes, but no acute effect. Furthermore, leptin appears to have opposite effects on basal and GHRH-stimulated GH secretion.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/metabolismo , Hormônio do Crescimento/metabolismo , Hipófise/metabolismo , Proteínas/farmacologia , Animais , Células Cultivadas , Leptina , Ovinos , Estimulação QuímicaRESUMO
Phosphodiesterase I (EC 3.1.4.1)/nucleotide pyrophosphatase (EC 3.6.1.9) enzymes are a family of type II transmembrane proteins that catalyze the cleavage of phosphodiester and phosphosulfate bonds of a variety of molecules, including deoxynucleotides, NAD, and nucleotide sugars. The human genes for two members of this family have been cloned and designated PC-1 (PDNP1) and PD-Ialpha/autotaxin (PDNP2). We have now cloned the third member of this family from a human prostate cDNA library and designated it human phosphodiesterase-Ibeta (PD-Ibeta). The PD-Ibeta cDNA contains a 2625-bp-long open reading frame which encodes an 875-amino-acid protein. COS-7 cells transfected with an expression vector, pBK-CMV, containing PD-Ibeta cDNA had high phosphodiesterase I activity compared to the mock-transfected cells. By using in situ hybridization to human metaphase chromosomes, we have assigned the locus for the PD-Ibeta (PDNP3) gene to the q22 region of human chromosome 6.
Assuntos
Diester Fosfórico Hidrolases/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 6/genética , Clonagem Molecular , DNA Complementar/genética , Feminino , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Masculino , Dados de Sequência Molecular , Fosfodiesterase I , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição TecidualRESUMO
OBJECTIVE: To date, mutations in the insulin receptor gene are the only clearly defined cause of extreme insulin resistance in man. Recently, however, some patients with severe insulin resistance have been reported to have marked over-expression of the transmembrane glycoprotein PC-1 in their cultured fibroblasts. This protein appears to act as an endogenous inhibitor of the insulin receptor tyrosine kinase which suggests that primary over-expression of PC-1 may play an aetiological role in some forms of insulin resistance. One sub-type of extreme insulin resistance in which the insulin receptor gene has been reported to be normal is pseudo-acromegalic insulin resistance. The main aim of this study was to determine whether overexpression of PC-1 might contribute to the severe insulin resistance exhibited by some patients with pseudo-acromegaly. DESIGN AND PATIENTS PC-1 phosphodiesterase activity and PC-1 protein and mRNA content were measured in cultured dermal fibroblast from three severely insulin resistant pseudo-acromegalic patients. These were compared with fibroblasts from normoinsulinaemic normoglycaemic controls and from subjects with known genetic defects in the insulin receptor or IRS-1. RESULTS: In the fibroblasts from pseudo-acromegalic insulin resistant subjects PC-1 activity and PC-1 protein and mRNA levels were indistinguishable from the normoinsulinaemic controls. Consistent with this observation, insulin receptor tyrosine kinase activity was similar in extracts from fibroblasts of pseudo-acromegalic subjects and normal controls. Surprisingly, subjects with insulin receptor or IRS-1 mutations had a profound reduction in PC-1 activity (p < or = 0.005), protein (p < or = 0.05) and mRNA levels (P < or = 0.005). CONCLUSIONS: The results indicate that PC-1 over-expression does not appear to contribute to the insulin resistant state of pseudo-acromegalic patients. The finding of normal insulin receptor tyrosine kinase activity in these subjects suggests that the site of defective insulin signalling is likely to be distal to the receptor. The unexpected finding that PC-1 activity, protein and mRNA were all dramatically reduced in patients with lesions early in the insulin signalling cascade provides further evidence for a link, albeit as yet poorly understood, between cellular insulin action and the expression of PC-1.
Assuntos
Fibroblastos/metabolismo , Resistência à Insulina/fisiologia , Glicoproteínas de Membrana/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases , Receptor de Insulina/antagonistas & inibidores , Pele/metabolismo , Células Cultivadas , Humanos , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Fosforilação , RNA Mensageiro/análise , SíndromeRESUMO
We have identified B10, a plasma membrane protein previously defined by a monoclonal antibody, as an alkaline phosphodiesterase I (APDE) expressed in the plasma membrane of rat hepatocytes and enterocytes, with a restricted apical distribution. B10 complementary DNA (cDNA) was cloned from a rat intestinal library screened with a polyclonal antibody directed to the hepatic protein. Two distinct B10 clones with an open reading frame of 2,625 bp were obtained that differed only by 12 bases in the coding region. One B10 clone had a single base difference with gp130RB13-6 cDNA, which was recently cloned in rat fetal brain. B10/gp130RB13-6 had 50% identity at the amino acid level with the plasma cell antigen PC-1, an APDE cloned in the mouse and in human. Anti-B10 antibodies immunoprecipitated 34% of the APDE activity in liver plasma membranes and over 95% of the APDE activity in intestinal cells. Most of the remaining activity in hepatocytes (44%) could be immunoprecipitated by antibodies directed to PC-1. APDE activity immunoprecipitated with anti-B10 antibodies was found in the apical rat liver plasma membrane fractions on a sucrose gradient whereas most of the remaining APDE activity was associated with the basolateral fractions, which contained PC-1. By immunofluorescence, B10 was localized to the apical surfaces of hepatocytes and enterocytes whereas PC-1 was present on the basolateral surfaces of hepatocytes. B10/gp130RB13-6 and rat PC-1 are a unique example of distinct molecules having similar enzymatic activity but different apical/basolateral location, and possibly different functions.