RESUMO
Wolfram syndrome (WFS; OMIM 222300) is an autosomal recessive neurodegenerative disorder defined by young-onset non-immune insulin-dependent diabetes mellitus and progressive optic atrophy. Linkage to markers on chromosome 4p was confirmed in five families. On the basis of meiotic recombinants and disease-associated haplotypes, the WFS gene was localized to a BAC/P1 contig of less than 250 kb. Mutations in a novel gene (WFS1) encoding a putative transmembrane protein were found in all affected individuals in six WFS families, and these mutations were associated with the disease phenotype. WFS1 appears to function in survival of islet beta-cells and neurons.
Assuntos
Cromossomos Humanos Par 4 , Proteínas de Membrana/genética , Mutação , Síndrome de Wolfram/genética , Adulto , Sequência de Bases , Criança , Clonagem Molecular , Feminino , Ligação Genética , Haplótipos , Humanos , Masculino , Repetições de Microssatélites , Dados de Sequência Molecular , Linhagem , Polimorfismo GenéticoRESUMO
AIMS/HYPOTHESIS: Glycogen synthase kinase 3ß (GSK-3ß) is an enzyme that is suppressed by insulin and when elevated results in insulin resistance in skeletal muscle and diabetes. Its role in beta cell development and function is little known. Because of the enzyme's anti-proliferative and pro-apoptotic properties, the hypothesis to be tested here was that beta cell specific deficiency of GSK-3ß in mice would result in enhanced beta cell mass and function. METHODS: Mice with beta cell deficiency of GSK-3ß (ß-Gsk-3ß [also known as Gsk3b](-/-)) were generated by breeding Gsk-3ß (flox/flox) mice with mice overexpressing the Cre recombinase gene under the control of the rat insulin 2 gene promoter (RIP-Cre mice), and glucose tolerance, insulin secretion, islet mass, proliferation and apoptosis were measured. Changes in islet proteins were investigated by western blotting. RESULTS: On a normal diet ß-Gsk-3ß ( -/- ) mice were found to have mild improvement of glucose tolerance and glucose-induced insulin secretion, and increased beta cell mass accompanied by increased proliferation and decreased apoptosis. On a high-fat diet ß-Gsk-3ß (-/-) mice exhibited improved glucose tolerance and expanded beta cell mass with increased proliferation relative to that in control mice, resisting fat-fed diabetes. Molecular mechanisms accounting for these phenotypic changes included increased levels of islet IRS1 and IRS2 proteins and phospho-Akt, suggesting enhanced signalling through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and increased islet levels of pancreas/duodenum homeobox protein 1 (PDX1). Inhibition of GSK3 in MIN6 cells in vitro led to increased IRS1 and IRS2 protein levels through inhibition of proteosomal degradation. CONCLUSIONS/INTERPRETATION: These results are consistent with a mechanism whereby endogenous GSK-3ß activity controls islet beta cell growth by feedback inhibition of the insulin receptor/PI3K/Akt signalling pathway.
Assuntos
Diabetes Mellitus Experimental/etiologia , Gorduras na Dieta/efeitos adversos , Resistência a Medicamentos/genética , Quinase 3 da Glicogênio Sintase/genética , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/crescimento & desenvolvimento , Animais , Células Cultivadas , Diabetes Mellitus Experimental/genética , Dieta Aterogênica , Ingestão de Alimentos/fisiologia , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Tamanho do Órgão/genética , Especificidade de Órgãos/genética , Ratos , Regulação para Cima/genéticaRESUMO
Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple the cellular metabolic state to electrical activity and are a critical link between blood glucose concentration and pancreatic insulin secretion. A mutation in the second nucleotide-binding fold (NBF2) of the sulfonylurea receptor (SUR) of an individual diagnosed with persistent hyperinsulinemic hypoglycemia of infancy generated KATP channels that could be opened by diazoxide but not in response to metabolic inhibition. The hamster SUR, containing the analogous mutation, had normal ATP sensitivity, but unlike wild-type channels, inhibition by ATP was not antagonized by adenosine diphosphate (ADP). Additional mutations in NBF2 resulted in the same phenotype, whereas an equivalent mutation in NBF1 showed normal sensitivity to MgADP. Thus, by binding to SUR NBF2 and antagonizing ATP inhibition of KATP++ channels, intracellular MgADP may regulate insulin secretion.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Insulina/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Receptores de Droga/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular Transformada , Chlorocebus aethiops , Cricetinae , Diazóxido/farmacologia , Humanos , Hiperinsulinismo/genética , Hipoglicemia/genética , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Mutação Puntual , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/genética , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/genética , Rubídio/metabolismo , Receptores de Sulfonilureias , TransfecçãoAssuntos
Diabetes Mellitus Tipo 2/genética , Animais , Mapeamento Cromossômico , Humanos , Ratos , Ratos MutantesRESUMO
The purpose of these experiments was to determine whether alterations in preproinsulin messenger (m)RNA activity could account for changes in insulin biosynthesis during fasting and refeeding. Rats were fasted 4 d and then fed for 6, 8, 24, or 48 h. With fasting, body weight decreased 25%, plasma glucose decreased from 6.1 to 2.2 mM, and pancreatic insulin content fell to 40% that of fed animals. Islet RNA decreased to 50% and protein to 55% that of control animals, while islet DNA content remained unchanged. After 6 h of refeeding, islet RNA content increased and was not significantly different from controls. Total islet and preproinsulin mRNA activity was estimated with an mRNA-dependent wheat germ cell-free protein synthesizing system. Preproinsulin and total protein synthesis was linearly dependent upon added RNA at concentrations up to 3 mug. Preproinsulin was identified by its mobility on SDS polyacrylamide gel electrophoresis and by hybrid arrested translation of preproinsulin mRNA. After an 18-h fast, islet mRNA activity decreased 33%; after 4 d mRNA activity decreased to 66% below that of control fed animals. Preproinsulin mRNA activity was decreased, but to a lesser extent, accounting for 20% of total islet protein in fed animals and 46% in the 4-d fasted animals. Total mRNA activity returned to control values after 8 h of refeeding and increased to 150% of controls at 24 and 48 h. Preproinsulin mRNA activity increased more rapidly on refeeding. By 8 h it was 160% of controls.To determine whether changes in preproinsulin mRNA activity were associated with changes in the amount of preproinsulin mRNA, nucleic acid hybridization analysis was performed. Pancreatic RNA from fed and fasted animals was electrophoresed on agarose gels, transferred to diazophenylthio paper, and hybridized to (32)P-labeled preproinsulin complementary (c)-DNA. This analysis demonstrated that changes in mRNA activity were associated with changes in the amount of hybridizable mRNA present. These studies are the first to demonstrate alterations of preproinsulin mRNA under any conditions, and the changes correlate with alterations in rates of insulin biosynthesis.
Assuntos
Alimentos , Proinsulina/metabolismo , Precursores de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Inanição/metabolismo , Animais , DNA/metabolismo , Insulina , Ilhotas Pancreáticas/metabolismo , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , Proteínas/metabolismo , RNA/metabolismo , RatosRESUMO
These studies of partial pancreatectomy assess pancreatic proinsulin messenger RNA (mRNA) levels as an index of in vivo insulin biosynthesis, and show relationships to glucose homeostasis. Rats were subjected to sham operation, 50% pancreatectomy (Px), or 90% Px, and were examined after 1, 3, or 14 wk. Proinsulin mRNA was measured by dot hybridization to complementary DNA. After 50% Px there was a nearly complete adaptation of proinsulin mRNA. After 90% Px a marked increase of proinsulin mRNA occurred, but it was insufficient and it was not maintained with time. The deficit in insulin production is related to development of hyperglycemia. Sham-operated controls showed no worsening of fasting or fed blood glucose or of intraperitoneal glucose tolerance within the period of observation. Total proinsulin mRNA and pancreatic insulin content rose in proportion to body weight. 50% Px produced no change from controls in body weight or blood glucose. The concentration of proinsulin mRNA in the 50% pancreatic remnant paralleled that of controls after 1 and 3 wk, but then increased after 14 wk, such that total proinsulin mRNA approached control levels. This adaptive response was reflected by changes in serum insulin, but not by pancreatic insulin content, which was only 30% of control after 14 wk. Intraperitoneal glucose tolerance was impaired mildly, and did not worsen with time after pancreatectomy. 90% Px led to elevated fed blood glucose and reduced serum insulin after 3 wk, and fasting hyperglycemia was seen after 14 wk. Proinsulin mRNA concentration in the 10% pancreatic remnant showed an adaptive increase after 1 and 3 wk, such that total proinsulin mRNA reached 40% of control. After 14 wk, however, remnant proinsulin mRNA concentration was no longer increased; total proinsulin mRNA and pancreatic insulin content were severely reduced. Intraperitoneal glucose tolerance was impaired more dramatically than with the 50% Px animals, and worsened with time after operation. These observations indicate ability to increase proinsulin mRNA levels as an adaptation to pancreatectomy. Insufficiency of this adaptation is associated with the development of hyperglycemia, and the loss of this adaptation correlates with a worsening of glucose tolerance.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Proinsulina/biossíntese , Fatores Etários , Animais , Peso Corporal , Insulina/sangue , Masculino , Tamanho do Órgão , Pâncreas/anatomia & histologia , Pancreatectomia , RNA Mensageiro/metabolismo , Ratos , Ratos EndogâmicosRESUMO
In the present study we examined mRNA and protein levels for the muscle/adipose tissue glucose transporter (GLUT-4) in various tissues of spontaneously obese mice (C57BL/KsJ, db/db) and their lean littermates (db/+). Obese (db/db) mice were studied at 5 wk of age, when they were rapidly gaining weight and were severely insulin resistant, evidenced by hyperglycemia (plasma glucose 683 +/- 60 vs. 169 +/- 4 mg/dl in db/+, P less than 0.05) and hyperinsulinemia (plasma insulin 14.9 +/- 0.53 vs. 1.52 +/- 0.08 ng/ml in db/+, P less than 0.05). The GLUT-4 mRNA was reduced in quadriceps muscle (67.5 +/- 8.5%, P = 0.02), but unaltered in adipose tissue (120 +/- 19%, NS), heart (95.7 +/- 6.1%, NS), or diaphragm (75.2 +/- 12.1%, NS) in obese (db/db) mice relative to levels in lean littermates. The GLUT-4 protein, measured by quantitative immunoblot analysis using two different GLUT-4 specific antibodies, was not different in five insulin-sensitive tissues including diaphragm, heart, red and white quadriceps muscle, and adipose tissue of obese (db/db) mice compared with tissue levels in lean littermates; these findings were consistent when measured relative to tissue DNA levels as an index of cell number. These data suggest that the marked defect in glucose utilization previously described in skeletal muscle of these young obese mice is not due to a decrease in the level of the major muscle glucose transporter. An alternate step in insulin-dependent activation of the glucose transport process is probably involved.
Assuntos
Resistência à Insulina , Proteínas de Transporte de Monossacarídeos/análise , Obesidade/metabolismo , Tecido Adiposo/análise , Animais , DNA/análise , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Camundongos , Camundongos Obesos , Proteínas de Transporte de Monossacarídeos/genética , Músculos/análise , RNA Mensageiro/análiseRESUMO
The phosphoinositide 3-kinase-Akt/PKB pathway mediates the mitogenic effects various nutrients and growth factors in cultured cells. To study its effects in vivo in pancreatic islet beta cells, we created transgenic mice that expressed a constitutively active Akt1/PKB alpha linked to an Insulin gene promoter. Transgenic mice exhibited a grossly visible increase in islet mass, largely due to proliferation of insulin-containing beta cells. Morphometric analysis verified a six-fold increase in beta cell mass/pancreas, a two-fold increase in 5-bromo-2'-deoxyuridine incorporation, a four-fold increase in the number of beta cells per pancreas area, and a two-fold increase in cell size in transgenic compared with wild-type mice at 5 weeks. At least part of the increase in beta cell number may be accounted for by neogenesis, defined by criteria that include beta cells proliferating from ductular epithelium, and by a six-fold increase in the number of single and doublet beta cells scattered throughout the exocrine pancreas of the transgenic mice. Glucose tolerance was improved, and fasting as well as fed insulin was greater compared with wild-type mice. Glucose-stimulated insulin secretion was maintained in transgenic mice, which were resistant to streptozotocin-induced diabetes. We conclude that activation of the Akt1/PKB alpha pathway affects islet beta cell mass by alteration of size and number.
Assuntos
Hiperinsulinismo/etiologia , Pâncreas/patologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas , Animais , Glicemia/metabolismo , Divisão Celular , Hiperplasia , Hipertrofia , Insulina/metabolismo , Secreção de Insulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Pâncreas/metabolismo , Proteínas Proto-Oncogênicas c-aktRESUMO
Previous studies have suggested that alteration in the expression of the insulin-regulatable glucose transporter of muscle (GLUT-4 protein) may be an important determinant of insulin action. In the present studies, we have examined GLUT-4 mRNA and protein concentrations in muscle after variations in the metabolic status of the intact animal (i.e., 7 d streptozotocin-induced diabetes, 7 d insulin-induced hypoglycemia, and 3 d fasting). These changes in glucose homeostasis were associated with the following changes in GLUT-4 gene products: a decrease of approximately 30% in both mRNA and protein with diabetes; a 50% increase in mRNA and a 2.4-fold increase in protein with insulin injection; and normal mRNA in spite of a 2.7-fold increase in protein with fasting. Fasted diabetics exhibited an increase of 50% in GLUT-4 mRNA and a 2.4-fold increase in protein relative to fed diabetics. In diabetic and insulin-injected groups, the changes in GLUT-4 protein were similar to changes in mRNA, but in fasting, GLUT-4 protein increased without a concomitant change in mRNA. Overall there was no correlation between muscle concentrations of GLUT-4 protein and mRNA. Muscle GLUT-4 protein concentration tended to correlate with plasma glucose (r = -0.57, P less than 0.001), but not with plasma insulin. These results indicate that (a) chronic changes in glucose homeostasis are associated with changes in expression of GLUT-4 protein in muscle; (b) GLUT-4 protein increased in fasted soleus muscle without change in mRNA, thereby differing from fasted adipocytes in which both GLUT-4 products diminish; and (c) no simple relationship exists between total muscle GLUT-4 protein content and whole-body insulin sensitivity.
Assuntos
Glicemia/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Músculos/fisiologia , Animais , Western Blotting , Jejum , Expressão Gênica , Homeostase , Hipoglicemia/fisiopatologia , Masculino , Proteínas de Transporte de Monossacarídeos/genética , RNA Mensageiro/genética , Ratos , Ratos EndogâmicosRESUMO
To examine the possible involvement of insulin and glucose in regulation of pancreatic islet gene expression, hyperinsulinemic (insulin infusion 4.1 mU/kg per min) clamps were performed for 12 h in rats at two different levels of glycemia (either 3 or 8 mM). A control group received a saline infusion for 12 h. At the end of the 12-h study period, pancreatic RNA was extracted, proinsulin and amylin mRNAs were measured on total RNA, and glucokinase and glucose transporter (GLUT-2) mRNAs were measured on poly(A)+ RNA by dot blot analysis. In insulin-infused hypoglycemic rats, there was a 58% decrement in proinsulin mRNA (P less than 0.01) relative to levels in controls, with no change in amylin, glucokinase, or islet GLUT-2 mRNAs. In insulin-infused hyperglycemic rats, there was a comparable decrement (44%, P less than 0.01) in proinsulin mRNA and a smaller decrement in GLUT-2 mRNA (32%, P less than 0.05), with no change in amylin or glucokinase mRNAs relative to levels in control animals. These studies suggest that insulin has a negative feedback inhibitory effect on its own synthesis. The mechanism of inhibition is unknown. It could be a direct effect of insulin on its own transcription, or alternatively an indirect effect mediated by humoral or neural factors. Sustained hyperinsulinemia may lead to suppression of normal islet beta cells and may contribute to the ultimate hypoinsulinemia of noninsulin-dependent diabetes mellitus.
Assuntos
Expressão Gênica/efeitos dos fármacos , Insulina/farmacologia , Amiloide/genética , Animais , Sequência de Bases , Retroalimentação , Insulina/sangue , Insulina/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Ilhotas Pancreáticas/metabolismo , Masculino , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , RNA Mensageiro/análise , Ratos , Ratos EndogâmicosRESUMO
To test the hypothesis that alterations in regulatory regions of the insulin gene occur in a subset of patients with non-insulin-dependent diabetes mellitus (NIDDM), the promoter region was studied by polymerase chain reaction (PCR) amplification directly from genomic DNA, followed by high-resolution polyacrylamide gel electrophoresis under nondenaturing conditions. By using this method a previously identified HincII polymorphism (GTTGAC to GTTGAG at position-56) in American Blacks was readily detected, indicating that single base changes could be observed. In the course of screening the insulin promoter from 40 American Black subjects with NIDDM, an apparent larger allele was found in two individuals. Both patients were shown to have in addition to a normal allele, a larger allele containing an 8-bp repeat, TGGTCTAA from positions -322 to -315 of the insulin promoter. To facilitate rapid screening for the 8-bp repeat, a high-resolution agarose gel electrophoretic analysis was adopted. DNA from American Black NIDDM subjects (n = 100) and nondiabetic subjects (n = 100) was PCR amplified and analyzed. The 8-bp repeat was present in five NIDDM subjects, and one nondiabetic subject. DNA from Mauritius Creoles, also of African ancestry, was analyzed, and the 8-bp repeat was present in 3 of 41 NIDDM subjects, and 0 of 41 nondiabetic subjects. Analysis of glucose metabolism in three presumed normal sibs of an NIDDM patient with an 8-bp repeat revealed that one sib had overt diabetes, and two sibs were glucose intolerant, but there was no consistent segregation of the insulin promoter variant with the diabetes phenotype. The variant promoter was not present in 35 Caucasian NIDDM patients or in 40 Pima Indians. To test the biological consequences of the 8-bp repeat sequence in the insulin promoter, a normal and variant promoter were subcloned into a luciferase plasmid, and reporter gene activity assessed by transient transfection into mouse insulinoma (beta TC1) and hamster insulinoma (HIT) cells. The promoter activity of the variant allele was found to be reduced to 37.9 +/- 10.3% of the activity of the normal promoter in HIT cells (P less than 0.01, n = 4), and 49.1 +/- 6.4% in beta TC1 cells (P less than 0.01, n = 6). These data thus suggest that a naturally occurring variant of the insulin promoter may contribute to the diabetes phenotype in 5-6% of Black NIDDM patients.
Assuntos
Diabetes Mellitus Tipo 2/genética , Insulina/genética , Alelos , Sequência de Bases , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida/métodos , Frequência do Gene , Humanos , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , Regiões Promotoras GenéticasRESUMO
The purpose of these experiments was to estimate insulin biosynthesis in vivo in a rat model for non-insulin-dependent diabetes. Insulin biosynthesis rates were determined in 4-wk-old animals that had been injected with 90 mg/kg of streptozotocin 2 d postpartum. Control and diabetic animals did not differ in body weight or fasting plasma glucose. Fed plasma glucose was significantly elevated (186 +/- 13 micrograms/dl vs. 139 +/- 7 mg/dl, P less than 0.05) and pancreatic insulin content was reduced (41 +/- 2 micrograms/g vs. 63 +/- 8 micrograms/g, P less than 0.05) in the diabetic rats. Insulin biosynthesis was estimated in vivo by measuring and comparing [3H]leucine incorporation into proinsulin with that into total pancreatic protein 45 min after injection. Insulin biosynthesis was 0.391 +/- 0.07% of pancreas protein synthesized in control rats and 0.188 +/- 0.015% (P less than 0.05) in diabetic rats. In animals of the same age, the fractional and absolute rate of pancreatic protein synthesis were determined. Total pancreatic protein synthesis was not reduced in streptozotocin treated animals (185.5 +/- 14.1%/d vs. 158.6 +/- 14.9%/d, NS) but was markedly reduced in control rats after a 48-h fast (to 70.8 +/- 5.5%/d, P less than 0.01). Because total pancreatic protein synthesis was not decreased in the diabetic rats, the decrease in the fraction of radiolabel incorporated into insulin seems to represent an absolute decrease in the rate of insulin biosynthesis in this animal model for diabetes. Through RNA blot hybridization with 32P-labeled cloned rat insulin complementary DNA, proinsulin messenger RNA (mRNA) was estimated as the rate of insulin biosynthesis in control and diabetic animals. There was a 61% reduction in proinsulin mRNA at 4 wk and an 85% reduction at 7 wk (P less than 0.001) in the diabetic animals. After streptozotocin injection in neonatal rats, there is marked beta-cell damage and hyperglycemia. Beta-cell regeneration occurs with return to normoglycemia, but with age hyperglycemia develops. The reduction in insulin synthesis and proinsulin mRNA seemed disproportionate with the more modest reduction in beta-cell number. The importance of these observations is that, in this animal model, diabetes is associated with a limited ability to regenerate beta-cell mass and to synthesize insulin. The relationship between the defect in glucose-stimulated insulin release and impaired insulin biosynthesis has yet to be determined.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Insulina/biossíntese , Pâncreas/metabolismo , Animais , Glicemia , Diabetes Mellitus Tipo 2/metabolismo , Proinsulina/biossíntese , Ratos , Ratos Endogâmicos , EstreptozocinaRESUMO
Variations in DNA sequences flanking the insulin gene were studied in relation to noninsulin-dependent diabetes mellitus (NIDDM) in 87 unrelated Pima Indians at least 35 yr of age. DNA was isolated from nuclei of peripheral blood leukocytes and digested with restriction endonucleases. Less variation in this region was found in Pima Indians than in other racial groups previously studied. Only two classes of alleles (classes 1 and 3) were found, and there was virtually no variation within classes. At least one class 3 allele was found in 47% of the 38 nondiabetic subjects and in 37% of the 49 with NIDDM (odds ratio = 0.65, P = 0.4, 95% confidence interval for the odds ratio = 0.25 to 1.67). Homozygosity for class 3 alleles, however, was found only in diabetics. There were no differences according to genotype in obesity, fasting or postload glucose or insulin concentrations, or in the relationships between insulin and glucose concentrations. 61% (11/18) of the diabetics with a class 3 allele were receiving drug treatment for diabetes compared with only 26% (8/31) of diabetics without a class 3 allele (P = 0.03). The insulin gene polymorphism probably plays no important role in the genesis of NIDDM in Pima Indians, nor does it influence the glucose or insulin concentrations or their relationship to each other, but the class 3 allele, especially when homozygous in this population, may influence the severity of the disease as indicated by need for drug treatment.
Assuntos
Glicemia/análise , Diabetes Mellitus Tipo 2/genética , Indígenas Norte-Americanos , Insulina/sangue , Insulina/genética , Polimorfismo Genético , Adulto , Alelos , Arizona , Sequência de Bases , DNA/análise , Diabetes Mellitus Tipo 2/tratamento farmacológico , Genótipo , Homozigoto , HumanosRESUMO
Reactive hypoglycemia was documented in ten postgastrectomy patients by a control oral glucose tolerance test (OGTT). Nine patients experienced nausea, flushing, and fatigue during the first hour of the test. Neuroglycopenic or adrenergic symptoms of hypoglycemia occurred in eight patients two to five hours after oral glucose. The oral administration of phenylephrine elixir, 15 mg., thirty minutes before a repeat OGTT, significantly raised thelowest plasma glucose from 37.5 +/- 2.8 mg./dl. to 45.2 +/- 3.8 mg./dl. (p less than 0.05) but did not affect the occurrence of either the early or the late symptoms. In contrast, propranolol, 10 mg., raised the lowest plasma glucose from 37.5 +/- 2.8 mg./dl. to 57 +/- 5.2 mg./dl. (p less than 0.02) and prevented the occurrence of early and late symptoms. Neither peak nor total plasma insulin levels were affected by either drug. The rate of glucose utilization, as determined by intravenous glucose tolerance tests, did not significantly change after the oral administration of either drug. It is concluded that propranolol ameliorated the symptoms and chemical hypoglycemia after oral glucose and merits more detailed study as a long-term therapy for this disorder.
Assuntos
Hipoglicemia/tratamento farmacológico , Fenilefrina/uso terapêutico , Síndromes Pós-Gastrectomia/etiologia , Propranolol/uso terapêutico , Adulto , Idoso , Glicemia/metabolismo , Fadiga/etiologia , Feminino , Glucose/metabolismo , Teste de Tolerância a Glucose , Humanos , Hipoglicemia/etiologia , Insulina/sangue , Masculino , Pessoa de Meia-Idade , Náusea/etiologiaRESUMO
These studies compared measurements of in vivo insulin secretion, insulin stores, and insulin synthesis. Rats were studied at 24 wk of age, either 1 or 20 wk after a sham operation (Sham) or 50% pancreatectomy (Px), reducing beta-cell number. By 20 wk after surgery, an adaptation to pancreatectomy was apparent from results of serial glucose tolerance tests, done in a preliminary protocol. Some of the rats also received dexamethasone (ShamDex and PxDex, respectively), imposing insulin resistance. Insulin secretion was assessed with the acute insulin response to arginine under basal (AIRbas) and maximum glucose-potentiated (AIRmax) states. Pancreatic insulin was measured, and insulin synthesis was estimated by measurement of proinsulin mRNA. At 1 wk after surgery, there was no difference among Sham and Px rats in AIRbas, but in the Px rats, expected reductions of AIRmax, pancreatic insulin, and proinsulin mRNA were found. ShamDex rats had a markedly augmented AIRbas and increased AIRmax and proinsulin mRNA. However, pancreatic insulin was reduced both in ShamDex and PxDex rats. At 20 wk after surgery, the predicted adaptation to Px was substantiated by AIRmax and proinsulin mRNA in Px rats not different from those in Sham rats, but pancreatic insulin in the Px rats remained low. AIRbas and proinsulin mRNA were augmented in ShamDex and PxDex rats, but pancreatic insulin was again reduced, and in PxDex rats, low AIRmax and fed hyperglycemia were seen. Linear correlations of AIRbas and AIRmax with proinsulin mRNA were observed over a roughly fourfold range of secretion and synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Insulina/metabolismo , Pâncreas/química , Proinsulina/genética , RNA Mensageiro/metabolismo , Adaptação Fisiológica , Animais , Arginina/farmacologia , Glicemia/análise , Glicemia/metabolismo , Peso Corporal , Dexametasona/farmacologia , Teste de Tolerância a Glucose , Insulina/análise , Insulina/sangue , Secreção de Insulina , Masculino , Tamanho do Órgão , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatectomia , Proinsulina/metabolismo , RNA Mensageiro/genética , Ratos , Ratos EndogâmicosRESUMO
C57BL/KsJ db/db mice develop hyperphagic obesity and nonketotic diabetes similar to non-insulin-dependent diabetes mellitus in humans. Initially the mice demonstrate an abundant beta-cell mass and hyperinsulinemia, which is followed by apparent beta-cell loss. As an index of insulin synthesis, this study assesses pancreatic proinsulin mRNA, measured by dot hybridization to cloned cDNA, during the development of diabetes in the mice. Changes in proinsulin mRNA from 5 to 13 wk of age are compared with serum insulin, pancreatic insulin content, and blood glucose. In control (+/db) mice, total proinsulin mRNA and pancreatic insulin content increased with age. Both changes were proportional to an increase in body weight. Obesity, hyperglycemia, and hyperinsulinemia were evident in diabetic (db/db) mice at 5 wk of age. Although pancreatic insulin content was comparable to that in the +/db controls at 5 wk, a fourfold relative elevation of proinsulin mRNA was observed. Despite an increase in body weight, proinsulin mRNA concentration and total proinsulin mRNA fell to levels similar to those of the control mice at 10 and 13 wk, associated with a loss of hyperinsulinemia, a mild decrease in pancreatic insulin content, and a marked increased in fasting blood glucose. A separate group of db/db mice was pair fed with the +/db controls from 4 to 13 wk. These diet-restricted diabetic mice were heavier than control mice and gained weight with age, but they weighed less than the unrestricted mice at all ages. Compared with the unrestricted db/db mice, a more modest fasting hyperglycemia was apparent, and a persistent hyperinsulinemia was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ilhotas Pancreáticas/análise , Camundongos Obesos/genética , Proinsulina/genética , RNA Mensageiro/análise , Animais , Glicemia/análise , Peso Corporal , Diabetes Mellitus Experimental/genética , Hiperglicemia/genética , Insulina/análise , Insulina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL/genética , Obesidade/genéticaRESUMO
Glucokinase, the major enzyme that phosphorylates glucose upon entry into liver and islet beta-cells, has been considered a prime candidate for inherited defects predisposing to NIDDM. Now that the human gene has been isolated, this question has been addressed directly. Polymorphic markers flanking the gene were identified. These markers (microsatellites) are composed of variable numbers of dinucleotide repeats that vary in size, resulting in different alleles. Variably sized alleles can be typed rapidly from genomic DNA of individuals by the PCR. Studies of inheritance of glucokinase genes have revealed significant linkage in families with early-onset NIDDM, or MODY, and mutations have been identified within the coding region of the gene in some families. These studies are extremely encouraging, as they indicate that genes can be identified even in this heterogeneous genetic disorder. This study considers the phenotypes that result from glucokinase defects and the relationship of MODY to NIDDM, and it estimates the role of glucokinase defects in NIDDM in general.
Assuntos
Diabetes Mellitus Tipo 2/genética , Glucoquinase/genética , Fatores Etários , Animais , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/enzimologia , Genes , Ligação Genética , Glucoquinase/metabolismo , Humanos , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido NucleicoRESUMO
Polymorphic sites adjacent to known genes can be used to examine the segregation of a disease relative to that gene in families, or to map the gene of interest relative to other loci. The polymorphic region 5' to the human insulin gene (5' FP) permits such analysis, but the three size classes previously identified are insufficient for many studies. More alleles are identified with restriction enzymes that generate small fragments (Pvu II). Nonetheless, sufficient polymorphism for informative family analyses is often not present. To facilitate such analyses, we searched for other polymorphisms in over 20 KB of DNA at the insulin locus in Pima Indians, American Blacks, and Caucasians. The previously described allelic variant at a Pst I site in the 3'-untranslated portion of the gene was not polymorphic in any race. An upstream Hinc II site (-62 BP) was present in only 48% of Black alleles, but was not polymorphic in Pima Indians or Caucasians. New polymorphisms were found at a Taq I site (-11,000 BP) and a Rsa I site (-13,000 BP). The Taq I site was present in 89% of Black alleles, 87% of Pima Indian alleles, and 84% of Caucasian alleles. In contrast, the Rsa I site was present in 60% of Black and Caucasian alleles, but in only 47% of Pima Indian alleles. The Hinc II, Rsa I, and Taq I sites show no obvious linkage with each other or the 5' FP. A fourth polymorphism, previously identified with Sac I, was found to be the creation of a new Sac I site at +2500 BP in 10% of Black alleles.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Insulina/genética , Polimorfismo Genético , Alelos , Povo Asiático , População Negra , DNA/genética , Enzimas de Restrição do DNA , Heterozigoto , Homozigoto , Humanos , Indígenas Norte-Americanos , Linhagem , População BrancaRESUMO
To determine whether a structural defect in glucokinase could be a primary cause of glucose intolerance in the common form of NIDDM, the prevalence of mutations in the gene in 60 American black NIDDM patients was investigated. First, by Southern blot analysis of DNA from a subset of randomly selected subjects (n = 20), no gross deletions, insertions, or rearrangements of the gene were detected. Next, the 5'-untranslated and coding regions of the gene were amplified directly from genomic DNA by the polymerase chain reaction. PCR products were screened for mutations by using single-strand conformational polymorphism analysis. A total of nine variants were identified, with two in the 5'-UT regions of islet exon 1, two in the 5'-UT region of liver exon 1, and five in the coding regions. For islet exon 1, 5 of 60 NIDDM patients had both variants in the 5'-UT region; and for liver exon 1, two variants each occurred in 1 of 60 NIDDM patients. The coding region variants included a missense mutation in islet exon 1, substitution of Ala11 (GCC) with Thr11 (ACC), found in 2 patients. The biological consequences of this mutation and the mutations in the 5'-UT portion of the gene have yet to be determined. The rest of the variants were third base pair changes of codons, i.e., silent. A common polymorphism, which was in linkage equilibrium with microsatellite repeats GCK1 and GCK2, was found in intron 9, and a variant in intron 2 in both alleles of 1 patient.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Diabetes Mellitus Tipo 2/genética , Variação Genética , Glucoquinase/genética , Ilhotas Pancreáticas/enzimologia , Fígado/enzimologia , Alelos , Sequência de Aminoácidos , Sequência de Bases , Diabetes Mellitus Tipo 2/enzimologia , Éxons , Frequência do Gene , Ligação Genética , Humanos , Íntrons , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , Sequências Repetitivas de Ácido NucleicoRESUMO
The purpose of these studies was to determine whether glucose, the principal regulator of insulin biosynthesis in mammals, controls synthesis through alterations in levels of proinsulin mRNA in whole animals. Rats were starved for 3 days and then either refed or injected with glucose or saline for 24 h. Glucose injection raised plasma glucose levels equivalent to levels seen with refeeding but provided less than 20% of caloric replacement. Pancreatic RNA was extracted and the relative concentration of proinsulin mRNA was determined by blot hybridization with a cloned rat proinsulin cDNA probe. In starved animals proinsulin mRNA levels were 15-20% that of fed controls. Glucose injection produced a specific three- to fourfold increase in proinsulin mRNA levels relative to total pancreatic RNA, within 24 h. The effect was measurable 2 h after glucose injection and appeared largely complete by 12 h. Actinomycin D blocked the glucose-induced increase in proinsulin mRNA. These studies demonstrate effects of changes of plasma glucose on levels of proinsulin mRNA. Their rapidity of onset and large magnitude are comparable to effects of glucose on rates of insulin biosynthesis in isolated islets and suggest that insulin biosynthesis is regulated at least in part by levels of proinsulin mRNA.