RESUMEN
INTRODUCTION: Mutation testing for the MEN1 gene is a useful method to diagnose and predict individuals who either have or will develop multiple endocrine neoplasia type 1 (MEN 1). Clinical selection criteria to identify patients who should be tested are needed, as mutation analysis is costly and time consuming. This study is a report of an Australian national mutation testing service for the MEN1 gene from referred patients with classical MEN 1 and various MEN 1-like conditions. RESULTS: All 55 MEN1 mutation positive patients had a family history of hyperparathyroidism, had hyperparathyroidism with one other MEN1 related tumour, or had hyperparathyroidism with multiglandular hyperplasia at a young age. We found 42 separate mutations and six recurring mutations from unrelated families, and evidence for a founder effect in five families with the same mutation. DISCUSSION: Our results indicate that mutations in genes other than MEN1 may cause familial isolated hyperparathyroidism and familial isolated pituitary tumours. CONCLUSIONS: We therefore suggest that routine germline MEN1 mutation testing of all cases of "classical" MEN1, familial hyperparathyroidism, and sporadic hyperparathyroidism with one other MEN1 related condition is justified by national testing services. We do not recommend routine sequencing of the promoter region between nucleotides 1234 and 1758 (Genbank accession no. U93237) as we could not detect any sequence variations within this region in any familial or sporadic cases of MEN1 related conditions lacking a MEN1 mutation. We also suggest that testing be considered for patients <30 years old with sporadic hyperparathyroidism and multigland hyperplasia.
Asunto(s)
Análisis Mutacional de ADN/métodos , Neoplasia Endocrina Múltiple Tipo 1/genética , Proteínas Proto-Oncogénicas/genética , Australia , ADN/genética , ADN/aislamiento & purificación , Mutación de Línea Germinal , Humanos , Hiperparatiroidismo/genética , Datos de Secuencia Molecular , Neoplasia Endocrina Múltiple Tipo 1/clasificación , MutaciónRESUMEN
BACKGROUND: Peroxisome proliferator activated receptor gamma (PPARgamma) is a ligand-activated transcription factor known to be central to both adipose tissue development and insulin action. Growth of adipose tissue requires differentiation of preadipocytes with acquisition of specific cellular functions including insulin sensitivity, leptin secretion and the capacity to store triglyceride. Dietary fatty acids and members of the thiazolidinedione class of compounds have been reported to influence adipogenesis at the transcriptional level. Here, we compare the effects of a dietary fatty acid, linoleic acid, and a thiazolidinedione, rosiglitazone, on biochemical and functional aspects of human preadipocyte differentiation in vitro. MATERIALS AND METHODS: Human omental and subcutaneous preadipocytes were subcultured 2-3 times and subsequently differentiated for 21 days in the presence of either linoleic acid or rosiglitazone. Differentiation was assessed using a number of biochemical and functional parameters. RESULTS: Omental and subcutaneous preadipocytes differentiated in the presence of linoleic acid showed marked cytoplasmic triacylglycerol accumulation however, no biochemical markers of differentiation (LPL expression, G3PDH gene expression and enzyme activity and leptin expression or secretion) were detected. In contrast, treatment of these cells with rosiglitazone induced full biochemical differentiation as judged by all markers assessed, despite comparatively little lipid accumulation. The rosiglitazone effects were subcutaneous depot-specific. Cells treated with linoleic acid showed decreased glucose uptake cf rosiglitazone-treated cells. A luciferase reporter assay demonstrated that rosiglitazone potently activates h-peroxisome proliferator activated receptor gamma while linoleic acid had no effect. CONCLUSIONS: These studies demonstrate that (a) human preadipocytes have the potential to accumulate triacylglycerol irrespective of their stage of biochemical differentiation; (b) while omental preadipocytes are refractory to biochemical differentiation in vitro, they are able to accumulate triacylglycerol; and (c) rosiglitazone and linoleic acid may exert their effects via different biochemical pathways.
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Adipocitos/citología , Tejido Adiposo/citología , Diferenciación Celular/efectos de los fármacos , Ácido Linoleico/uso terapéutico , Tiazoles/uso terapéutico , Tiazolidinedionas , Adipocitos/efectos de los fármacos , Tejido Adiposo/efectos de los fármacos , Adulto , Anciano , Diferenciación Celular/fisiología , Femenino , Humanos , Hipoglucemiantes/uso terapéutico , Masculino , Persona de Mediana Edad , Receptores Citoplasmáticos y Nucleares/metabolismo , Rosiglitazona , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismoRESUMEN
Estrogen influences regional adipose tissue distribution and the accompanying cardiovascular disease risk. To elucidate the mechanisms of this link further, we assessed whether human preadipocytes (PAs) expressed estrogen receptors (ERs) and whether there were any regional or gender differences in ER complement. Human PAs expressed the ERalpha gene but not ERbeta by reverse transcriptase-polymerase chain reaction, possessed ERa protein on Western blotting, and displayed specific 17beta-estradiol (E2) binding with calculated dissociation constants of 0.78 nM, 0.96 nM, and 1.19 nM and maximal binding capacities of 9.3 fmol/mg, 14.6 fmol/ mg, and 18.2 fmol/mg from three whole cell binding assays. There were no regional differences in ERalpha complement for males or females. There were no gender differences in ERalpha complement for subcutaneous or visceral samples. We conclude that ERa but not ERbeta is present in human PAs. This suggests that the effect of estrogen on adipose tissue deposition has a contribution from the direct effect of estrogen on human PAs via ERa.
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Adipocitos/metabolismo , Receptores de Estrógenos/genética , Células Madre/metabolismo , Adipocitos/química , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Constitución Corporal , Neoplasias de la Mama , Estradiol/metabolismo , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Epiplón , Receptores de Estrógenos/análisis , Receptores de Estrógenos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Caracteres Sexuales , Células Madre/química , Células Tumorales CultivadasRESUMEN
Adipogenesis is preceded by development of a microvascular network, and optimal functioning of adipose tissue as an energy store and endocrine organ is dependent on extensive vascularization. We have examined the role of endothelial cell-derived factors that influence the proliferation of human preadipocytes. Microvascular endothelial cells and preadipocytes were isolated from human omental and subcutaneous adipose tissue biopsies by use of a developed procedure of collagenase digest, immunoselection, and differential trypsinization. Conditioned medium from microvascular endothelial cell cultures promoted the proliferation of preadipocytes (P = <0.001) and (to a lesser extent) other cell types. No depot-specific differences in mitogenic capacity of microvascular endothelial cell medium or of preadipocyte response were observed. These results indicate that adipose tissue endothelial cells secrete soluble adipogenic factor(s).
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Adipocitos/citología , Tejido Adiposo/irrigación sanguínea , División Celular , Endotelio Vascular/fisiología , Células Madre/citología , Adulto , Anciano , Anticuerpos Monoclonales , Biopsia , Separación Celular , Células Cultivadas , Colágeno/metabolismo , Medios de Cultivo Condicionados , Femenino , Técnica del Anticuerpo Fluorescente , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Masculino , Microesferas , Persona de Mediana Edad , Epiplón , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Tripsina/metabolismoRESUMEN
Type 1 diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta-cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta-cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in APNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta-cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(+/+) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.
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Apoptosis/genética , ADN Glicosilasas , Diabetes Mellitus Tipo 1/genética , N-Glicosil Hidrolasas/genética , Animales , Diabetes Mellitus Tipo 1/etiología , Diabetes Mellitus Tipo 1/patología , Predisposición Genética a la Enfermedad , Islotes Pancreáticos/patología , Ratones , Ratones Noqueados , EstreptozocinaRESUMEN
Paget's disease of bone is a common condition characterized by bone pain, deformity, pathological fracture, and an increased incidence of osteosarcoma. Genetic factors play a role in the pathogenesis of Paget's disease but the molecular basis remains largely unknown. Susceptibility loci for Paget's disease of bone have been mapped to chromosome 6p21.3 (PDB1) and 18q21.1-q22 (PDB2) in different pedigrees. We have identified a large pedigree of over 250 individuals with 49 informative individuals affected with Paget's disease of bone; 31 of whom are available for genotypic analysis. The disease is inherited as an autosomal dominant trait in the pedigree with high penetrance by the sixth decade. Linkage analysis has been performed with markers at PDB1; these data show significant exclusion of linkage with log10 of the odds ratio (LOD) scores < -2 in this region. Linkage analysis of microsatellite markers from the PDB2 region has excluded linkage with this region, with a 30 cM exclusion region (LOD score < -2.0) centered on D18S42. These data confirm the genetic heterogeneity of Paget's disease of bone. Our hypothesis is that a novel susceptibility gene relevant to the pathogenesis of Paget's disease of bone lies elsewhere in the genome in the affected members of this pedigree and will be identified using a microsatellite genomewide scan followed by positional cloning.
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Cromosomas Humanos Par 18/genética , Cromosomas Humanos Par 6/genética , Ligamiento Genético/genética , Osteítis Deformante/genética , Adulto , Anciano , Anciano de 80 o más Años , Australia , Mapeo Cromosómico , Femenino , Heterogeneidad Genética , Predisposición Genética a la Enfermedad , Humanos , Escala de Lod , Masculino , Repeticiones de Microsatélite/genética , Persona de Mediana Edad , Osteítis Deformante/tratamiento farmacológico , Osteítis Deformante/fisiopatología , Linaje , FenotipoRESUMEN
Glucocorticoid excess causes visceral obesity and its accompanying insulin resistance, dyslipidemia and hypertension. Glucocorticoids enhance preadipocyte (PA) differentiation and increase their aromatase activity (oestrogen production) and there is regional variability in these PA processes. Therefore, we studied human PAs for the presence of, and any regional or gender differences in, glucocorticoid receptors (GRs). Confluent subcultured human subcutaneous (Sc) and visceral (Vis) PAs from both genders contained GRs as assessed by GR gene expression and specific glucocorticoid (dexamethasone) binding. The dissociation constant was similar to that of other human cells and there was no difference between Sc and Vis sites or between males and females. There was significantly less GR mRNA in Vis PAs compared with Sc PAs in females (P=0.008) but not in males. There was less glucocorticoid binding in Vis compared with Sc PAs in females, measured by maximal binding capacity (P=0.035) or single saturating dose glucocorticoid binding (Bssd) (P=0.019). There was no regional difference in specific glucocorticoid binding in males. There was a gender difference with fewer GRs in Vis PAs in females compared with males measured by Bssd (P=0.006). In summary, GRs are present in human PAs. There is a lower GR density in Vis compared with Sc PAs in females, and females have fewer GRs in Vis PAs compared with males. These differences are likely to affect regional aromatase activity and to contribute to the smaller visceral fat mass in females compared with males.
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Adipocitos/metabolismo , Receptores de Glucocorticoides/genética , Adipocitos/citología , Diferenciación Celular , Células Cultivadas , Dexametasona/metabolismo , Femenino , Expresión Génica , Humanos , Modelos Lineales , Masculino , Epiplón , Unión Proteica , Receptores de Glucocorticoides/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores Sexuales , Estadísticas no ParamétricasRESUMEN
The development of diabetes in non-obese diabetic (NOD) mice, which normally takes between 3 and 7 months, can be accelerated by cyclophosphamide (CY) injections, with rapid progression to diabetes within only 2-3 weeks. This insulin-dependent diabetes mellitus (IDDM) can be prevented or delayed in CY-treated NOD mice by nicotinamide (NA). The present study was undertaken to determine the mode of cell death responsible for the development of IDDM in CY-treated male NOD mice and to investigate the effect of NA on beta-cell death. Apoptotic beta cells were present within the islets of Langerhans in haematoxylin and eosin-stained sections of the pancreata harvested from 3- and 12-week-old male NOD mice, from 8 h until 14 days after a single intraperitoneal injection of CY (150 mg/kg body weight). The maximum amount of beta-cell apoptosis in 3-week-old animals occurred 1-2 days after CY treatment (20 apoptotic cells per 100 islets), after which time levels of apoptosis declined steadily throughout the 14-day period studied. The incidence of beta-cell apoptosis in 12-week-old male NOD mice occurred in two peaks; the first was recorded 8-24 h after CY treatment (30 apoptotic cells/100 islets), while the second, at 7 days (36 apoptotic cells per 100 islets), coincided with increased insulitis. Administration of NA 15 min before CY treatment, and thereafter daily, substantially reduced the amount of apoptosis and effectively eliminated (4 apoptotic cells per 100 islets) the second wave of beta-cell apoptosis seen at day 7 in 12-week-old animals given CY alone. These results show that apoptosis is the mode of beta-cell death responsible for the development of CY-induced IDDM and that prevention of IDDM by NA is associated with a reduction in beta-cell apoptosis.
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Apoptosis/efectos de los fármacos , Diabetes Mellitus Experimental/prevención & control , Diabetes Mellitus Tipo 1/prevención & control , Islotes Pancreáticos/patología , Niacinamida/uso terapéutico , Animales , Ciclofosfamida , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/inducido químicamente , Diabetes Mellitus Tipo 1/patología , Masculino , Ratones , Ratones Endogámicos NODRESUMEN
OBJECTIVES: To determine the apolipoprotein E (apoE) allelic frequencies and the effect of apoE genotype on lipid concentrations in indigenous Australian subjects. DESIGN: Cross-sectional study. SUBJECTS AND SETTING: 155 indigenous Australians (92 women and 63 men) of mean (+/- standard deviation) age 45 +/- 17 years (SD +/- 50) were recruited without regard to history of atherosclerotic disease, in collaboration with community-based health centres in five indigenous communities in south-east Queensland. For comparison, 113 subjects of European descent and similar age distribution from the Brisbane and Gold Coast regions were also studied. MAIN OUTCOME MEASURES: ApoE allelic frequency; apoE genotype; sex; age; diabetes status; body mass index; history of atherosclerotic vascular disease; and concentrations of total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol. RESULTS: The frequency of the apoE4 allele was found to be significantly higher in the indigenous subjects than in the subjects of European descent (P < 0.001). Among indigenous subjects, those with the apoE4 allele tended to have higher triglyceride concentrations and had significantly lower HDL-cholesterol concentrations than those with the apoE3/3 and 3/2 genotypes. CONCLUSIONS: ApoE allelic frequency is likely to be one of the cluster of factors contributing to the high cardiovascular mortality of indigenous Australians.
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Apolipoproteínas E/genética , Hiperlipidemias/genética , Indigencia Médica , Polimorfismo Genético , Adulto , Alelos , Arteriosclerosis/complicaciones , Australia , Índice de Masa Corporal , Estudios Transversales , Complicaciones de la Diabetes , Femenino , Genotipo , Humanos , Hiperlipidemias/sangre , Hiperlipidemias/complicaciones , Lípidos/sangre , Masculino , Persona de Mediana Edad , Prevalencia , QueenslandRESUMEN
OBJECTIVES: To determine plasma homocysteine levels in indigenous Australians living in urban areas, and the relationship of these levels with other risk factors in this population. DESIGN: Cross-sectional study. SUBJECTS AND SETTING: 365 urban indigenous Australian subjects, 153 men and 212 women, mean (SE) age 42 (1) years, ascertained without regard to history of atherosclerotic disease, in collaboration with community-based health centres in five indigenous communities in south-east Queensland, 1997-1998. MAIN OUTCOME MEASURES: Plasma homocysteine levels, age, sex, smoking history, metformin therapy, history of atherosclerotic vascular disease, serum creatinine level, red cell folate and serum vitamin B12 levels. RESULTS: 89 subjects (24%) had plasma homocysteine levels 15 mumol/L or above. Homocysteine levels were higher in men than in women (men: 14.4 mumol/L; 95% confidence interval [CI], 13.6-15.2; women: 11.9 mumol/L; 95% CI, 11.4-12.5) (P < 0.001); correlated with age (P < 0.001); higher in current smokers (P = 0.02); higher in subjects taking metformin therapy (P = 0.007); and higher in subjects with a history of atherosclerotic vascular disease (P < 0.001). Homocysteine levels were also correlated with serum levels of creatinine (P < 0.001), red cell folate (P < 0.001), and vitamin B12 (P < 0.001). CONCLUSIONS: These data indicate that the high plasma levels of homocysteine of Australian indigenous subjects are associated with a history of vascular disease, and correlated with, among other things, smoking, and folate and vitamin B12 nutritional deficiency. These are potentially reversible risk factors, and our data suggest that focusing public health initiatives on these issues may reduce the high prevalence of cardiovascular disease in the Australian indigenous population.
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Homocisteína/sangre , Hiperhomocisteinemia/sangre , Hiperhomocisteinemia/genética , Nativos de Hawái y Otras Islas del Pacífico , Salud Urbana , Adulto , Distribución por Edad , Enfermedades Cardiovasculares/etiología , Creatina/sangre , Estudios Transversales , Femenino , Deficiencia de Ácido Fólico/complicaciones , Humanos , Hiperhomocisteinemia/complicaciones , Masculino , Persona de Mediana Edad , Queensland , Factores de Riesgo , Distribución por Sexo , Fumar/efectos adversos , Deficiencia de Vitamina B 12/complicacionesRESUMEN
Streptozotocin (STZ) is believed to induce pancreatic beta cell death in mice by depleting the cell of NAD+NADH. The drug is known to cause a greater depletion of beta cell NAD+NADH in C57bl/6J mice than in Balb/c mice. To investigate the basis for this strain difference, we compared the effects of streptozotocin on poly(ADP-ribose)polymerase (PARP) activation - the major site of NAD consumption, and on mitochondrial activity - the major site of NAD production.%A significant strain difference was demonstrated in STZ-induced PARP activation (fmol NAD incorporated/min/microgram DNA+/-s.e.m.: Balb/c control 2.28+/-0.14, Balb STZ 3.11+/-0.25; C57bl/6J control 2.57+/-0.29, C57bl/6J STZ 4.17+/-0.24). In comparison, no strain difference could be demonstrated in hydrogen-peroxide-induced PARP activation. No strain differences could be detected in the activity of STZ-treated islet mitochondria as measured by determining ATP production (pmol/microgram protein/h+/-s. e.m.: Balb/c control 0.20+/-0.02, Balb/c STZ 0.15+/-0.02; C57bl/6J control 0.23+/- 0.03, C57bl/6J STZ 0.15+/-0.02) or by 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye reduction (change in optical density/mg protein+/-s.e.m.: Balb/c control 10.19+/-0.62, Balb/c STZ 6.01+/-1.17; C57bl/6J control 6. 15+/-0.98, C57bl/6J STZ 5.81+/-0.96).% The strain difference in STZ-induced NAD depletion appears to be due to a difference in NAD consumption and not a difference in a mitochondrial process involved in replacing decreasing NAD concentrations. It is unlikely that a strain difference in the enzymic activity of PARP is responsible for strain differences in the effects of STZ, as no strain differences in hydrogen-peroxide-induced PARP activation could be detected. Thus the greater PARP activation, NAD depletion and beta cell death observed in C57bl/6J islets may be due to greater levels of DNA damage or differences in the DNA excision repair processes.
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Diabetes Mellitus Experimental/inducido químicamente , Islotes Pancreáticos/efectos de los fármacos , Ratones Endogámicos BALB C/metabolismo , Ratones Endogámicos C57BL/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Estreptozocina/toxicidad , Adenosina Trifosfato/biosíntesis , Animales , Daño del ADN , Reparación del ADN , Diabetes Mellitus Experimental/enzimología , Susceptibilidad a Enfermedades , Resistencia a Medicamentos , Activación Enzimática , Peróxido de Hidrógeno/farmacología , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , NAD/deficiencia , NAD/metabolismo , Especificidad de la Especie , Estreptozocina/farmacologíaRESUMEN
NIDDM has a substantial genetic component, but the nature of the genetic susceptibility is largely unknown. Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous monogenic form of NIDDM characterized by an early age of onset and autosomal dominant inheritance, and linkage studies have identified genes that are mutated in different MODY pedigrees on chromosome 20 (MODY1 locus, hepatocyte nuclear factor-4alpha [HNF-4alpha] gene), chromosome 7 (MODY2 locus, glucokinase gene), and chromosome 12 (MODY3 locus, HNF-1alpha gene). We studied an extended pedigree in which multiple members are affected by late-onset NIDDM associated with insulin resistance and performed linkage analysis with four microsatellite markers in the MODY3 region of chromosome 12q. We found significant evidence for linkage between NIDDM and the MODY3 locus (logarithm of odds score 3.65 at theta = 0.008 telomeric to marker D12S321), but sequencing of the 10 exons and promoter of HNF-1alpha did not identify any causative mutation in this gene. Our results indicate that the region of chromosome 12q close to MODY3 harbors a novel susceptibility gene or genes for NIDDM.
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Cromosomas Humanos Par 12 , Proteínas de Unión al ADN , Diabetes Mellitus Tipo 2/genética , Predisposición Genética a la Enfermedad , Proteínas Nucleares , Factores de Transcripción/genética , Adulto , Anciano , Exones , Femenino , Haplotipos , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Resistencia a la Insulina , Escala de Lod , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Linaje , Regiones Promotoras GenéticasRESUMEN
Inbred strains of mice vary in their sensitivity to the diabetogenic effects of streptozotocin (STZ). To investigate the basis for this strain difference we exposed islet cells from two strains of mice that differ in sensitivity to the drug. We examined them morphologically and measured islet NAD + NADH content, streptozotocin metabolite accumulation, glucose transport capacity, Glut2 levels and medium nitrite accumulation. C57bl/6J mice were more sensitive to STZ than Balb/c mice as judged by the extent of pancreatic insulin depletion and beta cell death, in vivo and in vitro. The mode of cell death was necrosis. After a 30-min in vitro exposure to the drug the more sensitive C57bl/6J islets contained higher levels of streptozotocin metabolites and less NAD + NADH than the more resistant Balb/c islets. The lack of any strain differences in 3-O-methyl glucose transport, Glut2 levels and medium nitrite accumulation suggested that STZ transport and nitric oxide metabolism were not responsible for differences in STZ sensitivity and metabolite accumulation. Thus the strain differences in STZ sensitivity appears to be due to intracellular events within the beta cell occurring after STZ transport and before NAD + NADH depletion. STZ metabolite accumulation appears to be associated with STZ sensitivity. Further studies are warranted to determine if differential STZ metabolite accumulation is responsible for STZ sensitivity.
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Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Estreptozocina/metabolismo , Estreptozocina/farmacología , Animales , Glucemia/análisis , Supervivencia Celular/efectos de los fármacos , Resistencia a Medicamentos/genética , Insulina/metabolismo , Islotes Pancreáticos/patología , Masculino , Ratones , Ratones Endogámicos BALB C/genética , Ratones Endogámicos BALB C/metabolismo , Ratones Endogámicos C57BL/genética , Ratones Endogámicos C57BL/metabolismo , Ratones Endogámicos CBA/genética , Ratones Endogámicos CBA/metabolismo , Ratones Endogámicos , Microscopía Electrónica , NAD/metabolismo , Páncreas/metabolismo , Especificidad de la Especie , Factores de TiempoRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) production by adipocytes is elevated in obesity, as shown by increased adipose tissue TNF-alpha mRNA and protein levels and by increased circulating concentrations of the cytokine. Furthermore, TNF-alpha has distinct effects on adipose tissue including induction of insulin resistance, induction of leptin production, stimulation of lipolysis, suppression of lipogenesis, induction of adipocyte dedifferentiation, and impairment of preadipocyte differentiation in vitro. Taken together, these effects all tend to decrease adipocyte volume and number and suggest a role for TNF-alpha in limiting increase in fat mass. The aim of the present study was to determine if TNF-alpha could induce apoptosis in human adipose cells, hence delineating another mechanism by which the cytokine could act to limit the development of, or extent of, obesity. Cultured human preadipocytes and mature adipocytes in explant cultures were exposed in vitro to human TNF-alpha at varying concentrations for up to 24 h. Apoptosis was assessed using morphological (histology, nuclear morphology following acridine orange staining, electron microscopy) and biochemical (demonstration of internucleosomal DNA cleavage by gel electrophoresis and of annexin V staining using immunocytochemistry) criteria. In control cultures, apoptotic indexes were between 0 and 2.3% in all experiments. In the experimental systems, TNF-alpha induced apoptosis in both preadipocytes and adipocytes, with indexes between 5 and 25%. Therefore, TNF-alpha induces apoptosis of human preadipocytes and adipocytes in vitro. In view of the major metabolic role of TNF-alpha in human adipose tissue, and the knowledge that adipose tissue is dynamic (with cell acquisition via preadipocyte replication/differentiation and cell loss via apoptosis), these findings describe a further mechanism whereby adipose tissue mass may be modified by TNF-alpha.
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Adipocitos/fisiología , Apoptosis , Factor de Necrosis Tumoral alfa/farmacología , Naranja de Acridina , Adipocitos/ultraestructura , Anexina A5/análisis , Células Cultivadas , Colorantes , Medio de Cultivo Libre de Suero , Humanos , Microscopía Electrónica , Microscopía Fluorescente , Células Madre/química , Células Madre/fisiologíaRESUMEN
BACKGROUND: Inferior petrosal sinus sampling (IPSS) is a useful investigative technique in the differential diagnosis of ACTH-dependent Cushing's syndrome. Diagnostic accuracy is improved by the administration of corticotrophin releasing-factor (CRF) during the procedure to stimulate ACTH secretion. We hypothesized that, given the unavailability of CRF in Australia, stimulation of ACTH secretion from tumorous corticotrophs with metyrapone treatment before IPSS may be useful. AIMS: To describe our clinical experience with a novel diagnostic test, and to compare results between IPSS with and without metyrapone pre-treatment. SETTING: Metropolitan, Australian university teaching hospital. PATIENTS: 18 patients were studied on 21 occasions: three with Cushing's disease without metyrapone treatment prior to IPSS (M-), 11 with Cushing's disease with metyrapone pretreatment (M+), three with ectopic ACTH syndrome, and one with pseudo-Cushing's syndrome. TREATMENT: Patients received oral metyrapone, median dose 750 mg 6 hourly, for 24 h before IPSS. RESULTS: No major side effects were noted. Metyrapone increased serum 11-deoxycortisol concentration to a median of 400 nmol/l (range 36-1310) on the morning of the test. Radiological confirmation of correct catheter placement was shown in 36/42 inferior petrosal sinuses (86%). Median peak central: peripheral ACTH ratios were 9.8 for M- pituitary adenomas (range 5.7-13.6), 12.9 for the technically successful M+ pituitary adenomas (range 8-54.1), and 1.6 for M+ ectopic ACTH syndrome cases (range 1.2-3.4). Repeat studies in unoperated patients with ectopic ACTH syndrome showed ratios < 1.6. IPSS showed median peak ACTH concentrations of 190 ng/l for M- pituitary adenomas (range 83-205), 595 ng/l for the technically successful M+ pituitary adenomas (range 80-7630; P = 0.035 compared to M-), and 62 ng/l for M+ ectopic ACTH syndrome cases (range 47-220). IPSS correctly identified the pituitary source of ACTH production in all cases of Cushing's disease (except one technical failure where MRI revealed a lesion). MRI scanning correctly identified a lesion in 3/14 operated Cushing's disease cases. IPSS correctly lateralized 1/3 M- and 7/8 M+ Cushing's disease cases where the procedure was technically successful and surgical descriptions adequate. Pituitary exploration revealed a visible lesion in 75% of cases corresponding to the side predicted by IPSS; 'blind' hemi-hypophysectomy was performed on the side predicted from IPSS in the remainder. All cases of Cushing's disease were cured or improved following surgery, with a median follow-up of 2.8 years (range 0.7-5.9). CONCLUSIONS: Metyrapone pre-treated inferior petrosal sinus sampling is safe, and appears to induce high ACTH output from pituitary corticotroph adenomas. The technique has allowed accurate localization and treatment of pituitary corticotroph microadenomas.
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Hormona Adrenocorticotrópica/metabolismo , Síndrome de Cushing/diagnóstico , Metirapona , Muestreo de Seno Petroso , Síndrome de ACTH Ectópico/diagnóstico , Adenoma/diagnóstico , Adolescente , Hormona Adrenocorticotrópica/sangre , Adulto , Cortodoxona/sangre , Síndrome de Cushing/sangre , Síndrome de Cushing/fisiopatología , Diagnóstico Diferencial , Femenino , Humanos , Hidrocortisona/sangre , Masculino , Persona de Mediana Edad , Neoplasias Hipofisarias/diagnóstico , Estimulación QuímicaRESUMEN
The NOD/Lt mouse, a widely used model of human autoimmune IDDM, was used to establish the mode of beta-cell death responsible for the development of IDDM. Apoptotic cells were present within the islets of Langerhans in hematoxylin and eosin-stained sections of pancreases harvested from 3- to 18-week-old female NOD/Lt mice (a range of 11-50 apoptotic cells per 100 islets). Immunohistochemical localization of insulin to the dying cells confirmed the beta-cell origin of the apoptosis. Although some islets from age-matched control female NOD/scid mice contained apoptotic cells, virtually all of these cells were insulin negative as determined by immunohistochemistry. The small number of apoptotic insulin-positive cells identified in islets from NOD/scid mice (a range of 0-1 apoptotic cells per 100 islets) was not statistically significant, compared with the numbers recorded in NOD/Lt mice. All dying cells showed the morphological changes characteristic of cell death by apoptosis and stained positively with the TUNEL method for end-labeling DNA strand breaks. The maximum mean amount of beta-cell apoptosis occurring in NOD/Lt mice was at week 15 (50 apoptotic cells per 100 islets), which coincided with the earliest onset of diabetes as determined by blood glucose, urine glucose, and pancreatic immunoreactive insulin measurements. While there was no peak incidence of beta-cell apoptosis throughout the time period studied (weeks 3-18), the incidence of apoptosis decreased at week 18, by which time 50% of the animals had overt diabetes. The low levels of beta-cell apoptosis observed is indicative of a gradual deletion of the beta-cell population throughout the extensive preclinical period seen in this model and would be sufficient to account for the beta-cell loss resulting in IDDM. Apoptosis of beta-cells preceded the appearance of T-cells (CD3-positive by immunohistochemistry) in islets. Lymphocytic infiltration of islets (insulitis) was not detected until week 6. The results show that beta-cell apoptosis is responsible for the development of IDDM in the NOD/Lt mouse and that its onset precedes lymphocytic infiltration of the islets.
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Apoptosis , Diabetes Mellitus Tipo 1/etiología , Islotes Pancreáticos/fisiología , Ratones Endogámicos NOD , Animales , Glucemia/análisis , Femenino , Islotes Pancreáticos/ultraestructura , RatonesRESUMEN
The ability of beta cells to endure assaults by various environmental agents, including toxins and viruses, may be relevant to the development of diabetes. We have examined the mode of cell death caused by streptozotocin (STZ) in a murine pancreatic beta cell line, INS-1. Apoptosis was identified by detection of initial endonuclease-mediated DNA strand breaks by DNA gel electrophoresis. Apoptosis and necrosis were distinguished morphologically by light and electron microscopy. Higher rates of apoptosis, as compared to necrosis, were observed when cells were exposed to 15 mM STZ for 1 hr followed by a 24 hrs recovery period. Higher doses of STZ (30 mM) caused the cells to undergo necrosis (22%) as well as apoptosis (17%). These results suggest that the cytotoxic effect of STZ, at low doses, on beta cells involves the activation of the apoptotic pathway, whereas, at high doses, the mode of beta cell death is predominantly necrosis.
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Apoptosis/efectos de los fármacos , Islotes Pancreáticos/efectos de los fármacos , Estreptozocina/toxicidad , Animales , Línea Celular , Daño del ADN , Relación Dosis-Respuesta a Droga , Islotes Pancreáticos/patología , Islotes Pancreáticos/ultraestructura , Ratones , NecrosisRESUMEN
Although insulin-dependent diabetes mellitus (IDDM) results from irreversible loss of beta cells, the mode of cell death responsible for this loss has not previously been categorized. In this study, the multiple low-dose streptozotocin (stz) model (intraperitoneal injection of stz at a concentration of 40 mg/kg body weight per day for five consecutive days) was used to investigate beta-cell death during the development of IDDM in male C57B1/6 mice. Apoptotic cells were evident by light microscopy within the islets of Langerhans of treated animals from day 2 (the day of the second stz injection) until day 17. Immunohistochemical localization of insulin to the dying cells confirmed the beta-cell origin of the apoptosis. Two peaks in the incidence of beta-cell apoptosis occurred: the first at day 5, which corresponded to an increase in blood glucose concentration, and the second at day 11, when lymphocytic infiltration of the islets (insulitis) was maximal. Insulitis did not begin until day 9, by which time treated animals had developed overt diabetes as revealed by blood glucose and pancreatic immunoreactive insulin (IRI) measurements. Beta-cell apoptosis preceded the appearance of T-cells in the islets and continued throughout the period of insulitis. Thus, whether induced by stz or a subsequent immune response, apoptosis is the mode of cell death responsible for beta-cell loss in the multiple low-dose stz model of IDDM.
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Apoptosis , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/patología , Islotes Pancreáticos/patología , Animales , Glucemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animales de Enfermedad , Técnicas para Inmunoenzimas , Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , EstreptozocinaRESUMEN
A subset of growth-hormone (GH) producing pituitary adenomas harbour mutations at residues Arg201 and Gln227 of the alpha subunit of the stimulatory G protein (Gs alpha). One such mutation has been reported in a GH-producing tumour from a patient with multiple endocrine neoplasia type 1 (MEN 1) although mutations have not been reported in other tumours associated with MEN 1. We used PCR-induced restriction site analysis to screen for these mutations in 80 tumours of the pituitary, parathyroid and endocrine pancreas. Arg201 mutations were detected in 1 non-functioning and 4 GH-producing pituitary tumours. No mutations were found in any of the endocrine tumours tested at Arg179 of the Gi2 alpha subunit, a homologous residue to Arg201 of Gs alpha. Our results indicate oncogenesis in the majority of pituitary and parathyroid tumours is independent of mutations of Gs and Gi2 alpha.