RESUMEN
OBJECTIVE: Previous studies suggest that differences in molecular features of endometrial cancers between racial groups may contribute to the poorer survival in Blacks. The objective of this investigation was to determine whether gene expression among endometrial cancers is different between Blacks and Whites. METHODS: Fresh frozen tumors from 25 Black patients were matched by stage, grade, and histology to endometrial cancer specimens from 25 White patients. Each case was macrodissected to produce specimens possessing a minimum of 75% cancer cellularity. A subset of 10 matched pairs was also prepared using laser microdissection (LMD) to produce specimens possessing a minimum of 95% cancer cells. Total RNA isolated from each sample was analyzed using the Affymetrix Human Genome U133 Plus 2.0 arrays. Data were analyzed using principal component analysis and binary class comparison analyses. RESULTS: Unsupervised analysis of the 50 endometrial cancers failed to identify global gene expression profiles unique to Black or White patients. In a subset analysis of 10 matched pairs from Blacks and Whites prepared using LMD and macrodissection, unsupervised analysis did not reveal a unique gene expression profile associated with race in either set, but associations were identified that relate to sample preparation technique, histology and stage. CONCLUSIONS: Our microarray data revealed no global gene expression differences and identified few individual gene differences between endometrial cancers from Blacks and Whites. More comprehensive methods of transcriptome analysis could uncover RNAs that may underpin the disparity of outcome or prevalence of endometrial cancers in Blacks and Whites.
Asunto(s)
Población Negra/genética , Neoplasias Endometriales/etnología , Neoplasias Endometriales/genética , Población Blanca/genética , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Clasificación del Tumor , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Componente Principal , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/análisis , ARN Neoplásico/genéticaRESUMEN
Diflunisal, 5-(2',4'-difluorophenyl)salicylic acid, excreted in urine as its glucuronide, was given to normal humans (n = 6) along with a glucose load specifically labeled with 14C. Glucuronide excreted by each subject was reduced to its glucoside and glucose from it degraded to yield the distribution of 14 C in its six carbons. Randomization of the 14C from the specifically labeled glucose was taken as a measure of the extent to which glucose was deposited indirectly (i.e., glucose----lactate----glucose----6-P----glycogen), rather than directly (i.e., glucose----glucose-6-P----glycogen). The maximum contribution to glycogen formation by the direct pathway was estimated to be 65 +/- 1%, on the assumption that glucuronide and glycogen are derived from the same hepatic pool of glucose-6-P in liver. Evidence that supports that assumption was obtained by comparing the randomization of 14C in the urinary glucuronide with that in glucose in blood from the hepatic vein of four of the subjects before and after they were given glucagon. Other evidence supporting the assumption was obtained by comparing in two subjects 3H/14C ratios in glucose from hepatic vein blood before and after glucagon administration with that in urinary glucuronide, having labeled the uridine diphosphate (UDP)-glucose in their livers with 14C by giving them 1-[14C]galactose and their circulating glucose with 3H by giving a 5-[3H]glucose-labeled load. It is concluded that glucuronide formation in humans can be used to trace glucose metabolism in the liver, and that in humans the indirect pathway of glucose metabolism is active.
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Carbohidratos de la Dieta/metabolismo , Glucosa/metabolismo , Glucógeno Hepático/biosíntesis , Administración Oral , Adulto , Radioisótopos de Carbono , Diflunisal , Femenino , Glucosa/administración & dosificación , Humanos , MasculinoRESUMEN
A method is introduced for estimating the contribution of gluconeogenesis to glucose production. 2H2O is administered orally to achieve 0.5% deuterium enrichment in body water. Enrichments are determined in the hydrogens bound to carbons 2 and 6 of blood glucose and in urinary water. Enrichment at carbon 6 of glucose is assayed in hexamethylenetetramine, formed from formaldehyde produced by periodate oxidation of the glucose. Enrichment at carbon 2 is assayed in lactate formed by enzymatic transfer of the hydrogen from glucose via sorbitol to pyruvate. The fraction gluconeogenesis contributes to glucose production equals the ratio of the enrichment at carbon 6 to that at carbon 2 or in urinary water. Applying the method, the contribution of gluconeogenesis in healthy subjects was 23-42% after fasting 14 h, increasing to 59-84% after fasting 42 h. Enrichment at carbon 2 to that in urinary water was 1.12 +/- 0.13. Therefore, the assumption that hydrogen equilibrated during hexose-6-P isomerization was fulfilled. The 3H/14C ratio in glucose formed from [3-3H,3-14C]lactate given to healthy subjects was 0.1 to 0.2 of that in the lactate. Therefore equilibration during gluconeogenesis of the hydrogen bound to carbon 6 with that in body water was 80-90% complete, so that gluconeogenesis is underestimated by 10-20%. Glycerol's contribution to gluconeogenesis is not included in these estimates. The method is applicable to studies in humans of gluconeogenesis at safe doses of 2H2O.
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Ayuno/metabolismo , Gluconeogénesis , Espectrometría de Masas , Adulto , Glucemia/metabolismo , Agua Corporal/metabolismo , Deuterio/metabolismo , Femenino , Humanos , Marcaje Isotópico , Masculino , Metenamina/metabolismo , Persona de Mediana Edad , Modelos Biológicos , Orina/químicaRESUMEN
Healthy subjects ingested 2H2O and after 14, 22, and 42 h of fasting the enrichments of deuterium in the hydrogens bound to carbons 2, 5, and 6 of blood glucose and in body water were determined. The hydrogens bound to the carbons were isolated in formaldehyde which was converted to hexamethylenetetramine for assay. Enrichment of the deuterium bound to carbon 5 of glucose to that in water or to carbon 2 directly equals the fraction of glucose formed by gluconeogenesis. The contribution of gluconeogenesis to glucose production was 47 +/- 49% after 14 h, 67 +/- 41% after 22 h, and 93 +/- 2% after 42 h of fasting. Glycerol's conversion to glucose is included in estimates using the enrichment at carbon 5, but not carbon 6. Equilibrations with water of the hydrogens bound to carbon 3 of pyruvate that become those bound to carbon 6 of glucose and of the hydrogen at carbon 2 of glucose produced via glycogenolysis are estimated from the enrichments to be approximately 80% complete. Thus, rates of gluconeogenesis can be determined without corrections required in other tracer methodologies. After an overnight fast gluconeogenesis accounts for approximately 50% and after 42 h of fasting for almost all of glucose production in healthy subjects.
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Ayuno/metabolismo , Gluconeogénesis , Glucosa/biosíntesis , Adulto , Glucemia/metabolismo , Cromatografía Líquida de Alta Presión , Deuterio , Femenino , Glicerol/metabolismo , Humanos , Masculino , Técnica de Dilución de Radioisótopos , Factores de Tiempo , XilosaRESUMEN
Predicting peak pathogen loadings can provide a basis for watershed and water treatment plant management decisions that can minimize microbial risk to the public from contact or ingestion. Artificial neural network models (ANN) have been successfully applied to the complex problem of predicting peak pathogen loadings in surface waters. However, these data-driven models require substantial, multiparameter databases upon which to train, and missing input values for pathogen indicators must often be estimated. In this study, ANN models were evaluated for backfilling values for individual observations of indicator bacterial concentrations in a river from 44 other related physical, chemical, and bacteriological data contained in a multi-year database. The ANN modeling approach provided slightly superior predictions of actual microbial concentrations when compared to conventional imputation and multiple linear regression models. The ANN model provided excellent classification of 300 randomly selected, individual data observations into two defined ranges for fecal coliform concentrations with 97% overall accuracy. The application of the relative strength effect (RSE) concept for selection of input variables for ANN modeling and an approach for identifying anomalous data observations utilizing cross validation with ANN model are also presented.
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Bacterias/crecimiento & desarrollo , Heces/química , Modelos Estadísticos , Redes Neurales de la Computación , Agua/química , Heces/microbiología , PredicciónRESUMEN
Global gene expression patterns in breast cancer cells after treatment with oxidants (hydrogen peroxide, menadione, and t-butyl hydroperoxide) were investigated in three replicate experiments. RNA collected after treatment (at 1, 3, 7, and 24 h) rather than after a single time point, enabled an analysis of gene expression patterns. Using a 17,000 microarray, template-based clustering and multidimensional scaling analysis of the gene expression over the entire time course identified 421 genes as being either up- or down-regulated by the three oxidants. In contrast, only 127 genes were identified for any single time point and a 2-fold change criteria. Surprisingly, the patterns of gene induction were highly similar among the three oxidants; however, differences were observed, particularly with respect to p53, IL-6, and heat-shock related genes. Replicate experiments increased the statistical confidence of the study, whereas changes in gene expression patterns over a time course demonstrated significant additional information versus a single time point. Analyzing the three oxidants simultaneously by template cluster analysis identified genes that heretofore have not been associated with oxidative stress.
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Neoplasias de la Mama/genética , Expresión Génica/efectos de los fármacos , Oxidantes/farmacología , Neoplasias de la Mama/metabolismo , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo/genética , Reproducibilidad de los Resultados , Activación Transcripcional , Células Tumorales Cultivadas , Vitamina K 3/farmacología , terc-Butilhidroperóxido/farmacologíaRESUMEN
The uptake and phosphorylation of 2-deoxy-D-glucose by isolated adipocytes of the rat was determined by a method of rapid flotation through oil coupled with separation of sugar from sugar phosphate by chromatography on Dowex-1-formate. Uptake of the sugar is rapid and linear over 5 min, with a gradual decline thereafter; by 1 h, no further uptake is observed. Initially only 2-deoxy-glucose phosphate is observed within the cells; by 1 h, however, free 2-deoxy-glucose accumulates to levels approximately those in the medium. Phosphorylation ceases when intracellular levels of 2-deoxyglucose phosphate are about 50 mM regardless of the medium concentration of 2-deoxyglucose; this does not represent feedback inhibition of hexokinase, since the enzyme in fat cell homogenates is not inhibited by 50 mM 2-deoxyglucose 6-phosphate. Accumulation of deoxyglucose 6-phosphate is associated with a marked decline in intracellular ATP levels. Fat cell respiration is also depressed by approximately 50 per cent after a 1 h preincubation with 10 or 20 mM 2-deoxyglucose. Intracellular ATP levels and O2 uptake are only partially corrected by the addition of pyruvate to the incubation medium. Since no glucose was present in the medium, and intracellular concentrations of glycogen are known to be small in adipose tissue, it is proposed that accumulation of 2-deoxyglucose 6-phosphate within fat cells has a direct inhibitory effect on cell respiration unrelated to inhibition of glycolysis. No increase in intracellular free fatty acids was observed to explain this, and under the conditions of the incubations it is unlikely that Pi availability was rate limiting. The exact locus of inhibition is unknown.
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Tejido Adiposo/metabolismo , Desoxiazúcares/metabolismo , Desoxiglucosa/metabolismo , Adenosina Trifosfato/metabolismo , Tejido Adiposo/citología , Animales , Ácidos Grasos no Esterificados/metabolismo , Glucofosfatos/farmacología , Hexoquinasa/metabolismo , Masculino , Metilglucósidos/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Fosfatos/metabolismo , Piruvatos/farmacología , RatasRESUMEN
We have previously reported changes in the chemical composition of cell surface membranes in diabetic rats (Chandramoulis, V. and Carter, Jr., J. R. (1975) Diabetes 24, 257-262 [1]). To examine the possible implications of these changes for cell surface structures, we have measured the binding of labeled lectins and desialylated glycoproteins to plasma membranes prepared from the livers of streptozotocin--diabetic and control rats. Lectins were chosen which have affinities for different carbohydrate moieties. The binding of ricin and concanavalin A to liver cell membranes from the diabetic rats was significantly reduced, but no change in the binding of wheat germ agglutinin was noted. Binding of desialylated thyrozine--binding globulin, previously shown to be dependent on membrane sialic acid residues, ws strongly suggest that insulin deficiency leads to generalized changes in cell surfaced glycoproteins, at least in this animal model of diabetes.
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Membrana Celular/metabolismo , Diabetes Mellitus/metabolismo , Lectinas , Hígado/metabolismo , Animales , Unión Competitiva , Concanavalina A , Diabetes Mellitus/inducido químicamente , Globulinas/metabolismo , Cinética , Masculino , Unión Proteica , Ratas , Receptor de Insulina , Ácidos Siálicos , Estreptozocina , Proteínas de Unión a Tiroxina/metabolismoRESUMEN
We have examined the effect of chronic diabetes mellitus upon cell membrane composition and turnover in streptozotocin-treated rats and control animals maintained for four to eight weeks. Liver plasma membranes, prepared from diabetic animals, showed enhanced activities of alkaline phosphatase and glucose-6-phosphatase and depressed 5'-nucleotidase when compared with controls. Studies of the nonprotein constituents of liver plasma membranes and red cell "ghosts" showed similar changes in both tissues: sialic acid and cholesterol content were reduced in the membranes of diabetic animals, while phospholipids (total and individual classes) and neutral sugars were unchanged. To look for changes in relative turnover rates of individual membrane proteins, we combined a double-label in-vivo technic using [3H] and [14C] leucine with polyacrylamide gel separation of membrane proteins. No significant differences were observed between control and diabetic animals. In chronically diabetic animals, cell membranes may show significant changes in overall composition with no significant changes in the rate of protein turnover.
Asunto(s)
Membrana Celular/ultraestructura , Diabetes Mellitus/patología , Fosfatasa Alcalina/metabolismo , Animales , Carbohidratos/análisis , Membrana Celular/análisis , Membrana Celular/enzimología , Colesterol/análisis , Enfermedad Crónica , Diabetes Mellitus/inducido químicamente , Complejo IV de Transporte de Electrones/metabolismo , Eritrocitos/análisis , Eritrocitos/ultraestructura , Glucosa-6-Fosfatasa/metabolismo , Leucina/metabolismo , Hígado/ultraestructura , Masculino , Microsomas Hepáticos/análisis , Nucleotidasas/metabolismo , Fosfolípidos/análisis , Proteínas/metabolismo , Ratas , Ácidos Siálicos/análisis , EstreptozocinaRESUMEN
Pancreatic islets from healthy (control) and neonatally streptozocin-induced diabetic (STZ-D) rats, a model for non-insulin-dependent diabetes mellitus, were incubated with 3H2O and 5.5 or 16.7 mM glucose. At 5.5 mM glucose, no detectable [3H]glucose was formed. At 16.7 mM, 2.2 patom.islet-1.h-1 of 3H was incorporated into glucose by the control islets and 5.4 patom.islet-1.h-1 by STZ-D islets. About 75% of the 3H was bound to carbon-2 of the glucose. Glucose utilization was 35.3 pmol.islet-1.h-1 by the control and 19.0 pmol.islet-1.h-1 by the STZ-D islets. Therefore, 4.5% of the glucose-6-phosphate formed by the control islets and 15.7% by the STZ-D islets was dephosphorylated. This presumably occurred in the beta-cells of the islets catalyzed by glucose-6-phosphatase. An increased glucose cycling, i.e., glucose----glucose-6-phosphate----glucose, in islets of STZ-D rats may contribute to the decreased insulin secretion found in these animals.
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Diabetes Mellitus Experimental/metabolismo , Glucosa/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Femenino , Glucosa/farmacología , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Masculino , Técnica de Dilución de Radioisótopos , Ratas , Ratas Endogámicas , Valores de Referencia , TritioRESUMEN
Contributions of renal glucose production to whole-body glucose turnover were determined in healthy individuals by using the arteriovenous balance technique across the kidneys and the splanchnic area combined with intravenous infusion of [U-13C6]glucose, [3-(3)H]glucose, or [6-(3)H]glucose. In the postabsorptive state, the rate of glucose appearance was 11.5 +/- 0.6 micromol x kg(-1) x min(-1). Hepatic glucose production, calculated as the sum of net glucose output (9.8 +/- 0.8 micromol x kg(-1) x min(-1)) and splanchnic glucose uptake (2.2 +/- 0.3 micromol x kg(-1) x min(-1)) accounted for the entire rate of glucose appearance. There was no net exchange of glucose across the kidney and no significant renal extraction of labeled glucose. The renal contribution to total glucose production calculated from the arterial, hepatic, and renal venous 13C-enrichments (glucose M+6) was 5 +/- 2%. In the 60-h fasted state, the rate of glucose appearance was 8.2 +/- 0.3 micromol x kg(-1) x min(-1). Hepatic glucose production, estimated as net splanchnic output (5.8 +/- 0.7 micromol x kg(-1) x min(-1)) plus splanchnic uptake (0.6 +/- 0.3 micromol x kg(-1) x min(-1)) accounted for 79% of the rate of glucose appearance. There was a significant net renal output of glucose (0.9 +/- 0.3 micromol x kg(-1) x min(-1)), but no significant extraction of labeled glucose across the kidney. The renal contribution to whole-body glucose turnover calculated from the 13C-enrichments was 24 +/- 3%. We concluded that 1) glucose production by the human kidney in the postabsorptive state, in contrast to recent reports, makes at most only a minor contribution (approximately 5%) to blood glucose homeostasis, but that 2) after 60-h of fasting, renal glucose production may account for 20-25% of whole-body glucose turnover.
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Ingestión de Alimentos/fisiología , Ayuno/fisiología , Glucosa/biosíntesis , Riñón/metabolismo , Hígado/metabolismo , Adulto , Humanos , Masculino , Valores de Referencia , Factores de TiempoRESUMEN
Impaired glucose effectiveness (i.e., a diminished ability of glucose per se to facilitate its own metabolism), increased gluconeogenesis, and endogenous glucose release are, together with insulin resistance and beta-cell abnormalities, established features of type 2 diabetes. To explore aspects of the pathophysiology behind type 2 diabetes, we assessed in a group of healthy people prone to develop type 2 diabetes (n = 23), namely first-degree relatives of type 2 diabetic patients (FDR), 1) endogenous glucose release and fasting gluconeogenesis measured using the 2H2O technique and 2) glucose effectiveness. The FDR group was insulin resistant when compared with an age-, sex-, and BMI-matched control group without a family history of type 2 diabetes (n = 14) (M value, clamp: 6.07 +/- 0.48 vs. 8.06 +/- 0.69 mg x kg(-1) lean body weight (lbw) x min(-1); P = 0.02). Fasting rates of gluconeogenesis (1.28 +/- 0.06 vs. 1.41 +/- 0.07 mg x kg(-1) lbw x min(-1); FDR vs. control subjects, P = 0.18) did not differ in the two groups and accounted for 53 +/- 2 and 60 +/- 3% of total endogenous glucose release. Glucose effectiveness was examined using a combined somatostatin and insulin infusion (0.17 vs. 0.14 mU x kg(-1) x min(-1), FDR vs. control subjects), the latter replacing serum insulin at near baseline levels. In addition, a 360-min labeled glucose infusion was given to simulate a prandial glucose profile. After glucose infusion, the integrated plasma glucose response above baseline (1,817 +/- 94 vs. 1,789 +/- 141 mmol/l per 6 h), the ability of glucose to simulate its own uptake (1.50 +/- 0.13 vs. 1.32 +/- 0.16 ml x kg(-1) lbw x min(-1)), and the ability of glucose per se to suppress endogenous glucose release did not differ between the FDR and control group. In conclusion, in contrast to overt type 2 diabetic patients, healthy people at high risk of developing type 2 diabetes are characterized by normal glucose effectiveness at near-basal insulinemia and normal fasting rates of gluconeogenesis.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Ingestión de Alimentos/fisiología , Ayuno/metabolismo , Gluconeogénesis , Glucosa/fisiología , Resistencia a la Insulina/fisiología , Adulto , Glucemia/análisis , Femenino , Predisposición Genética a la Enfermedad , Glucosa/metabolismo , Glucosa/farmacología , Hormonas/sangre , Humanos , Masculino , Concentración Osmolar , Valores de ReferenciaRESUMEN
Based on our earlier work, a 2.5-fold increase in insulin secretion should completely inhibit hepatic glucose production through the hormone's direct effect on hepatic glycogen metabolism. The aim of the present study was to test the accuracy of this prediction and to confirm that gluconeogenic flux, as measured by three independent techniques, was unaffected by the increase in insulin. A 40-min basal period was followed by a 180-min experimental period in which an increase in insulin was induced, with euglycemia maintained by peripheral glucose infusion. Arterial and hepatic sinusoidal insulin levels increased from 10 +/- 2 to 19 +/- 3 and 20 +/- 4 to 45 +/- 5 microU/ml, respectively. Net hepatic glucose output decreased rapidly from 1.90 +/- 0.13 to 0.23 +/- 0.16 mg. kg(-1). min(-1). Three methods of measuring gluconeogenesis and glycogenolysis were used: 1) the hepatic arteriovenous difference technique (n = 8), 2) the [(14)C]phosphoenolpyruvate technique (n = 4), and 3) the (2)H(2)O technique (n = 4). The net hepatic glycogenolytic rate decreased from 1.72 +/- 0.20 to -0.28 +/- 0.15 mg. kg(-1). min(-1) (P < 0.05), whereas none of the above methods showed a significant change in hepatic gluconeogenic flux (rate of conversion of phosphoenolpyruvate to glucose-6-phosphate). These results indicate that liver glycogenolysis is acutely sensitive to small changes in plasma insulin, whereas gluconeogenic flux is not.
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Gluconeogénesis/fisiología , Glucosa/metabolismo , Insulina/fisiología , Glucógeno Hepático/metabolismo , Hígado/metabolismo , Animales , Glucemia/metabolismo , Radioisótopos de Carbono/farmacocinética , Óxido de Deuterio/farmacocinética , Perros , Femenino , Glucagón/sangre , Hiperinsulinismo/sangre , Hiperinsulinismo/metabolismo , Insulina/sangre , Lactatos/sangre , Hígado/efectos de los fármacos , Masculino , Modelos Biológicos , Fosfoenolpiruvato/metabolismo , Técnica de Dilución de RadioisótoposRESUMEN
Effects of free fatty acids (FFAs) on endogenous glucose production (EGP) and gluconeogenesis (GNG) were examined in healthy subjects (n = 6) during stepwise increased Intralipid/heparin infusion (plasma FFAs 0.8+/-0.1, 1.8+/-0.2, and 2.8+/-0.3 mmol/l) and during glycerol infusion (plasma FFAs approximately 0.5 mmol/l). Rates of EGP were determined with D-[6,6-2H2]glucose and contributions of GNG from 2H enrichments in carbons 2 and 5 of blood glucose after 2H2O ingestion. Plasma glucose concentrations decreased by approximately 10% (P < 0.01), whereas plasma insulin increased by approximately 47% (P = 0.02) after 9 h of lipid infusion. EGP declined from 9.3+/-0.5 (lipid) and 9.0+/-0.8 pmol x kg(-1) x min(-1) (glycerol) to 8.4+/-0.5 and 8.2+/-0.7 micromol x kg(-1) x min(-1), respectively (P < 0.01). Contribution of GNG similarly rose (P < 0.01) from 46+/-4 and 52+/-3% to 65+/-8 and 78+/-7%. To exclude interaction of FFAs with insulin secretion, the study was repeated at fasting plasma insulin (approximately 35 pmol/l) and glucagon (approximately 90 ng/ml) concentrations using somatostatin-insulin-glucagon clamps. Plasma glucose increased by approximately 50% (P < 0.005) during lipid but decreased by approximately 12% during glycerol infusion (P < 0.005). EGP remained unchanged over the 9-h period (9.9+/-1.2 vs. 9.0+/-1.1 micromol x kg(-1) x min(-1)). GNG accounted for 62+/-5 (lipid) and 60+/-6% (glycerol) of EGP at time 0 and rose to 74+/-3% during lipid infusion only (P < 0.05 vs. glycerol: 64+/-4%). In conclusion, high plasma FFA concentrations increase the percent contribution of GNG to EGP and may contribute to increased rates of GNG in patients with type 2 diabetes.
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Ácidos Grasos no Esterificados/sangre , Gluconeogénesis , Glucosa/biosíntesis , Absorción , Adulto , Glucemia/análisis , Péptido C/sangre , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Emulsiones Grasas Intravenosas/farmacología , Femenino , Glicerol/sangre , Glicerol/farmacología , Heparina/farmacología , Hormonas/farmacología , Humanos , Insulina/sangre , Masculino , Concentración Osmolar , Somatostatina/farmacologíaRESUMEN
Several problems limit quantification of gluconeogenesis. We applied in vitro 2H-nuclear magnetic resonance (NMR) spectroscopy to simultaneously measure 2H in all glucose carbons for direct assessment of gluconeogenesis. This method was compared with 2H measurement in carbons 5 and 2 using gas chromatography-mass spectrometry (hexamethylenetetramine [HMT]) and with in vivo 13C magnetic resonance spectroscopy (MRS). After 14 h of fasting, and following 2H2O ingestion, blood was obtained from nine healthy and seven type 2 diabetic subjects. Glucose was purified, acetylated, and analyzed for 2H in carbons 1-6 with 2H-NMR. Using 5:2 ratios, gluconeogenesis increased (P < 0.05) over time and mean gluconeogenesis was lower in control subjects than in type 2 diabetic patients (63 +/- 3 vs. 75 +/- 2%, P < 0.01). 13C-MRS revealed higher hepatic glycogenolysis in control subjects (3.9 +/- 0.4 vs. 2.3 +/- 0.2 micromol.kg(-1).min(-1)) yielding mean contribution of gluconeogenesis of 65 +/- 3 and 77 +/- 2% (P < 0.005). Measurement of gluconeogenesis by 2H-NMR correlated linearly with 13C-MRS (r = 0.758, P = 0.0007) and HMT (r = 0.759, P = 0.0007). In an additional protocol, 2H enrichments demonstrated a fast decline of gluconeogenesis from approximately 100 to approximately 68% (P < 0.02) within 4 h of galactose infusion after 40-44 h of fasting. Thus, in vitro 2H-NMR offers an alternative approach to determine fractional gluconeogenesis in good agreement with standard methods and allows monitoring of rapid metabolic alterations.
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Fenómenos Fisiológicos Sanguíneos , Gluconeogénesis , Espectroscopía de Resonancia Magnética , Adulto , Sangre/metabolismo , Isótopos de Carbono , Deuterio , Galactosa/administración & dosificación , Glucógeno/metabolismo , Humanos , Infusiones Intravenosas , Hígado/metabolismo , MasculinoRESUMEN
To examine the mechanism by which metformin lowers endogenous glucose production in type 2 diabetic patients, we studied seven type 2 diabetic subjects, with fasting hyperglycemia (15.5 +/- 1.3 mmol/l), before and after 3 months of metformin treatment. Seven healthy subjects, matched for sex, age, and BMI, served as control subjects. Rates of net hepatic glycogenolysis, estimated by 13C nuclear magnetic resonance spectroscopy, were combined with estimates of contributions to glucose production of gluconeogenesis and glycogenolysis, measured by labeling of blood glucose by 2H from ingested 2H2O. Glucose production was measured using [6,6-2H2]glucose. The rate of glucose production was twice as high in the diabetic subjects as in control subjects (0.70 +/- 0.05 vs. 0.36 +/- 0.03 mmol x m(-2) min(-1), P < 0.0001). Metformin reduced that rate by 24% (to 0.53 +/- 0.03 mmol x m(-2) x min(-1), P = 0.0009) and fasting plasma glucose concentration by 30% (to 10.8 +/- 0.9 mmol/l, P = 0.0002). The rate of gluconeogenesis was three times higher in the diabetic subjects than in the control subjects (0.59 +/- 0.03 vs. 0.18 +/- 0.03 mmol x m(-2) min(-1) and metformin reduced that rate by 36% (to 0.38 +/- 0.03 mmol x m(-2) x min(-1), P = 0.01). By the 2H2O method, there was a twofold increase in rates of gluconeogenesis in diabetic subjects (0.42 +/- 0.04 mmol m(-2) x min(-1), which decreased by 33% after metformin treatment (0.28 +/- 0.03 mmol x m(-2) x min(-1), P = 0.0002). There was no glycogen cycling in the control subjects, but in the diabetic subjects, glycogen cycling contributed to 25% of glucose production and explains the differences between the two methods used. In conclusion, patients with poorly controlled type 2 diabetes have increased rates of endogenous glucose production, which can be attributed to increased rates of gluconeogenesis. Metformin lowered the rate of glucose production in these patients through a reduction in gluconeogenesis.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/antagonistas & inhibidores , Hipoglucemiantes/uso terapéutico , Metformina/uso terapéutico , Calorimetría Indirecta , Diabetes Mellitus Tipo 2/diagnóstico , Femenino , Gluconeogénesis/efectos de los fármacos , Gluconeogénesis/fisiología , Glucosa/biosíntesis , Glucosa/metabolismo , Glucógeno/metabolismo , Humanos , Hígado/metabolismo , Espectroscopía de Resonancia Magnética , Masculino , Persona de Mediana EdadRESUMEN
Pancreatic islets from fed 7-month old lean and obese hyperglycemic mice (ob/ob) were incubated with 3H2O and 5.5 mM or 16.7 mM glucose. Incorporation of 3H into the medium glucose was taken as the measure of glucose-6-P hydrolysis to glucose. Glucose utilization was measured from the yield of 3H2O from [5-3H]glucose. Only 3-4% of the glucose phosphorylated was dephosphorylated by the lean mouse islets irrespective of the glucose concentration. In contrast, the ob/ob mouse islets at 5.5 mM glucose dephosphorylated 18% of the glucose phosphorylated and 30% at 16.7 mM. Thus, the islets of hyperglycemic mice demonstrate increased glucose cycling as compared to the islets of normoglycemic lean mice.
Asunto(s)
Glucosa/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Glucosa-6-Fosfato , Glucofosfatos/metabolismo , Hidrólisis , Cinética , Ratones , Ratones Obesos , Fosforilación , TritioRESUMEN
Antisera prepared by immunization of rabbits with human T4-binding globulin (TBG) contained two populations of antibodies: one directed against determinants of the native molecule, and the other directed against antigenic sites present only in denatured TBG. These two populations of antibodies were present in all nine antisera prepared in this or other laboratories that were tested. Exploiting this property of anti-TBG sera and using radioiodinated denatured TBG as a tracer, a RIA was developed which measures specifically denatured TBG in the presence of native TBG. The RIA for measuring denatured TBG used purified native TBG, which was denatured by reduction and pyridylethylation (RP-TBG) before labeling with 125I. Native TBG was measured using the same antiserum, but the 125I-labeled tracer was unmodified TBG. The sensitivity of the native TBG RIA was 0.25 ng purified native TBG. Equivalent amounts of native TBG in serum, desialylated TBG, and deglycosylated TBG produced superimposeable standard curves. The cross-reactivity with RP-TBG was less than 0.02%. The denatured TBG RIA had a sensitivity of 1 ng, and superimposeable curves were produced with equivalent concentrations of RP-TBG and heat-denatured native TBG. The cross-reactivity of 0.8% with native and deglycosylated TBG was, at least in part, due to denatured TBG in the purified preparations. The specificity of the two RIAs is due to the existence of distinct and exclusive antigenic determinants in native TBG and denatured TBG which are probably located on the surface of the tertiary structure and internally at the primary structure of the molecule, respectively. Heat and acid pH treatments of serum produced a progressive loss in immunoreactive native TBG, proportional to the loss of T4-binding capacity. A reciprocal and quantitative increase in denatured TBG, as measured in the denatured TBG RIA, was found. T4 partially protected the native TBG from denaturation. Denatured TBG was detected in sera from normal adults. The mean value was 6.05 +/- 2.25 (+/- SD) micrograms/dl (n = 11). Similar values were found in 8 pregnant women, 5 men with familial partial TBG deficiency, and 15 hypothyroid 7 hepatic, and 8 renal failure patients.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Proteínas de Unión a Tiroxina/análisis , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Femenino , Calor , Humanos , Concentración de Iones de Hidrógeno , Recién Nacido , Masculino , Embarazo , Conformación Proteica , Desnaturalización Proteica , Conejos , RadioinmunoensayoRESUMEN
Hepatic fructose-6-phosphate (fructose-6-P) cycling and pentose cycle activity were quantified in hyperthyroid patients. A measure of the fructose-6-P cycle was the incorporation of 14C, on administering [3-3H,6-14C]galactose, into carbon 1 of blood glucose and the 3H/14C ratio in blood glucose. The measure of the pentose cycle was the randomization of 14C to carbon 1 of blood glucose on administering [2-14C]galactose. [2-3H]Galactose was also administered, so the 3H/14C ratio in blood glucose measured the extent of equilibration of glucose-6-P with fructose-6-P. Patients given [3-3H,6-14C]galactose were restudied when euthyroid. Of the 14C from [3-3H,6-14C]galactose, 7.7-9.5% was in carbon 1 of glucose in both states. 3H/14C ratios were also the same in both states. Fructose-6-P cycling was estimated to be 13 +/- 1% the rate of glucose turnover in the euthyroid and 15 +/- 1% that in the hyperthyroid state. The pentose cycle contributed about 2% to glucose utilization, similar to previous estimates in healthy humans. As in healthy individuals, about 25% of 3H was retained in the conversion of [2-3H]glucose-6-P to glucose. Thus, the fractions of glucose turnover participating in hepatic fructose-6-P and pentose cycling are similar in hyperthyroid and healthy subjects. As a result, augmented fructose-6-P cycling does not substantially contribute to increased hepatic oxygen consumption in hyperthyroidism.
Asunto(s)
Fructosafosfatos/metabolismo , Hipertiroidismo/metabolismo , Pentosas/metabolismo , Adulto , Glucemia/análisis , Femenino , Galactosa/administración & dosificación , Galactosa/metabolismo , Glucosa/metabolismo , Humanos , Hígado/metabolismo , Matemática , Persona de Mediana Edad , Consumo de Oxígeno , Distribución AleatoriaRESUMEN
OBJECTIVES: One-third of practices signing-out to The Children's Hospital Call Center in Denver, Colorado, choose to do second-level physician (SLP) triage for calls judged by the Center to require after-hours referral (AHR). We examined: 1) the effect of SLP triage on the rate of AHRs and 2) reasons for physicians' decisions. DESIGN: From January 1998 to August 1998 all calls from patients using a 5-member suburban pediatric practice judged by the Call Center to require AHR were referred to the practice's on-call physician who did SLP triage and completed a questionnaire. RESULTS: There were 955 eligible calls, 22% (N = 216) of which were initially given an urgent disposition by Call Center nurses. Physician questionnaires were completed for 97% (N = 209). Of patients initially triaged for AHR, 49% (N = 103) were subsequently given an AHR, 17% (N = 35) a next day office referral, and 34% (N = 71) home care and advice. Reasons for not urgently referring included the following: 1) medical problem didn't require urgent evaluation (95%, N = 99); 2) change in the patient's condition; (40% N = 43); 3) prior knowledge of family's ability to evaluate and care for the patient (40%, N = 43); and 4) knowledge of the patient's medical history (18%, N = 19). After SLP triage the overall urgent referral rate was 11%. CONCLUSIONS: Signing out to a Call Center decreased physicians' after-hours calls by 77% and SLP triage halved the number of urgent after-hours referrals.