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OBJECTIVE: To study alterations in plasma lipid profile and oxidative damage to lipoprotein fractions (LF) and their fatty acids during an early insulin-resistant and increased oxidative state developed by a fructose-rich diet (FRD). METHODS AND RESULTS: Wistar rats were fed a commercial diet with (FRD) or without (CD) 10% fructose in the drinking water. After 3 weeks, plasma glucose, triglyceride (TG), insulin (I), fructosamine (F), free fatty acids (FFA) and lipid profile (total cholesterol [TC] and HDL-C, LDL-C and VLDL-C sub-fractions) were determined. The insulin sensitivity HOMA index was assessed. FRD-fed rats had higher plasma TG, I, and F levels; increased HOMA; decreased HDL-C and LDL-C; augmented VLDL-C and TC/HDL-C, and TG/HDL-C atherogenic risk scores. LF of FRD rats had increased oxidative damage on the fatty acyl profile and in copper-induced lipoperoxidation. CONCLUSIONS: Fructose feeding early increases the atherogenic risk inducing an insulin resistant-glycoxidative state that affects plasma lipid profiles.

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The possible contribution of early changes in lipid composition, function, and antioxidant status of abdominal adipose tissue (AAT) induced by a fructose-rich diet (FRD) to the development of insulin resistance (IR) and oxidative stress (OS) was studied. Wistar rats were fed with a commercial diet with (FRD) or without 10% fructose in the drinking water for 3 weeks. The glucose (G), triglyceride (TG), and insulin (I) plasma levels, and the activity of antioxidant enzymes, lyposoluble antioxidants, total glutathione (GSH), lipid peroxidation as TBARS, fatty acid (FA) composition of AAT-TG as well as their release by incubated pieces of AAT were measured. Rats fed with a FRD have significantly higher plasma levels of G, TG, and I. Their AAT showed a marked increase in content and ratios of saturated to monounsaturated and polyunsaturated FAs, TBARS, and catalase, GSH-transferase and GSH-reductase, together with a decrease in superoxide dismutase and GSH-peroxidase activity, and total GSH, alpha-tocopherol, beta-carotene and lycopene content. Incubated AAT from FRD released in vitro higher amount of free fatty acids (FFAs) with higher ratios of saturated to monounsaturated and polyunsaturated FAs. Our data suggest that FRD induced an early prooxidative state and metabolic dysfunction in AAT that would favor the overall development of IR and OS and further development of pancreatic beta-cell failure; therefore, its early control would represent an appropriate strategy to prevent alterations such as the development of type 2 diabetes.

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Postprandial glucose and triglyceride increments after a mixed meal are more prolonged in people with type 1 and 2 diabetes or with impaired glucose tolerance than in normal individuals. Evidence in the literature suggests that these transient increases represent an additional and independent risk for chronic hyperglycemia to induce endothelial dysfunction, an important fact for the development of diabetic vascular complications. This article presents the more relevant mechanisms by which acute postprandial hyperglycemia and hyperlipidemia have been proved to determine the risk of reactive oxygen species overproduction, an increased synthesis of non enzymatic early-glycated and nitrated proteins, and a more atherogenic lipoprotein profile. Recent recommendations suggest that care for this transient glycoxidative stress should be associated with fasting glucose or HbA1c care, to reduce the risk of macro- and microvascular complications in people with diabetes.

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The aim of this study was to develop an oviparous model suitable for studying the differential effects and mechanisms by which a high concentration of extracellular glucose and other sugars produce diabetes complications, particularly body growth retardation during development. Hence, we studied the experimental conditions necessary to obtain measurable effects of high sugar concentrations (5-mM glucose, mannitol, fructose and galactose) upon body growth and development of Bufo arenarum embryos and larvae, and upon the activity of aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), and alkaline phosphatase (APP). Unfed animals kept in glucose showed lower body weight than controls at all stages, a condition only observed at stage 26 for animals kept in galactose and fructose. All animals reached the same stage of development regardless of the solution in which they were kept. Glucose and fructose significantly decreased the activity of all enzymes tested, while galactose only affected GGT activity. The model provides the first experimental evidence for the deleterious effect exerted in vivo by different sugars upon developing embryos and larvaes of Bufo arenarum. The results prove that this model might help to elucidate the effects and the pathogenic mechanisms of hyperglycemia upon growth and development of embryos exposed to environments with high sugar concentrations. It might also become a useful tool for testing the effectiveness of drugs designed to prevent the deleterious effect of such exposure.

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Chronic hyperglycemia induces an increase in the non enzymatic glycation of circulating and structural proteins together with a glucose-generated oxidative and carbonyl stress, known as glycoxidation. The physicochemical characteristics and the metabolism of lipoproteins are altered by glycation/glycoxidation and resemble those of other body proteins, except for the fact that there is a simultaneous glycoxidation of both protein and phospholipid components generating an oxidative stress that increases lipoxidation. Information gathered during the last few years suggests that, among lipoproteins, modified LDL would principally contribute to developing diabetic micro-macrovascular complications. The control and the prevention of the progress of such complications are difficult to attain due to the irreversibility of glycoxidation. As glycation/glycoxidation is related to mean blood glucose, the goal in diabetes treatment must be the achievement of a close to normal metabolic control. This review summarizes advances in the importance of lipoprotein glycation/glycoxidation in diabetes mellitus.

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Chronic hyperglycemia induces an increase in the non enzymatic glycation of circulating and structural proteins together with a glucose-generated oxidative and carbonyl stress, known as glycoxidation. The physicochemical characteristics and the metabolism of lipoproteins are altered by glycation/glycoxidation and resemble those of other body proteins, except for the fact that there is a simultaneous glycoxidation of both protein and phospholipid components generating an oxidative stress that increases lipoxidation. Information gathered during the last few years suggests that, among lipoproteins, modified LDL would principally contribute to developing diabetic micro-macrovascular complications. The control and the prevention of the progress of such complications are difficult to attain due to the irreversibility of glycoxidation. As glycation/glycoxidation is related to mean blood glucose, the goal in diabetes treatment must be the achievement of a close to normal metabolic control. This review summarizes advances in the importance of lipoprotein glycation/glycoxidation in diabetes mellitus.

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El objetivo del estudio fue determinar la frecuencia y características clínicas y microbiológicas de las infecciones del tracto urinario (ITU) en pacientes diabéticos (DM) y no diabéticos (NoDM) ambulatorios. Los pacientes se evaluaron a través del análisis de urocultivos (UC) y de una encuesta individual relevada en el momento del análisis. Los UC se efectuaron en tres laboratorios microbiológicos con metodología estandarizada y la encuesta se registró en un formulario ad hoc. Se incluyeron 2.379 pacientes, de los cuales 208 eran DM(8.7). El porcentaje de UC positivos fue significativamente mayor en los DM (34.1) que en los NoDM (20.7; X² p=0.000001). La presencia de UC positivo fue mayor en mujeres DM (35.7) que en las NoDM (20.4;X²p=0.002). En pacientes con UC positivo el antecedente de ITU fue mas frecuente en los DM (62) que en los NoDM(44). No hubo diferencias significativas con respecto a la presencia de síntomas en ambos grupos, siendo la disuria el síntoma más frecuente. No hubo diferencias significativas en la frecuencia y tipo de microorganismos aislados en ambos grupos. El análisis de la sensibilidad de los antimicrobianos mostró alta resistencia a aminopenicilina (AMN) y trimetoprima-sulfametoxazol(TMS) y una actividad aceptable de la cefalexina(CEF). Estos resultados confirman la mayor frecuencia de ITU en los DM, especialmente en mujeres,y su mayor recurrencia. La AMN y la TMS no deberían utilizarse como tratamiento empírico de las ITU ambulatorias por su baja actividad, siendo la CEF una buena elección. Las fluorquinolonas presentaron el menor índice de resistencia, aunque mayor al descripto

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El objetivo del estudio fue determinar la frecuencia y características clínicas y microbiológicas de las infecciones del tracto urinario (ITU) en pacientes diabéticos (DM) y no diabéticos (NoDM) ambulatorios. Los pacientes se evaluaron a través del análisis de urocultivos (UC) y de una encuesta individual relevada en el momento del análisis. Los UC se efectuaron en tres laboratorios microbiológicos con metodología estandarizada y la encuesta se registró en un formulario ad hoc. Se incluyeron 2.379 pacientes, de los cuales 208 eran DM(8.7). El porcentaje de UC positivos fue significativamente mayor en los DM (34.1) que en los NoDM (20.7; X² p=0.000001). La presencia de UC positivo fue mayor en mujeres DM (35.7) que en las NoDM (20.4;X²p=0.002). En pacientes con UC positivo el antecedente de ITU fue mas frecuente en los DM (62) que en los NoDM(44). No hubo diferencias significativas con respecto a la presencia de síntomas en ambos grupos, siendo la disuria el síntoma más frecuente. No hubo diferencias significativas en la frecuencia y tipo de microorganismos aislados en ambos grupos. El análisis de la sensibilidad de los antimicrobianos mostró alta resistencia a aminopenicilina (AMN) y trimetoprima-sulfametoxazol(TMS) y una actividad aceptable de la cefalexina(CEF). Estos resultados confirman la mayor frecuencia de ITU en los DM, especialmente en mujeres,y su mayor recurrencia. La AMN y la TMS no deberían utilizarse como tratamiento empírico de las ITU ambulatorias por su baja actividad, siendo la CEF una buena elección. Las fluorquinolonas presentaron el menor índice de resistencia, aunque mayor al descripto(AU)

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El objetivo de este estudio fue establecer en nuestra población valores de referencia para fructosamina en embarazadas normales durante el primero, segundo y tercer trimestre de gestación y en niños de primera (<1-6 años) y segunda (6-12) infancia. El límite superior de fructosamina (expresado como 97,5 percentilo) para adultos de nuestro medio (271 µmol/L) no difiere del hallado en estudios multicéntricos. En cambio son menores los límites de normalidad para embarazadas de segundo (231 µmo/L) y tercer (221 µmol/L) trimestre de embarazo, y niños de primera infancia (239 µmol/L). Lo mismo ocurre al expresar los valores normalizados por proteinemia. Exiten correlaciones inversas significativasentre la concentración de fructosamina y a) el progreso de la edad de gestación(r=0,64; p<0,001; n= 147), y b) el incremento de la edad en los niños de 0 a 12 años (r=0,63; p<0,001; n= 137). Estos resultados demuestran la necesidad de definir rangos de normalidad independientes para los adultos, los niños de primera infancia y las embarazadas de acuerdo a su edad de gestación

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El objetivo de este estudio fue establecer en nuestra población valores de referencia para fructosamina en embarazadas normales durante el primero, segundo y tercer trimestre de gestación y en niños de primera (<1-6 años) y segunda (6-12) infancia. El límite superior de fructosamina (expresado como 97,5 percentilo) para adultos de nuestro medio (271 Amol/L) no difiere del hallado en estudios multicéntricos. En cambio son menores los límites de normalidad para embarazadas de segundo (231 Amo/L) y tercer (221 Amol/L) trimestre de embarazo, y niños de primera infancia (239 Amol/L). Lo mismo ocurre al expresar los valores normalizados por proteinemia. Exiten correlaciones inversas significativasentre la concentración de fructosamina y a) el progreso de la edad de gestación(r=0,64; p<0,001; n= 147), y b) el incremento de la edad en los niños de 0 a 12 años (r=0,63; p<0,001; n= 137). Estos resultados demuestran la necesidad de definir rangos de normalidad independientes para los adultos, los niños de primera infancia y las embarazadas de acuerdo a su edad de gestación (AU)

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Se valoró microalbuminuria (MA), simultáneamente con una tira reactiva (TR), semicuantitativa, un método inmunoturbidimétrico (IT), cuantitativo y el readioinmunoanálisis (RIA). El objetivo fue validar los métodos ensayados y verificar la influencia del tiempo y forma de recolección de la muestra. TR mostró buena concordancia para ocho observadores en 11 muestras de concentración entre 5-100 mg/L. Los CV intra e interensayo fueron: IT=4,7 y 4,9 ; RIA=6,6 y 9. Se hallaron los siguientes coeficientes de correlación (n=130):r=0.83,p<10-6(TA vs RIA y TR vs IT) y r=0.93, p<1-6(IT vs RIA). TR sería adecuado para la determinación semicuantitativa rápida de MA. Permitiría seleccionar con relativa especificidad y buena sensibilidad (falsos positivos 23 y falsos negativos 2, pacientes que luego podrían estudiarse con técnicas cuantitativas de mayor exactitud. IT tiene alta precisión y exactitud y su correlación con RIA es altamente significativa. En relación con la muestra de orina, la más conveniente sería la de 24 h, especialmente para los diabéticos. Ni la temperatura ni el recipiente de recolección afectan el resultado

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Se valoró microalbuminuria (MA), simultáneamente con una tira reactiva (TR), semicuantitativa, un método inmunoturbidimétrico (IT), cuantitativo y el readioinmunoanálisis (RIA). El objetivo fue validar los métodos ensayados y verificar la influencia del tiempo y forma de recolección de la muestra. TR mostró buena concordancia para ocho observadores en 11 muestras de concentración entre 5-100 mg/L. Los CV intra e interensayo fueron: IT=4,7 y 4,9 ; RIA=6,6 y 9. Se hallaron los siguientes coeficientes de correlación (n=130):r=0.83,p<10-6(TA vs RIA y TR vs IT) y r=0.93, p<1-6(IT vs RIA). TR sería adecuado para la determinación semicuantitativa rápida de MA. Permitiría seleccionar con relativa especificidad y buena sensibilidad (falsos positivos 23 y falsos negativos 2, pacientes que luego podrían estudiarse con técnicas cuantitativas de mayor exactitud. IT tiene alta precisión y exactitud y su correlación con RIA es altamente significativa. En relación con la muestra de orina, la más conveniente sería la de 24 h, especialmente para los diabéticos. Ni la temperatura ni el recipiente de recolección afectan el resultado (AU)

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The perimeter, cell area and volume density (Vvi) of B cells and exocytotic images present in these cells were measured in rat pancreas perfused with 3.3 or 16.6 mM glucose. Four minutes after the beginning of 16.6-mM glucose perfusion and coincident with the appearance at the apex of the first phase of insulin secretion, all these parameters underwent a significant increase. The changes observed in the perimeter, the cell area and the Vvi of B cells suggest an increase in their surface area. An imbalance in the rate of endocytosis:exocytosis processes with a relative predominance of the latter would increase the length of the plasma membrane and could be responsible, at least partly, for the changes in the B cell size.

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Insulin secretion and B-cell calcium distribution, assessed with the pyroantimonate precipitation technique, were studied in rat pancreases perfused with glucose (3.3 or 16.6 mM) alone or together with verapamil or trifluoperazine (TFP). Total calcium pyroantimonate precipitates (CPP), and those bound to every B cell structure, at every sampling period, were larger at 16.6 mM glucose concentration. The largest percentage of CPP was located, at early stages of the glucose stimulatory period, mainly within the clear halo of the B granules, while later on they shifted to the plasma membrane. Verapamil and TFP diminished the second phase of glucose-induced insulin release and greatly affected the above mentioned pattern of B cell CPP distribution. The main changes consist in a diminution in the total number of CPP all throughout the perfusion-time as well as an alteration in the percentage distribution of the CPP within the different B cell organelles, i.e., an early diminution in the CPP present in the B granules and of those attached to the plasma membrane and to the mitochondria at the end of the perfusion, were the most striking changes observed. The results suggest that during the glucose stimulus, different B cell structures take over the control of available calcium within the cell, following a chronological sequence. Such sequence might be determined by the different Ca2(+)-set point of those structures. Intracellular provision of calcium might be sufficient to maintain the early phase of insulin secretion when the cation entrance is blocked. Conversely, this substitution might not be enough to sustain the second phase of insulin release. The different amounts of CPP in every B cell organelle, besides their buffering capacity to control free Ca2+ availability, might also be coupled to a regulatory role of the cation upon their respective metabolic functions. In some cases, this latter effect may be the main role for the cation distribution. Our results support the concept that the level of free calcium, acting as the coupler for the stimulus:secretion process, might be regulated by different calcium pools located in the B cell.

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Insulin secretion and the pattern of calcium distribution in B cells, assessed with the pyroantimonate precipitation technique, were simultaneously studied in rat pancreases perfused with 3.3 and 16.6 mM glucose solutions of pH 7.4 and 7.8. We have previously demonstrated the blocking effect of the latter pH upon glucose-induced insulin secretion. Glucose (16.6 mM) caused an increase in the total number of calcium pyroantimonate precipitates (CPP), as well as their number bound to different B cell structures, at every sampling period studied, with respect to the 3.3 mM glucose experiments. Extracellular alkalosis strongly inhibited both phases of the B cell response to the glucose stimulus, and greatly affected the distribution of CPP in the cells with respect to the pH 7.4 ones. During the first phase of glucose induced-insulin secretion, most of the CPP appeared within B granules at pH 7.4, while on the development of the second phase of secretion, they appeared mainly attached to the cell plasma membranes. Conversely, in pH 7.8 experiments, at the first minutes of the glucose challenge, CPP appeared principally located in the cytoplasm, being almost absent from the plasma membrane during the second phase of insulin secretion. These observations suggest that during the glucose stimulus, the cell calcium distribution within the B cells followed a clear chronological sequence. Such sequence might be determined, at least in part, according to the different Ca2(+)-set points of the different B cell structures. In our case, the extracellular alkalosis might interfere with the normal intracellular calcium fluxes, which in consequence might impair release of insulin by affecting several B cell functions.

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Insulin secretion and B-cell calcium distribution, assessed with the pyroantimonate precipitation technique, were studied in rat pancreases perfused with glucose (3.3 or 16.6 mM) alone or together with verapamil or trifluoperazine (TFP). Total calcium pyroantimonate precipitates (CPP), and those bound to every B cell structure, at every sampling period, were larger at 16.6 mM glucose concentration. The largest percentage of CPP was located, at early stages of the glucose stimulatory period, mainly within the clear halo of the B granules, while later on they shifted to the plasma membrane. Verapamil and TFP diminished the second phase of glucose-induced insulin release and greatly affected the above mentioned pattern of B cell CPP distribution. The main changes consist in a diminution in the total number of CPP all throughout the perfusion-time as well as an alteration in the percentage distribution of the CPP within the different B cell organelles, i.e., an early diminution in the CPP present in the B granules and of those attached to the plasma membrane and to the mitochondria at the end of the perfusion, were the most striking changes observed. The results suggest that during the glucose stimulus, different B cell structures take over the control of available calcium within the cell, following a chronological sequence. Such sequence might be determined by the different Ca2(+)-set point of those structures. Intracellular provision of calcium might be sufficient to maintain the early phase of insulin secretion when the cation entrance is blocked. Conversely, this substitution might not be enough to sustain the second phase of insulin release. The different amounts of CPP in every B cell organelle, besides their buffering capacity to control free Ca2+ availability, might also be coupled to a regulatory role of the cation upon their respective metabolic functions. In some cases, this latter effect may be the main role for the cation distribution. Our results support the concept that the level of free calcium, acting as the coupler for the stimulus:secretion process, might be regulated by different calcium pools located in the B cell.

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Insulin secretion and the pattern of calcium distribution in B cells, assessed with the pyroantimonate precipitation technique, were simultaneously studied in rat pancreases perfused with 3.3 and 16.6 mM glucose solutions of pH 7.4 and 7.8. We have previously demonstrated the blocking effect of the latter pH upon glucose-induced insulin secretion. Glucose (16.6 mM) caused an increase in the total number of calcium pyroantimonate precipitates (CPP), as well as their number bound to different B cell structures, at every sampling period studied, with respect to the 3.3 mM glucose experiments. Extracellular alkalosis strongly inhibited both phases of the B cell response to the glucose stimulus, and greatly affected the distribution of CPP in the cells with respect to the pH 7.4 ones. During the first phase of glucose induced-insulin secretion, most of the CPP appeared within B granules at pH 7.4, while on the development of the second phase of secretion, they appeared mainly attached to the cell plasma membranes. Conversely, in pH 7.8 experiments, at the first minutes of the glucose challenge, CPP appeared principally located in the cytoplasm, being almost absent from the plasma membrane during the second phase of insulin secretion. These observations suggest that during the glucose stimulus, the cell calcium distribution within the B cells followed a clear chronological sequence. Such sequence might be determined, at least in part, according to the different Ca2(+)-set points of the different B cell structures. In our case, the extracellular alkalosis might interfere with the normal intracellular calcium fluxes, which in consequence might impair release of insulin by affecting several B cell functions.

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The acute ultrastructural changes induced by glucose upon the B cells were studied in normal rat pancreas perfused with 3.3 and 16.6 mmol/l glucose. A significant increment in the volume of the RER, microtubule, mitochondria, lysosomes and B granules was induced by 16.6 mmol/l glucose, while no significant changes were detected in the total B cell volume or in the size of the nucleus, cytoplasm and Golgi complex. The number of secretory granules was greatly reduced in B cells obtained from pancreas perfused with 16.6 mmol/l glucose, while its diameter was significantly enhanced. In these cells both, the number of pale granules as well as those attached to the cell membrane, were increased. All these data suggest that the increase in the extracellular glucose concentration produces not only the classical biphasic secretion on insulin, but also induces significant and measurable changes in the volume of several B cell organelles. Such ultrastructural changes correlate well with the well-known effect of glucose upon the metabolism of these cells.

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The acute ultrastructural changes induced by glucose upon the B cells were studied in normal rat pancreas perfused with 3.3 and 16.6 mmol/l glucose. A significant increment in the volume of the RER, microtubule, mitochondria, lysosomes and B granules was induced by 16.6 mmol/l glucose, while no significant changes were detected in the total B cell volume or in the size of the nucleus, cytoplasm and Golgi complex. The number of secretory granules was greatly reduced in B cells obtained from pancreas perfused with 16.6 mmol/l glucose, while its diameter was significantly enhanced. In these cells both, the number of pale granules as well as those attached to the cell membrane, were increased. All these data suggest that the increase in the extracellular glucose concentration produces not only the classical biphasic secretion on insulin, but also induces significant and measurable changes in the volume of several B cell organelles. Such ultrastructural changes correlate well with the well-known effect of glucose upon the metabolism of these cells.

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Quantitative changes in the distribution of intracellular calcium in A cells from perfused rat pancreas in relation to the secretory state of A cells were studied with the pyroantimonate technique for calcium precipitation. A cells stimulated with a 3.3 mM glucose concentration in the perfusate presented numerous calcium precipitates attached to cell membranes, nucleus, cytoplasm and secretion granules. Independently of the length of treatment, inhibition of glucagon release with 16.6 mM glucose decreased the calcium precipitates in every cell organelle studied. These results suggest that intracellular calcium rearrangement might be important in coupling stimulus to secretion in A cells, as it has been demonstrated for pancreatic B cells.

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