Deletion of protein kinase Cε in mice has limited effects on liver metabolite levels but alters fasting ketogenesis and gluconeogenesis.

AIMS/HYPOTHESIS: Protein kinase Cε (PKCε) is emerging as a key mediator of lipid-induced insulin resistance in liver and hepatic lipid metabolism itself. We investigated whether PKCε plays a role in other metabolic processes, to further examine its suitability as a therapeutic target. METHODS: We measured amino acid, organic acid and sugar levels by liquid and gas chromatography-mass spectrometry of liver extracts from chow and fat-fed wild-type (WT) and PKCε-deficient (Prkce(-/-)) mice. Fed and fasting glucose, ketone and fatty acid levels were measured in blood. Triacylglycerol levels and gluconeogenic and ketogenic enzyme expression were measured in liver. The effect of fasting on epididymal fat pad mass was also determined. RESULTS: Metabolomic analysis indicated that the short-term high-fat diet affected over 20 compounds, including a 50% reduction in the glucogenic amino acid alanine. Prkce deletion resulted only in a reduction of 4-hydroxyproline and aspartate and an increase in glutamate. However, upon fasting, Prkce(-/-) mice were better able to maintain blood glucose levels and also exhibited lower levels of the ketone ß-hydroxybutyrate compared with WT mice. Upon fasting, Prkce deletion also resulted in lower liver and plasma lipids and a smaller reduction in fat pad mass. CONCLUSIONS/INTERPRETATION: Metabolomic analysis provided new insights into the effects of a high-fat diet on liver metabolite levels. Glucose homeostasis under fasting conditions is improved in Prkce(-/-) mice, which, in turn, may reduce the mobilisation of lipid from adipose tissue, reducing the availability of ketogenic substrate in the liver. Together with the protection against fat-diet-induced glucose intolerance previously observed in the fed state, these findings indicate PKCε as a unique therapeutic target for the improvement of glucose homeostasis.

Time-dependent effects of Prkce deletion on glucose homeostasis and hepatic lipid metabolism on dietary lipid oversupply in mice.

AIMS/HYPOTHESIS: We examined the time-dependent effects of deletion of the gene encoding protein kinase C epsilon (Prkce) on glucose homeostasis, insulin secretion and hepatic lipid metabolism in fat-fed mice. METHODS: Prkce(-/-) and wild-type (WT) mice were fed a high-fat diet for 1 to 16 weeks and subjected to i.p. glucose tolerance tests (ipGTT) and indirect calorimetry. We also investigated gene expression and protein levels by RT-PCR, quantitative protein profiling (isobaric tag for relative and absolute quantification; iTRAQ) and immunoblotting. Lipid levels, mitochondrial oxidative capacity and lipid metabolism were assessed in liver and primary hepatocytes. RESULTS: While fat-fed WT mice became glucose intolerant after 1 week, Prkce(-/-) mice exhibited normal glucose and insulin levels. iTRAQ suggested differences in lipid metabolism and oxidative phosphorylation between fat-fed WT and Prkce(-/-) animals. Liver triacylglycerols were increased in fat-fed Prkce(-/-) mice, resulting from altered lipid partitioning which promoted esterification of fatty acids in hepatocytes. In WT mice, fat feeding elevated oxygen consumption in vivo and in isolated liver mitochondria, but these increases were not seen in Prkce(-/-) mice. Prkce(-/-) hepatocytes also exhibited reduced production of reactive oxygen species (ROS) in the presence of palmitate. After 16 weeks of fat feeding, however, the improved glucose tolerance in fat-fed Prkce(-/-) mice was instead associated with increased insulin secretion during ipGTT, as we have previously reported. CONCLUSIONS/INTERPRETATION: Prkce deletion ameliorates diet-induced glucose intolerance via two temporally distinct phenotypes. Protection against insulin resistance is associated with changes in hepatic lipid partitioning, which may reduce the acute inhibitory effects of fatty acid catabolism, such as ROS generation. In the longer term, enhancement of glucose-stimulated insulin secretion prevails.

Saturated- and n-6 polyunsaturated-fat diets each induce ceramide accumulation in mouse skeletal muscle: reversal and improvement of glucose tolerance by lipid metabolism inhibitors.

Lipid-induced insulin resistance is associated with intracellular accumulation of inhibitory intermediates depending on the prevalent fatty acid (FA) species. In cultured myotubes, ceramide and phosphatidic acid (PA) mediate the effects of the saturated FA palmitate and the unsaturated FA linoleate, respectively. We hypothesized that myriocin (MYR), an inhibitor of de novo ceramide synthesis, would protect against glucose intolerance in saturated fat-fed mice, while lisofylline (LSF), a functional inhibitor of PA synthesis, would protect unsaturated fat-fed mice. Mice were fed diets enriched in saturated fat, n-6 polyunsaturated fat, or chow for 6 wk. Saline, LSF (25 mg/kg x d), or MYR (0.3 mg/kg x d) were administered by mini-pumps in the final 4 wk. Glucose homeostasis was examined by glucose tolerance test. Muscle ceramide and PA were analyzed by mass spectrometry. Expression of LASS isoforms (ceramide synthases) was evaluated by immunoblotting. Both saturated and polyunsaturated fat diets increased muscle ceramide and induced glucose intolerance. MYR and LSF reduced ceramide levels in saturated and unsaturated fat-fed mice. Both inhibitors also improved glucose tolerance in unsaturated fat-fed mice, but only LSF was effective in saturated fat-fed mice. The discrepancy between ceramide and glucose tolerance suggests these improvements may not be related directly to changes in muscle ceramide and may involve other insulin-responsive tissues. Changes in the expression of LASS1 were, however, inversely correlated with alterations in glucose tolerance. The demonstration that LSF can ameliorate glucose intolerance in vivo independent of the dietary FA type indicates it may be a novel intervention for the treatment of insulin resistance.

Muscle atrophy in titin M-line deficient mice.

We investigated the response to deletion of the titin M-line region in striated muscle, using a titin knockout model and a range of techniques that include histology, in situ hybridization, electron microscopy, and 2D gel analysis. We found that the loss of titin's kinase domain and binding sites for myomesin and MURF-1 causes structural changes in the sarcomere that proceed from the M-line to the Z-disc and ultimately result in disassembly of the sarcomere. Disassembly goes along with central localization of nuclei (a hallmark for muscular dystrophy), up-regulation of heat-shock proteins, and induction of proteasome activity. While fiber type composition does not change in soleus and extensor digitorum longus muscle, fiber size is reduced. Animals die from complications of muscle atrophy at five weeks of age. In addition to the structural importance of the titin M-line region in any striated muscle, our data show how differences in M-line composition between heart and skeletal muscle affect sarcomere stability and function.

Subtipos no clásicos de diabetes mellitus / Non classical subtypes of diabetes mellitus

Background: Some adult, obese and diabetic patients, initiate their disease with a severe diabetic ketoacidosis without a precipitating factor and do not require insulin thereafter. These patients are classified as having a non classical diabetes mellitus. Aim: To study the clinical, immunological, genetic and metabolic features of patients with non classical diabetes mellitus. Patients and methods: Ten patients (9 men, aged 45ñ12 years old) with non classical diabetes mellitus were studied. Anti islet and anti glutamic acid decarboxylase antibodies (ICA and anti GAD), HLA DQ a arginine 52 and non aspartic ß57 were measured. Insulin secretion was measured by C peptide after glucagon injection and with the minimal model of Bergman. The latter model was also used to determine insulin sensitivity. Results: Three patients were immunologically classified as type 1, since they had positive ICA or antiGAD antibodies and type 1 genetics (neutral or susceptible HLA DQ a and ß). They had insulin secretion after glucagon stimulus (C peptide ranging from 2.2 to 7.5 pmol/ml), but an almost absent response to a glucose load. They were also insulin resistant (a sensitivity index ranging from 0.05 to 1.67 x 10-4 min/µU x ml). These three cases could be categorized as latent type 1. The other seven patients were ICA negative and antiGAD negative. Five had a susceptible HLA genotype for type 1 diabetes and two were neutral. All had insulin secretion after glucagon stimulation and a variable response to glucose. Six were insulin resistant (sensitivity index ranging from 0.32 to 1.29 x 10-4 min/µU x ml). One patient was insulin sensitive (sensitivity index of 3.83 x 10-4 min/µU x ml). Therefore all these patients were classified as type two diabetics with an atypical debut. Conclusions: Not all diabetics presenting with a severe diabetic ketoacidosis are type I. Among these, there are subjects with a latent type 1 diabetes or with an atypical type 2 diabetes

Contribución del manejo nutricional al tratamiento de las porfirias hepáticas / Nutritional management into the treatment of hepatic porphyrias

Las porfirias son un conjunto de enfermedades metabólicas debidas a alteraciones, genéticas o adquiridas, de la actividad de enzimas de la vía de síntesis de las porfirinas. Los pacientes con porfirias de tipo agudo en remisión, deben someterse a una dieta personalizada hipograsa con un alto contenido de carbohidratos y un adecuado aporte de fibra dietaria y nutrientes esenciales. Los pacientes con porfirias agudas deben evitar la ingesta de alcohol, los ayunos prolongados y los ejercicios extenuantes. El tratamiento de elección de las crisis porfíricas es la administración por vía enteral y/o parenteral de una sobrecarga de carbohidratos. Los pacientes con porfirias con alteración cutáneas, deben seguir un régimen dietario similar al de aquellos con profirias agudas en fase de remisión. En la protoporfiria eritrocitaria se debe asegurar un adecuado aporte de hierro, en cambio en la porfiria cutánea tarda debe restringirse su ingesta, especialmente si es hemínico, así como también el consumo de alcohol

Diabetes mellitus descompensada y acidosis metabólica hiperclorémica: asociación de patologías en un caso / Association of hyperglycemia and hyperchloremic acidosis in a diabetic patient

Diabetic ketoacidosis is manifested by elevated blood glucose levels, ketosis and metabolic acidosis with increased anion gap. A transitory hyperchloremic acidosis. with normal anion gap, can appear. We report a 21 years old female with a type 2 diabetes mellitus, admitted to the emergency room of a general hospital with hyperglycemia, absence of ketonemia, severe hypokalemia and hyperchloremic metabolic acidosis. Initially, she was diagnosed and treated as a severe diabetic ketoacidosis. Normal blood glucose levels were rapidly achieved but electrolyte and acid base alterations persisted, leading to the suspicion that another associated condition was causing the acidosis and hypokalemia. Urinary pH and anion gap measurement, the study of renal acidification and a bicarbonate overload test lead to the diagnosis of a distal renal tubular acidosis, secondary to a Sjögren syndrome, that was confirmed with a Schirmer test and positive anti Ro antibodies. In this diabetic patient, the acute hyperglycemia intensified the hypokalemia of her distal renal tubular acidosis and unchained the acute metabolic condition

Nueva clasificación y criterios diagnósticos de la diabetes mellitus / The new classification and diagnostic criteria for diabetes mellitus

Se presenta y comenta la nueva clasificación y criterios diagnósticos de diabetes mellitus (DM), preparado por un grupo de expertos de la Asociación Americana de Diabetes. La nueva clasificación tiene un fundamento etiopatogénico, la antigua clasificación OMS 1985 era de índole terapéutico. Las llamadas diabetes insulinodependientes y diabetes no insulinodependiente, pasan a denominarse tipo 1 y tipo 2, respectivamente. Se mantienen las clases diabetes gestacional e intolerancia a la glucosa, agregándose a esta última el estado de intolerancia a la glucosa de ayuno. Para la pesquisa y diagnóstico de diabetes mellitus se recomienda la glicemia de ayuno y se fija la cifra >126 mg/dl (7,0 mmol/1) como nivel de anormalidad, antiguamente >140 mg/dl. Este cambio se realizó, debido a que los individuos con glicemias de ayunas entre los valores anotados desarrollan complicaciones crónicas. También se consideran diabéticos a los sujetos con una glicemia aleatoria en cualquier momento del día >200 mg/dl y la tercera alternativa diagnóstico corresponde a esta misma cifra, pero obtenida a las dos horas de una carga de 75 g de glucosa. En ausencia de síntomas debe repetirse el examen para confinnar el diagnóstico. Se establece como glicemias normales en ayunas a las menores de 110 mg/dl, los valores intermedios, entre 100 y 126 mg/dl, constituyen el estado de intolerancia a la glucosa de ayuno. No se modifican las cifras de intolerancia a la glucosa, las que corresponden a glicemias >140 y menor de 200 mg/dl a las dos horas post carga. Se mantuvieron los criterios diagnósticos del National Diabetes Data Group para diabetes gestacional, recomendándose realizar pesquisa en todas las mujeres con riesgo entre las semanas 24 y 28 de embarazo, con una glicemia una hora después de ingerir 50 g de glucosa, a cualquier hora del día. Las embarazadas con valores de glicemia >140 mg/dl deben ser sometidas a una prueba de tolerancia con 100g de glucosa y cuatro muestras horarias. El Comité considera la existencia de un grupo de gestantes de bajo riesgo en quienes no es necesario realizar pesquisa. El Grupo de Expertos, al igual que el informe de la OMS 1985, concluye que para la prueba de tolerancia a la glucosa oral sólo se requiere la glicemia de ayunas y la de las dos horas después de 75g de glucosa. Las glicemias intermedias no son necesarias para la práctica clínica. Se resalta además...

DNA-damage detection in man after radiation exposure--the comet assay--its possible application for human biomonitoring.

The exposure of human beings to ionizing radiation is still of great concern to occupational and environmental medicine. The goal of this workshop is to identify a panel of biological markers that could be used in humans after exposure to ionizing radiation. The comet assay or single cell gel (SCG) assay is a new method that allows efficient determination of single-strand breaks (SSB) and double-strand breaks (DSB), as well as alkali-labile sites in the DNA of single cells. In order to demonstrate the practicability of the comet assay for the detection of DNA damage caused by low doses of ionizing radiation, we exposed human peripheral blood cells to radiation in vitro. The extent of DNA damage in blood cells irradiated with x-rays (0.05-1 Gy) was significantly increased above the control values even at 0.05 Gy and shows a clear dose-relationship. To investigate the repair kinetics for x-ray-induced DNA damage following acute and chronic (fractionated) irradiation, we exposed peripheral blood to 1 Gy and examined the tail moment at different time intervals. The effect of one acute dose is repaired within two h, whereas the effect of fractionated irradiation gives a totally different result. The tail moment of the initial damage increased indicating an accumulation of the damage, and the repair activity clearly decreased. Until now, there was no data available concerning DNA damage in vivo. For this reason, we explored patients subjected to radioiodine therapy as well as a Chernobyl liquidator.(ABSTRACT TRUNCATED AT 250 WORDS)

Does physical activity induce DNA damage?

The single cell gel electrophoresis (SCG) assay (comet assay) is a sensitive technique for detecting the presence of DNA strand-breaks and alkali-labile damage in individual cells. This technique was used to study peripheral blood cells from three volunteers after physical activity. The test subjects had to run on a treadmill and were checked for blood pressure and ECG, lactate concentration and creatine kinase activity. Blood was taken before and several times during and after the run. In a first multiple step test, the volunteers ran as long as possible with increasing speed. In a second test they had to run for 45 min with a fixed individual speed which was defined to ensure an aerobic metabolism. In the first test, the white blood cells of all subjects showed increased DNA migration in the SCG assay. The effect was seen 6 h after the end of the exercise and reached its maximum 24 h later. After 72 h, DNA migration decreased to about control level. The distribution of DNA migration among cells clearly demonstrated that the majority of white blood cells exhibited increased DNA migration and that the effect was not only due to a small fraction of damaged cells. From the same blood samples, blood cultures were set up to study a possible effect on the frequency of sister chromatid exchanges (SCE), another indicator for genotoxic effects. However, there was no significant increase in SCE in any of the cultures. In the second exercise, during aerobic metabolism, the effect on DNA migration was not seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Early effects of benzene exposure in mice. Hematological versus genotoxic effects.

Female BDF1 mice were exposed to 100, 300 and 900 ppm benzene 6 h/day, 5 days/week, up to 8 weeks. Hematological studies included peripheral blood data, T4 and T8 lymphocyte counts in the blood and the spleen, hemopoietic stem and progenitor cell assays in the marrow (CFU-S, CFU-C, BFU-E, CFU-E). The single cell gel assay ("comet assay") was applied in parallel with cells from the peripheral blood, bone marrow, spleen and liver. The results showed minor changes in the stem and progenitor cells and the development of a slight anemia at 4 and 8 weeks, in agreement with reported data. New was the increase of the T4/T8 ratio in the peripheral blood (not in the spleen) at the end of the first week of exposure to 300 and 900 ppm. The results of the "comet assay" indicate a much higher sensitivity to this test system (strand breaks and alkali labile sites of DNA). The tail moment indicative of the damage to DNA increased as early as 3 days with 300 ppm in the peripheral blood cells. Furthermore, the liver cells did react to a much higher extent than the other cells tested. With 100 ppm significant changes were seen in the liver after 5 days, but not in the blood. The repair, studied 24 and 48 h after the end of the exposure, was almost complete after 5-day exposure period in the blood and the liver, but not after 4 weeks of exposure with 300 ppm in the blood, and 100 and 300 ppm in the liver.

Catástrofe natural y salud mental de la población: niveles de ansiedad postraumática / Natural disasters and mental health of the population: anxiety postraumatic level

Una situación poco estudiada en nuestro país es el impact o que produce una catástrofe natural en la salud mental de una población. La erupción del volcán Lonquimay, IX Región, iniciada el 25 de Dic. de 1988, con todas sus posteriores consecuencias, es una buena oportunidad para realizar un estudio de esta índole. Se confeccionaron encuestas basadas en la "Escala de Ansiedad de Hamilton" y fueron aplicadas a la población de Lonquimay y grupos controles. En los resultados existen diferencias significativas en cuanto a niveles de ansiedad. En Lonquimay se observó un 37% de personas con ansiedad menor y 52% con ansiedad mayor y en los grupos controles un 40% de ansiedad menor y sólo un 25% de ansiedad mayor

Automedicación con lindano: consultantes ambulatorios Hospital Curacautín / Lindano self medication: ambulatory patients at Curacautín Hospital

Para conocer el nivel de automedicación con Lindano en nuestro medio se encuesta a 200 consultantes ambulatorios espontáneos, mayores de 14 años, tomados al azar, entre Noviembre y Diciembre de 1988, en el Hospital de Curacautín, IX Región. Se observa 32,5% de automedicación. Las principales causas invocadas fueron: dificultad al acceso de atención en los Servicios de Salud, negativa de uno o más familiares para concurrir al tratamiento familiar recomendación por terceras personas. El 90,5% de la muestra pertenece a los índices 1,2 y 3 de estratificación social por lo que es representativa de lo que ocurre en la población de escasos recursos. Se destaca que la mayoría de los que se automedicaron obtuvieron el Lindano en farmacias particulares por lo que es necesario un mayor control en la comercialización y educar a la población respecto a el uso correcto y los riesgos de la automedicación