The fat cell epigenetic signature in post-obese women is characterized by global hypomethylation and differential DNA methylation of adipogenesis genes.

BACKGROUND/OBJECTIVES: Obese subjects have increased number of enlarged fat cells that are reduced in size but not in number in post-obesity. We performed DNA methylation profiling in fat cells with the aim of identifying differentially methylated DNA sites (DMS) linked to adipose hyperplasia (many small fat cells) in post-obesity. SUBJECTS/METHODS: Genome-wide DNA methylation was analyzed in abdominal subcutaneous fat cells from 16 women examined 2 years after gastric bypass surgery at a post-obese state (body mass index (BMI) 26±2 kg m(-2), mean±s.d.) and from 14 never-obese women (BMI 25±2 kg m(-2)). Gene expression was analyzed in subcutaneous adipose tissue from nine women in each group. In a secondary analysis, we examined DNA methylation and expression of adipogenesis genes in 15 and 11 obese women, respectively. RESULTS: The average degree of DNA methylation of all analyzed CpG sites was lower in fat cells from post-obese as compared with never-obese women (P=0.014). A total of 8504 CpG sites were differentially methylated in fat cells from post-obese versus never-obese women (false discovery rate 1%). DMS were under-represented in CpG islands and surrounding shores. The 8504 DMS mapped to 3717 unique genes; these genes were over-represented in cell differentiation pathways. Notably, 27% of the genes linked to adipogenesis (that is, 35 of 130) displayed DMS (adjusted P=10(-8)) in post-obese versus never-obese women. Next, we explored DNA methylation and expression of genes linked to adipogenesis in more detail in adipose tissue samples. DMS annotated to adipogenesis genes were not accompanied by differential gene expression in post-obese compared with never-obese women. In contrast, adipogenesis genes displayed differential DNA methylation accompanied by altered expression in obese women. CONCLUSIONS: Global CpG hypomethylation and over-representation of DMS in adipogenesis genes in fat cells may contribute to adipose hyperplasia in post-obese women.

A microdialysis method for the in situ investigation of the action of large peptide molecules in human skeletal muscle: detection of local metabolic effects of insulin.

The possibility of using microdialysis catheters with a large pore size dialysis membrane (100 kDa) to investigate the action of macromolecules perfused into the interstitial space of peripheral tissues was explored. This was made possible by increasing the colloid osmotic pressure of the perfusate with 40 g/l of dextran-70 to prevent perfusate loss across the dialysis membranes. Microdialysis catheters were inserted into the quadriceps femoris muscle of 13 human subjects. With different perfusion flow rates (1. 33, 0.66, 0.33 and 0.16 microl/min) the recorded concentrations of glucose, lactate, and urea were in agreement with values previously obtained using a conventional membrane with a smaller pore size (20 kDa) [Rosdahl H, Hamrin K, Ungerstedt U, Henriksson. J Am J Physiol 1998;274:E936-45.]. When insulin was added to the perfusate, the concentration of glucose was significantly reduced, indicating that insulin diffuses across the dialysis membrane and has cellular effects that can be simultaneously recorded. The present findings are the first documentation on the use of microdialysis to study the local metabolic action of large peptide molecules in human tissues and may open new avenues for in-vivo metabolic research.

Manganese taken up into the CNS via the olfactory pathway in rats affects astrocytes.

Manganese (Mn), administered intranasally in rats, is effectively taken up in the CNS via the olfactory system. In the present study, Mn (as MnCl(2)) dissolved in physiological saline, was instilled intranasally in rats at doses of 0 (control), 10, 250, or 1000 microg. At the start of the experiment each rat received an intranasal instillation. Some rats were killed after one week without further treatment (the 1-w group), whereas the remaining rats received further instillations after one and two weeks and were killed after an additional week (the 3-w group). The brains were removed and either used for ELISA-determination of the astrocytic proteins glial fibrillary acidic protein (GFAP) and S-100b or histochemical staining of GFAP and S-100b, microglia (using an antibody against the iba1-protein) and the neuronal marker Fluoro-Jade. There were no indications that the Mn induced neuronal damage. On the other hand, the ELISA showed that both GFAP and S-100b decreased in the olfactory cortex, the hypothalamus, the thalamus, and the hippocampus of the 3-w group. The only effect observed in the 1-w group was a decrease of S-100b in the olfactory cortex at the highest dose. The immunohistochemistry showed no noticeable reduction in the number of astrocytes. We assume that the decreased levels of GFAP and S-100b are due to an adverse effect of Mn on the astrocytes, although this effect does not result in astrocytic demise. In the 3-w group, exposed to the highest dose of Mn, increased levels of GFAP and S-100b were observed in the olfactory bulbs, but these effects are probably secondary to a Mn-induced damage of the olfactory epithelium. Our results indicate that the astrocytes are the initial targets of Mn toxicity in the CNS.

Influence of adrenergic agonists on the release of amino acids from rat skeletal muscle studied by microdialysis.

The microdialysis technique was used to study the effects of adrenergic agonists on the release of amino acids from rat skeletal muscle. The release was monitored indirectly by measurements of interstitial concentrations. To distinguish metabolic from vasoactive effects, the adrenaline and isoprenaline results were compared with those of vasopressin, alpha-agonists and adenosine. As determined by the microdialysis ethanol technique, adrenaline, alpha-agonists and vasopressin induced vasoconstriction, whereas isoprenaline and adenosine induced vasodilatation. The lactate-to-pyruvate ratio increased fourfold with adrenaline (P < 0.001) and by 54% with isoprenaline (P < 0.05), whereas no change was observed with alpha-agonists and adenosine. Vasopressin induced a fivefold increase in the lactate-to-pyruvate ratio (P < 0.001), but with an unchanged pyruvate concentration, indicating that the effect may have been secondary to ischaemia. Adrenaline induced a twofold and vasopressin a 34% increase in the concentration of alanine (P < 0.001), whereas isoprenaline, adenosine and alpha-agonists had no significant effect. Adrenaline-perfusion induced an initial anabolic effect as evidenced by a reduced concentration of tyrosine. A significant decrease in the glutamate-to-glutamine ratio was observed with adrenaline and isoprenaline (22 and 27%, P < 0.01) whereas alpha-agonists, vasopressin and adenosine were without effect. In conclusion, the present study showed that adrenaline, via a beta-adrenergically mediated activation of glycogenolysis, possibly further stimulated by ischaemia, induced an increased release of alanine from skeletal muscle. The study indicates a beta-adrenergic stimulation on the glutamine synthetase step and a short lasting anabolic effect of adrenaline. Differences in the magnitude of the effects of adrenaline and isoprenaline could be related to their different vasoactive properties.

Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal muscle.

The molecular signaling mechanisms by which muscle contractions lead to changes in glucose metabolism and gene expression remain largely undefined. We assessed whether exercise activates MAP kinase proteins (ERK1/2, SEK1, and p38 MAP kinase) as well as Akt and PYK2 in skeletal muscle from healthy volunteers obtained during and after one-leg cycle ergometry at approximately 70% VO2max. Exercise led to a marked increase in ERK1/2 phosphorylation, which rapidly decreased to resting levels upon recovery. Exercise increased phosphorylation of SEK1 and p38 MAP kinase to a lesser extent than ERK1/2. In contrast to ERK1/2, p38 MAP kinase phosphorylation was increased in nonexercised muscle upon cessation of exercise. Phosphorylation of the transcription factor CREB was increased in nonexercised muscle upon cessation of exercise. Exercise did not activate Akt or increase tyrosine phosphorylation of PYK2. Thus, exercise has divergent effects on parallel MAP kinase pathways, of which only p38 demonstrated a systemic response. However, our data do not support a role of Akt or PYK2 in exercise/contraction-induced signaling in human skeletal. Activation of the different MAP kinase pathways by physical exercise appears to be important in the regulation of transcriptional events in skeletal muscle.

Transport and subcellular distribution of nickel in the olfactory system of pikes and rats.

Occupational exposure to nickel by inhalation may result in impaired olfactory sense. Recent studies have shown that nickel is transported from the olfactory epithelium along the axons of the primary olfactory neurons to the brain. In the present study 63Ni2+ was applied in the olfactory chambers of pikes (Esox lucius) and the rate at which the metal was transported in the primary olfactory neurons was determined by beta-spectrometry. The results showed a wave of 63Ni2+ in the olfactory nerves, which slowly moved toward the olfactory bulbs. The maximal 63Ni2+ transport rate corresponding to the movement of the base of the wave front was found to be about 0.13 mm/h at the experimental temperature (10 degrees C). This rate of 63Ni2+ transport falls into the class of slow axonal transport. Radioluminography of tape sections of a pike given 63Ni2+ in the right olfactory chamber showed a selective labeling of the right olfactory nerve. The subcellular distribution of 63Ni2+ in the olfactory nerves and the olfactory epithelium of the pikes was studied in tissues subjected to homogenizations and centrifugations, and these methods were also used to examine the subcellular distribution of 63Ni2+ in tissues of the olfactory system of rats given the metal intranasally. It was found that the 63Ni2+, in both the pike and the rat, was present in the cytosol and also in association with various particulate cell constituents. Gel filtrations of the cytosols showed that the 63Ni2+ mainly was eluted at a Ve/Vo ratio corresponding to a MW of about 250. The same coefficient was obtained in gel filtrations performed with 63Ni2+ mixed with histidine in vitro. It is likely that the cytosolic nickel may be bound to histidine or possibly to other amino acids which are similar in size to histidine. Additionally, in the olfactory tissues of the rat the 63Ni2+ was partly present in the cytosol in association with a component with a MW of about 25,000. It is concluded that (i) 63Ni2+ is transported in the primary olfactory neurons by means of slow axonal transport, (ii) in this process the metal is bound to both particulate and soluble cytosolic constituents, and (iii) the metal shows this subcellular distribution also in other parts of the olfactory system.

Polymorphisms of the genes encoding cruzipain, the major cysteine proteinase of Trypanosoma cruzi, in the region encoding the C-terminal domain.

Forty-eight cDNA clones obtained from different developmental stages of Trypanosoma cruzi and all encoding the C-terminal domain of the major cysteine proteinase (cruzipain) have been sequenced. A number of polymorphisms were detected, seven of them resulting in amino acid replacements. The predicted pI values of the corresponding gene products varied between 7.05 and 8.12. These changes in amino acid sequence, together with previously reported variations in carbohydrate composition at the only N-glycosylation site in the C-terminal domain, may account for most of the heterogeneities found in the mature enzyme.

Uptake of nickel into the brain via olfactory neurons in rats.

Intranasal instillation of nickel ([63]Ni2+) in rats resulted in an uptake of the metal in the olfactory epithelium and a migration along primary olfactory neurons to the glomeruli of the olfactory bulb. The metal was then seen to pass to the interior of the bulb and further to the olfactory peduncle, the olfactory tubercle and the rostral parts of the prepiriform, frontal and cingulate corticis. These results indicate that (63)Ni2+ slowly passes to secondary and tertiary olfactory neurons. Intraperitoneal injection of (63)Ni2+ resulted in a low uptake in the brain, without preferential labelling of the olfactory pathways. Inhalation of nickel compounds can impair the olfactory system. An uptake of nickel in the olfactory neurons may underly these lesions.

Uptake of manganese and cadmium from the nasal mucosa into the central nervous system via olfactory pathways in rats.

In the olfactory epithelium the primary olfactory neurones are in contact with the environment and via the axonal projections they are also connected to the olfactory bulbs of the brain. Therefore, the primary olfactory neurones provide a pathway by which foreign materials may gain access to the brain. In the present study we used autoradiography and gamma spectrometry to show that intranasal instillation of manganese (54Mn2+) in rats results in initial uptake of the metal in the olfactory bulbs. The metal was then seen to migrate via secondary and tertiary olfactory pathways and via further connections into most parts of the brain and also to the spinal cord. Intranasal instillation of cadmium (109Cd2+) resulted in uptake of the metal in the anterior parts of the olfactory bulbs but not in other areas of the brain. This indicates that this metal is unable to pass the synapses between the primary and secondary olfactory neurones in the bulbs. Intraperitoneal administration of 54Mn2+ or 109Cd2+ showed low uptake of the metals in the olfactory bulbs, an uptake not different from the rest of the brain. Manganese is a neurotoxic metal which in man can induce an extrapyramidal motor system dysfunction associated with occupational inhalation of manganese-containing dusts or fumes. We propose that the neurotoxicity of inhaled manganese is related to an uptake of the metal into the brain via the olfactory pathways. In this way manganese can circumvent the blood-brain barrier and gain direct access to the central nervous system.

Genes for cysteine proteinases from Trypanosoma rangeli.

PCR amplification of genomic DNA from the American trypanosome, Trypanosoma rangeli, using as primers oligonucleotides derived from the gene of cruzipain, the major cysteine proteinase (CP) from Trypanosoma cruzi, allowed the production of a probe which was used to obtain three clones encoding a CP with 70% overall identity with cruzipain. The genes are organized in tandem, with a monomere size of approximately 2 kbp, located on two chromosomes which, in some parasite isolates, have a high molecular mass (higher than 5.7 Mbp), and in others are much smaller (about 500 kbp). The low expression of this CP at the protein level correlates well with the low level of specific mRNA found in Northern blots.

Metabolic enzyme activity in the quadriceps femoris muscle in patients with severe chronic obstructive pulmonary disease.

Eighteen patients with severe COPD and seven healthy control subjects 64.0 +/- 2.2 and 66.8 +/- 1.4 yr of age, respectively (mean +/- SEM), were investigated. Arterial blood gas analysis, dynamic lung volumes, and muscle biopsy specimens from the quadriceps femoris muscle were performed. The muscle biopsies were analyzed for citrate synthase (CS), succinic acid dehydrogenase (SDH), 3-hydroxyacyl-CoA dehydrogenase (HAD), phosphofructokinase (PFK), and lactate dehydrogenase (LDH) activities and related to protein content. The PFK activity was higher in the COPD group than in the control group (+34%, p < 0.05). CS showed a group difference in the opposite direction (-29%, p < 0.05). LDH activity followed PFK and tended to be higher in the patient group (+27%, NS), whereas SDH (-31%, NS) and HAD (-28%, NS) mirrored the CS results. Muscle protein concentration tended to be lower in the COPD group (-14%, NS). There were no significant changes in enzyme activity after 7 mo of long-term oxygen therapy (n = 6). These results indicate adaptation in the form of augmented glycolysis (PFK), and decreased aerobic metabolism (CS) in the quadriceps femoris muscle in patients with advanced COPD.

A gene family encoding heterogeneous histone H1 proteins in Trypanosoma cruzi.

A gene family encoding a set of histone H1 proteins in Trypanosoma cruzi is described. The sequence of 3 genomic and 4 cDNA clones revealed the presence of several motifs characteristic of histone H1, although heterogeneity at the polypeptide level was evident. The clones encode histone H1 proteins of an unusually small size (74-97 amino acids), which lack the globular domain found in histone H1 of higher eukaryotes. All histone H1 mRNAs from T. cruzi are polyadenylated, although no typical polyadenylation signal was found. Furthermore, the genes encoding the histone H1 proteins in T. cruzi are found in a tandem array containing 15-20 gene copies per haploid genome. This tandem array is located on a large chromosome of 2.2 Mb.

Interstitial glucose and lactate balance in human skeletal muscle and adipose tissue studied by microdialysis.

1. Microdialysis was used to gain insight into the substrate exchanges in the interstitial space of skeletal muscle and adipose tissue. Probes were inserted in the quadriceps femoris muscle and para-umbilical subcutaneous adipose tissue of thirteen subjects and microdialysis was performed at different flow rates (1-4 microliters min-1) and during changes in tissue blood flow. 2. When ethanol (5 mM) is included in the perfusion solution, the ethanol clearance from the probe is a measure of tissue blood flow. Blood flow changes induced by adenosine or vasopressin perfusion, by exercise or by circulatory occlusion resulted in ethanol clearance values of 69-139% of the basal level. The ethanol clearance was higher in skeletal muscle than in adipose tissue (32-62%, P < 0.001), a difference compatible with a higher blood flow in muscle tissue. 3. The fraction of the interstitial glucose concentration that was recovered with the microdialysis was similar in skeletal muscle (the absolute values being 1.70 +/- 0.14 mM at 1 microliter min-1 and 0.59 +/- 0.05 mM at 4 microliters min-1) and adipose tissue (1.89 +/- 0.20 mM at 1 microliter min-1; 0.54 +/- 0.05 mM at 4 microliters min-1) and correlated inversely with the tissue ethanol clearance, both in the basal state and during changes in tissue blood flow (muscle: r = -0.56 to -0.67; adipose tissue r = -0.72 to -0.95). Coefficients of variation were 6-8% (glucose) and 11-16% (lactate) and were similar during isometric exercise. The reproducibility of the technique (comparison of two contralateral probes; perfusion flow rate 4 microliters min-1) was 5.3-8.3% (ethanol) and 23.9-20.8% (glucose) in muscle (n = 6) and adipose tissue (n = 4) respectively. 4. The skeletal muscle dialysate lactate concentration (1 microliter min-1: 1.16 +/- 0.2 mM) was higher than in adipose tissue (0.76 +/- 0.08 mM, P < 0.05), where the absolute amount of lactate that could be removed from the tissue (at 4 microliters min-1) was only half of that in skeletal muscle (0.8 +/- 0.11 vs. 1.76 +/- 0.23 nmol min-1, P < 0.05). The dialysate lactate level was not affected in either tissue by large changes in the interstitial glucose concentration indicating that in neither tissue is blood glucose a significant source of lactate formation. 5. The blood flow effects on the dialysate glucose concentration are the likely consequence of probe glucose drainage artificially shifting the balance between the supply and consumption of interstitial glucose.(ABSTRACT TRUNCATED AT 400 WORDS)

[Evaluation of 4 immunobiochemical/molecular methods for the identification of Trypanosoma cruzi and Trypanosoma rangeli strains]. / Evaluación de cuatro métodos immunobioquímico/moleculares en la identificación de cepas de Trypanosoma cruzi y Trypanosoma rangeli.

In American man can be infected with two trypanosomes: Trypanosoma cruzi, the etiological agent of Chagas' disease and Trypanosoma rangeli, a suspected nonpathogenic parasite. In this communication are presented 4 methods in order to improve the current knowledge about the specific identification of these parasites. Using the SDS-PAGE technique it was possible to differentiate between T. rangeli. and T. cruzi based in at less 4 protein bands with a relative molecular weights of 93, 77-73, 63 and 54-52 KDa. These polypeptides were found only in T. rangeli electrophoretic profiles. An ELISA test showed that the antigenic composition found in the enzyme cisteine proteinase (cruzipain) is specific for T. cruzi epimastigotes. Antigenic analysis by Western blot assay, proved that T. rangeli and not T. cruzi present antigenic bands with a Mr of 142, 63, 54, 51, 49, 43, 39 and 24 KDa. Finally, using the Southern blot procedure, it was confirmed that SAPA, a DNA sequence originally identified in the T. cruzi, genome, is absent in T. rangeli nuclear DNA. These initial observations revealed that it is possible to identify both parasites using the described methods, however further works are required to clarify the biochemical, immunological and molecular relationship between T. rangeli and T. cruzi.

Evaluación de cuatro métodos immunobioquímico/moleculares en la identificación de cepas de Trypanosoma cruzi y Trypanosoma rangeli / Evaluation of 4 immunobiochemical/molecular methods for the identification of Trypanosoma cruzi and Trypanosoma rangeli strains

In American man can be infected with two trypanosomes: Trypanosoma cruzi, the etiological agent of Chagas' disease and Trypanosoma rangeli, a suspected nonpathogenic parasite. In this communication are presented 4 methods in order to improve the current knowledge about the specific identification of these parasites. Using the SDS-PAGE technique it was possible to differentiate between T. rangeli. and T. cruzi based in at less 4 protein bands with a relative molecular weights of 93, 77-73, 63 and 54-52 KDa. These polypeptides were found only in T. rangeli electrophoretic profiles. An ELISA test showed that the antigenic composition found in the enzyme cisteine proteinase (cruzipain) is specific for T. cruzi epimastigotes. Antigenic analysis by Western blot assay, proved that T. rangeli and not T. cruzi present antigenic bands with a Mr of 142, 63, 54, 51, 49, 43, 39 and 24 KDa. Finally, using the Southern blot procedure, it was confirmed that SAPA, a DNA sequence originally identified in the T. cruzi, genome, is absent in T. rangeli nuclear DNA. These initial observations revealed that it is possible to identify both parasites using the described methods, however further works are required to clarify the biochemical, immunological and molecular relationship between T. rangeli and T. cruzi

The ethanol technique of monitoring local blood flow changes in rat skeletal muscle: implications for microdialysis.

We have investigated the feasibility of monitoring local skeletal muscle blood flow in the rat by including ethanol in the perfusion medium passing through a microdialysis probe placed in muscle tissue. Ethanol at 5, 55, or 1100 mM did not directly influence local muscle metabolism, as measured by dialysate glucose, lactate, and glycerol concentrations. The clearance of ethanol from the perfusion medium can be described by the outflow/inflow ratio ([ethanol]collected dialysate/[ethanol]infused perfusion medium), which was found to be similar (between 0.36 and 0.38) at all ethanol perfusion concentrations studied. With probes inserted in a flow-chamber, this ratio changed in a flow-dependent way in the external flow range of 5-20 microliters min-1. The ethanol outflow/inflow ratio in vivo was significantly (P less than 0.001) increased (to a maximum of 127 +/- 2.8% and 144 +/- 7.4% of the baseline, mean +/- SEM) when blood flow was reduced by either leg constriction or local vasopressin administration, and significantly (P less than 0.001) reduced (to 62 +/- 6.4% and 43 +/- 4.4% of baseline) with increases in blood flow during external heating or local 2-chloroadenosine administration, respectively. Dialysate glucose concentrations correlated negatively with the ethanol outflow/inflow ratio (P less than 0.01) and consequently decreased (to 46 +/- 7.6% and 56 +/- 5.6% of baseline) with constriction and vasopressin administration and increased (to 169 +/- 32.5% and 262 +/- 16.7% of baseline) following heating and 2-chloroadenosine administration. Dialysate lactate concentrations were significantly increased (approximately 2-fold, P less than 0.001) during all perturbations of blood flow. In conclusion, this technique makes it possible to monitor changes in skeletal muscle blood flow; however, methods of quantification remain to be established. The fact that blood flow changes were found to significantly affect interstitial glucose and lactate concentrations as revealed by microdialysis indicates that this information is critical in microdialysis experiments.

The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is encoded by multiple polymorphic tandemly organized genes located on different chromosomes.

We demonstrate that cruzipain, the major cysteine proteinase of Trypanosoma cruzi epimastigotes, is encoded by a large number of tandemly arranged genes. Restriction enzyme analysis of 20 clones containing complete repeat units of the gene, as well as sequencing of 2 of these clones, and comparison with previously published partial sequences, indicated that the sequence is conserved among the repeat units, although polymorphisms clearly exist. The repeat units contain an intergenic region of 528 bp and coding regions for pre- and pro-enzyme, a central domain and a C-terminal extension. The predicted amino acid sequences of these regions indicated a sequence identity of 30, 60, 70 and 36%, respectively, when the T. cruzi sequence was compared with the sequence of a similar cysteine proteinase from Trypanosoma brucei. Studies by pulsed field gel electrophoresis, complemented with restriction analysis, indicated that the clusters are located on 2-4 different chromosomes in several parasite isolates.

Hyaluronan in human skeletal muscle of lower extremity: concentration, distribution, and effect of exercise.

The concentration and localization of hyaluronan (HYA) were determined in biopsy specimens from resting human quadriceps femoris and anterior tibial muscles. The influence of physical exercise on HYA concentrations in the quadriceps femoris muscle and in blood was also evaluated. A sensitive radioassay was used for the quantification of HYA. The distribution of the glycosaminoglycan was demonstrated using a histochemical method that involved microwave-aided fixation and an HYA-binding protein. At rest, the muscle HYA concentration was 34.9 +/- 23.6 (SD) micrograms/g muscle wet wt with a large interindividual variation. Exercise had no significant effect on the muscle HYA concentration. The serum HYA concentration increased from 35.9 +/- 22.7 to 53.4 +/- 57.1 micrograms/l during exercise, but 30 min after the exercise the HYA concentration was significantly lower (19.1 +/- 6.3 micrograms/l) than the initial preexercise value. In resting skeletal muscles of the lower extremity, HYA was heterogeneously distributed in the perimysium and endomysium. Perivascular and perineural connective tissues were distinctly HYA positive.