Comparative structural analyses of purified glycogen particles from rat liver, human skeletal muscle and commercial preparations

Glycogen is a cellular energy store that is crucial for whole body energy metabolism, metabolic regulation and exercise performance. To understand glycogen structure we have purified glycogen particles from rat liver and human skeletal muscle tissues and compared their biophysical properties with those found in commercial glycogen preparations. Ultrastructural analysis of commercial liver glycogens fails to reveal the classical α-rosette structure but small irregularly shaped particles. In contrast, commercial slipper limpet glycogen consists of β-particles with similar branching and chain lengths to purified rat liver glycogen together with a tendency to form small α-particles, and suggest it should be used as a source of glycogen for all future studies requiring a substitute for mammalian liver glycogen.

In vivo cardiac glucose metabolism in the high-fat fed mouse: comparison of euglycemic-hyperinsulinemic clamp derived measures of glucose uptake with a dynamic metabolomic flux profiling approach

Rationale Cardiac metabolism is thought to be altered in insulin resistance and type 2 diabetes (T2D). Our understanding of the regulation of cardiac substrate metabolism and insulin sensitivity has largely been derived from ex vivo preparations which are not subject to the same metabolic regulation as in the intact heart in vivo. Studies are therefore required to examine in vivo cardiac glucose metabolism under physiologically relevant conditions. Objective To determine the temporal pattern of the development of cardiac insulin resistance and to compare with dynamic approaches to interrogate cardiac glucose and intermediary metabolism in vivo. Methods and results Studies were conducted to determine the evolution of cardiac insulin resistance in C57Bl/6 mice fed a high-fat diet (HFD) for between 1 and 16 weeks. Dynamic in vivo cardiac glucose metabolism was determined following oral administration of [U-¹³C] glucose. Hearts were collected after 15 and 60 min and flux profiling was determined by measuring ¹³C mass isotopomers in glycolytic and tricarboxylic acid (TCA) cycle intermediates. Cardiac insulin resistance, determined by euglycemic-hyperinsulinemic clamp, was evident after 3 weeks of HFD. Despite the presence of insulin resistance, in vivo cardiac glucose metabolism following oral glucose administration was not compromised in HFD mice. This contrasts our recent findings in skeletal muscle, where TCA cycle activity was reduced in mice fed a HFD. Similar to our report in muscle, glucose derived pyruvate entry into the TCA cycle in the heart was almost exclusively via pyruvate dehydrogenase, with pyruvate carboxylase mediated anaplerosis being negligible after oral glucose administration. Conclusions Under experimental conditions which closely mimic the postprandial state, the insulin resistant mouse heart retains the ability to stimulate glucose metabolism.

Avaliação do extrato de Momordica charantia em Salmonella typhimurium: Mutagenicidade e antimutagenicidade

In the last years some natural products has been described as supressors of the mutagenic process in bacteria, the antimutagenics. The literature reference that in most of the countries, the population makes use of medicinal plants. The plant Momordica charantia (Cucurbitaceae) is original from Africa being used popularly as purgative, antirheumatic and for skin problems, burns and hemorrhoids. The present work had as objective to evaluate the mutagenic and antimutagenic activities of the ethanolic extract of M. charantia in Salmonella/microsome assays using TA100, TA98 and TA102 strains. It was verified that the extract did not present mutagenic activity when evaluated in different concentrations (0.64, 1.27, 2.55 and 3.84 mg/plate) but acted as antimutagenic agent against the mutations induced by the sodium azide (TA100,-S9), 4-nitro-phenylenediamine (TA98, -S9), daunomycin (TA102, +S9) 2-anthramine (TA100 and TA98, +S9) and 2-aminofluorene (TA102, +S9). When the metabolic activation (+S9) was used, the percentage of inhibition of the mutagenicity varied in the range of 31%-96%, while in absence of metabolizing system (-S9), the maximum percentage of inhibition of the mutagenicity was 44%. In that way, we can conclude that the metabolites found in the extract has potential to protect the genetic material against the damages induced by different chemical agents.

Efeito da cadência de pedalada sobre as respostas metabólica e cardiovascular durante o exercício incremental e de carga constante em indivíduos ativos

The main purpose of this study was to analyze the effect of the pedaling cadence (500 × 100 rpm) on the heart rate (HR) and the blood lactate response during incremental and constant workload exercises in active individuals. Nine active male individuals (20.9 ± 2.9 years old; 73.9 ± 6.5 kg; 1.79 ± 0.9 m) were submitted to two incremental tests, and to 6-8 constant workload tests to determine the intensity corresponding to the maximal steady state lactate (MLSSintens) in both cadences. The maximal power (Pmax) attained during the incremental test, and the MLSSintens were significantly lower at 100 rpm (240.9 ± 12.6 W; 148.1 ± 154.W) compared to 50 rpm (263.9 ± 18.6 W; 186.1 ± 21.2 W), respectively. The HRmax did not change between cadences (50 rpm = 191.1 ± 8.8 bpm; 100 rpm = 192.6 ± 9.9 bpm). Regardless the cadence, the HRmax percentage (70, 80, 90, and 100%) determined the same lactate concentrations during the incremental test. However, when the intensity was expressed in Pmax percentage or in absolute power, the lactate and the HR values were always higher at highest cadences. The HR corresponding to MLSSintens was similar between cadences (50 rpm = 162.5 ± 9.1 bpm; 100 rpm = 160.4 ± 9.2 bpm). Based on these results, it can be conclude that regardless the cadence employed (50 × 100 rpm), the use of the HR to individualize the exercise intensity indicates similar blood lactate responses, and this relationship is also kept in the exercise of constant intensity performed at MLSSintens. On the other hand, the use of the Pmax percentages depend on the cadence used, indicating different physiological responses to a same percentage.

Diabetes and pregnancy: An update of the problem

Pregnancies complicated by diabetes account for about 7% of all pregnancies attended by the Brazilian Unified Healthcare System (SUS) and are one of the main causes of maternal/perinatal morbidity and mortality in Brazil. Considering the importance of this topic, this article presents an update of diabetes classification, diagnostic criteria, maternal/perinatal outcomes, and both clinical and obstetric prenatal care. Even though there is no consensus about screening and diagnostic standards, the investigation of hyperglycemia in all risk pregnancies is recommended. The importance of adequate metabolic control is emphasized in order to improve maternal and neonatal outcomes. Finally, the development of educational programs is encouraged, viewing not only good gestational outcome but also long-term changes in the lifestyle of these women. © by São Paulo State University.

Treatment of atherosclerotic renovascular disease

Treatment of atherosclerotic renovascular disease is controversial and revascularization is not a beneficial approach to all patients. Conditions as progressive deterioration of renal function, refractory hypertension or accelerated cardiovascular disease, especially recurrent pulmonary edema, could profit from renal angioplasty with stent placement. Surgical revascularization is a good option for patients who will need concomitant surgical corrections of abdominal aortic lesions. Treatment of all other patients must be individualized. Medical therapy is indicated for all patients with atherosclerotic renovascular disease. Observational studies pointed out to the beneficial effect of controlling blood pressure (<130/80 mm Hg), glucose and lipids profile, lifestyle modifications, specific use of platelet antiaggregant therapy, Angiotensin Conversion Enzyme Inhibitors (ACEI) and statins. All others cardiovascular risk factors must be controlled. The evaluation and management of other systemic atherosclerotic vascular lesions is important, especially coronary, carotid and abdominal aortic. This paper presents a review of evidences to rationale the atherosclerotic renovascular disease treatment. © 2008 Bentham Science Publishers Ltd.

Use of different carbon sources in cultivation of baker's yeast for production of glycerol-3-phosphate dehydrogenase

The physiological state of yeast cells changes during culture growth as a consequence of environmental changes (nutrient limitations, pH and metabolic products). Cultures that grow exponentially are heterogeneous cell populations made up of cells regulated by different metabolic and/or genetic control systems. The strain of baker's yeast selected by plating commercial compressed yeast was used for the production of glycerol-3- phosphate dehydrogenase. Glycerol-3-phosphate dehydrogenase (GPD) has been widely used in the enzyme assays with diverse compounds of industrial interest, such as glycerol or glycerol phosphate, as well as a number of important bioanalytical applications. Each cell state determines the level of key enzymes (genetic control), fluxes through metabolic pathways (metabolic control), cell morphology and size. The present study was carried out to determine the effects of environmental conditions and carbon source on GPD production from baker's yeast. Glucose, glycerol, galactose and ethanol were used as carbon sources. Glycerol and ethanol assimilations required agitation, which was dependent on the medium volume in the fermentation flask for the greatest accumulation of intracellular GPD. Enzyme synthesis was also affected by the initial pH of the medium and inoculum size. The fermentation time required for a high level of enzyme formation decreased with the inoculum size. The greatest amount of enzyme (0.45 U/ml) was obtained with an initial pH of 4.5 in the medium containing ethanol or glycerol. The final pH was maintained in YP-ethanol, but in the YP-glycerol the final pH increased to 6.9 during growth.

A randomized clinical trial of high volume peritoneal dialysis versus extended daily hemodialysis for acute kidney injury patients

Background: Acute kidney injury (AKI) requiring dialysis in critically ill patients is associated with an in-hospital mortality rate of 50-80 %. Extended daily hemodialysis (EHD) and high volume peritoneal dialysis (HVPD) have emerged as alternative modalities. Methods: A double-center, randomized, controlled trial was conducted comparing EHD versus HVPD for the treatment for AKI in the intensive care unit (ICU). Four hundred and seven patients were randomized and 143 patients were analyzed. Principal outcome measure was hospital mortality, and secondary end points were recovery of renal function and metabolic and fluid control. Results: There was no difference between the two groups in relation to median ICU stay [11 (5.7-20) vs. 9 (5.7-19)], recovery of kidney function (26.9 vs. 29.6 %, p = 0.11), need for chronic dialysis (9.7 vs. 6.5 %, p = 0.23), and hospital mortality (63.4 vs. 63.9 %, p = 0.94). The groups were different in metabolic and fluid control. Blood urea nitrogen (BUN), creatinine, and bicarbonate levels were stabilized faster in EHD group than in HVPD group. Delivered Kt/V and ultrafiltration were higher in EHD group. Despite randomization, there were significant differences between the groups in some covariates, including age, pre-dialysis BUN, and creatinine levels, biased in favor of the EHD. Using logistic regression to adjust for the imbalances in group assignment, the odds of death associated with HVPD was 1.4 (95 % CI 0.7-2.4, p = 0.19). A detailed investigation of the randomization process failed to explain the marked differences in patient assignment. Conclusions: Despite faster metabolic control and higher dialysis dose and ultrafiltration with EHD, this study provides no evidence of a survival benefit of EHD compared with HVPD. The limitations of this study were that the results were not presented according to the intention to treat and it did not control other supportive management strategies as nutrition support and timing of dialysis initiation that might influence outcomes in AKI. © 2012 Springer Science+Business Media Dordrecht.

Análise comparativa da expressão protéica em glândulas pós-faríngeas de saúva-limão, Atta sexdens rubropilosa Forel, 1908 (Hymenoptera: Formicidae) submetidas à suplementação lipídica

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Unravelling the Neospora caninum secretome through the secreted fraction (ESA) and quantification of the discharged tachyzoite using high-resolution mass spectrometry-based proteomics

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Oxidized low-density lipoproteins and their antibodies: Relationships with the reverse cholesterol transport and carotid atherosclerosis in adults without cardiovascular diseases

Background: Metabolic predictors and the atherogenicity of oxidized LDL (oxLDL) and the specific antibodies against oxLDL (oxLDL Ab) are unclear and controversial. Methods: In 107 adults without atherosclerotic manifestations, we measured oxLDL and oxLDL Ab, and also the activities of CETP. PLTP, lipases and the carotid intima-media thickness (cIMT). Comparisons were performed for the studied parameters between the lowest and the highest tertile of oxLDL and oxLDL Ab, and the relationships between studied variables were evaluated. Results: Subjects with higher oxLDL Ab present reduced hepatic lipase activity and borderline increased cIMT. In the highest oxLDL tertile, besides the higher levels of total cholesterol, LDL-C and apoB100, we found reduced CETP activity and higher cIMT. A significant correlation between oxLDL Ab and cIMT, independent of oxLDL, and a borderline correlation between oxLDL and cIMT independent of oxLDL Ab were found. In the multivariate analysis, apoAl was a significant predictor of oxLDL Ab, in contrast to regulation of oxLDL by apoB100, PLTP and inverse of CETP. Conclusions: In adults without atherosclerotic disease, the metabolic regulation and carotid atherosclerosis of oxLDLAb and oxLDL groups, characterized a dual trait in oxLDL Ab, as a contributor to carotid atherosclerosis, much less so than oxidized LDL, and with a modest atheroprotective role. (C) 2012 Elsevier B.V. All rights reserved.

Endocrine changes and liver mRNA abundance of somatotropic axis and insulin system constituents during negative energy balance at different stages of lactation in dairy cows

The liver has an important role in metabolic regulation and control of the somatotropic axis to adapt successfully to physiological and environmental changes in dairy cows. The aim of this study was to investigate the adaptation to negative energy balance (NEB) at parturition and to a deliberately induced NEB by feed restriction at 100 days in milk. The hepatic gene expression and the endocrine system of the somatotropic axis and related parameters were compared between the early and late NEB period. Fifty multiparous cows were subjected to 3 periods (1=early lactation up to 12 wk postpartum, 2=feed restriction for 3 wk beginning at around 100 days in milk with a feed-restricted and a control group, and 3=subsequent realimentation period for the feed-restricted group for 8 wk). In period 1, plasma growth hormone reached a maximum in early lactation, whereas insulin-like growth factor-I (IGF-I), leptin, the thyroid hormones, insulin, and the revised quantitative insulin sensitivity check index increased gradually after a nadir in early lactation. Three days after parturition, hepatic mRNA abundance of growth hormone receptor 1A, IGF-I, IGF-I receptor and IGF-binding protein-3 (IGFBP-3) were decreased, whereas mRNA of IGFBP-1 and -2 and insulin receptor were upregulated as compared with wk 3 antepartum. During period 2, feed-restricted cows showed decreased plasma concentrations of IGF-I and leptin compared with those of control cows. The revised quantitative insulin sensitivity check index was lower for feed-restricted cows (period 2) than for control cows. Compared with the NEB in period 1, the changes due to the deliberately induced NEB (period 2) in hormones were less pronounced. At the end of the 3-wk feed restriction, the mRNA abundance of IGF-I, IGFBP-1, -2, -3, and insulin receptor was increased as compared with the control group. The different effects of energy deficiency at the 2 stages in lactation show that the endocrine regulation changes qualitatively and quantitatively during the course of lactation.

No evidence for a local renin-angiotensin system in liver mitochondria

The circulating, endocrine renin-angiotensin system (RAS) is important to circulatory homeostasis, while ubiquitous tissue and cellular RAS play diverse roles, including metabolic regulation. Indeed, inhibition of RAS is associated with improved cellular oxidative capacity. Recently it has been suggested that an intra-mitochondrial RAS directly impacts on metabolism. Here we sought to rigorously explore this hypothesis. Radiolabelled ligand-binding and unbiased proteomic approaches were applied to purified mitochondrial sub-fractions from rat liver, and the impact of AngII on mitochondrial function assessed. Whilst high-affinity AngII binding sites were found in the mitochondria-associated membrane (MAM) fraction, no RAS components could be detected in purified mitochondria. Moreover, AngII had no effect on the function of isolated mitochondria at physiologically relevant concentrations. We thus found no evidence of endogenous mitochondrial AngII production, and conclude that the effects of AngII on cellular energy metabolism are not mediated through its direct binding to mitochondrial targets.

Hyperketonemia during lipopolysaccharide-induced mastitis affects systemic and local intramammary metabolism in dairy cows

Hyperketonemia interferes with the metabolic regulation in dairy cows. It is assumed that metabolic and endocrine changes during hyperketonemia also affect metabolic adaptations during inflammatory processes. We therefore studied systemic and local intramammary effects of elevated plasma β-hydroxybutyrate (BHBA) before and during the response to an intramammary lipopolysaccharide (LPS) challenge. Thirteen dairy cows received intravenously either a Na-DL-β-OH-butyrate infusion (n = 5) to achieve a constant plasma BHBA concentration (1.7 ± 0.1 mmol/L), with adjustments of the infusion rates made based on immediate measurements of plasma BHBA every 15 min, or an infusion with a 0.9% NaCl solution (control; n = 8) for 56 h. Infusions started at 0900 h on d 1 and continued until 1700 h 2 d later. Two udder quarters were challenged with 200 μg of Escherichia coli LPS and 2 udder quarters were treated with 0.9% saline solution as control quarters at 48 h after the start of infusion. Blood samples were taken at 1 wk and 2h before the start of infusions as reference samples and hourly during the infusion. Mammary gland biopsies were taken 1 wk before, and 48 and 56 h (8h after LPS challenge) after the start of infusions. The mRNA abundance of key factors related to BHBA and fatty acid metabolism, and glucose transporters was determined in mammary tissue biopsies. Blood samples were analyzed for plasma glucose, BHBA, nonesterified fatty acid, urea, insulin, glucagon, and cortisol concentrations. Differences were not different for effects of BHBA infusion on the mRNA abundance of any of the measured target genes in the mammary gland before LPS challenge. Intramammary LPS challenge increased plasma glucose, cortisol, glucagon, and insulin concentrations in both groups but increases in plasma glucose and glucagon concentration were less pronounced in the Na-DL-β-OH-butyrate infusion group than in controls. In response to LPS challenge, plasma BHBA concentration decreased in controls and decreased also slightly in the BHBA-infused animals because the BHBA concentration could not be fully maintained despite a rapid increase in BHBA infusion rate. The change in mRNA abundance of citrate synthase in LPS quarters was significant between the 2 treatment groups. The results indicate that elevated circulating BHBA concentration inhibits gluconeogenesis before and during immune response to LPS challenge, likely because BHBA can replace glucose as an energy source.

Genomic knock-out of rancRNAs in the archaeon H. volcanii

As translation is the final step in gene expression it is particularly important to understand the processes involved in translation regulation. It was shown in the last years that a class of RNA, the non-protein-coding RNAs (ncRNAs), is involved in regulation of gene expression via various mechanisms [1]. Herein included is the prominent example of gene silencing caused by micro RNAs (miRNAs) and small interfering RNAs (siRNAs). Almost all of these ncRNA discovered so far target the mRNA in order to modulate protein biosynthesis, this is rather unexpected considering the crucial role of the ribosome during gene expression. However, recent data from our laboratory showed that there is a new class of RNAs among the well-studied ncRNAs that target the ribosome itself [2,3]. These so called ribosome-associated ncRNAs (rancRNAs) have an impact on translation regulation, mainly by interfering / modulating the rate of protein biosynthesis. Recent studies show the presence of small regulatory RNAs (sRNAs) in archaea which are involved in many biological processes including stress response and metabolic regulation [4]. To date the biological function and the targets of these archaeal sRNAs are only described for a few examples. There are reports of sRNAs binding to the 5’ as well as to the 3’ of mRNAs [5,6]. In addition to these findings, a tRNA derived fragment (tRF) of Valine tRNA was found in a genomic screen of RNAs associated with the ribosome in H. volcanii in our laboratory [3]. This Valine tRF seems to be processed in a stress-dependent manner and showed in vitro binding to the ribosome and inhibited in vitro translation. These results showed that Valine tRF is capable to regulate translation in H. volcanii by targeting the ribosome. The main goal of this project is to identify and describe novel potential regulatory rancRNAs in H. volcanii with the focus on intergenic candidates. Northern blot analyses already revealed interactions with the ribosome and showed differential expression patterns in response to stress conditions. To investigate the biological relevance of some of the ribosome-associated ncRNA candidates, knock-out and phenotypic characterization studies are done. The genomic knock out of a hypothetical ORF (198nt), where one putative rancRNA candidate (46nt) named IG33 was detected in the library at the beginning of the ORF, showed interesting growth phenotype under specific stress conditions. Furthermore a strain with an introduced start to stop codon mutation in this hypothetical ORF still shows the same phenotype indicating that rather the missing protein than the missing sRNA causes this growth phenotype.