Insulin‐ and Exercise‐Stimulated Skeletal Muscle Blood Flow and Glucose Uptake in Obese Men

Objective: Insulin resistance in obese subjects results in the impaired use of glucose by insulin‐sensitive tissues, e.g., skeletal muscle. In the present study, we determined whether insulin resistance in obesity is associated with an impaired ability of exercise to stimulate muscle blood flow, oxygen delivery, or glucose uptake. Research Methods and Procedures: Nine obese (body mass index = 36 ± 2 kg/m²) and 11 age‐matched nonobese men (body mass index = 22 ± 1 kg/m²) performed one‐legged isometric exercise during hyperinsulinemia. Rates of femoral muscle blood flow, oxygen consumption, and glucose uptake were measured simultaneously in both legs using [¹⁵O]H₂O, [¹⁵O]O₂, [¹⁸F]fluoro‐deoxy‐glucose, and positron emission tomography. Results: The obese subjects exhibited resistance to insulin stimulation of glucose uptake in resting muscle, regardless of whether glucose uptake was expressed per kilogram of femoral muscle mass (p = 0.001) or per the total mass of quadriceps femoris muscle. At similar workloads, oxygen consumption, blood flow, and glucose uptake were lower in the obese than the nonobese subjects when expressed per kilogram of muscle, but similar when expressed per quadriceps femoris muscle mass. Discussion: We conclude that obesity is characterized by insulin resistance of glucose uptake in resting skeletal muscle regardless of how glucose uptake is expressed. When compared with nonobese individuals at similar absolute workloads and under identical hyperinsulinemic conditions, the ability of exercise to increase muscle oxygen uptake, blood flow, and glucose uptake per muscle mass is blunted in obese insulin‐resistant subjects. However, these defects are compensated for by an increase in muscle mass.     

Mechanisms of Glycine Release in Mouse Brain Stem Slices

In the brain stem glycine is associated with multiple sensory and visceral regulations, being involved in, for instance, cardiovascular, respiratory and auditory functions. We here studied the mechanisms of the release of preloaded [³H]glycine from mouse brain stem slices in a superfusion system. A depolarizing concentration of K⁺ ions (50 mM) evoked glycine release, but in the absence of Ca²⁺ the effect was attenuated, indicating that a part of the evoked release represents Ca²⁺-dependent exocytosis. The Ca²⁺-independent release was enhanced by omission of Na⁺ and Cl⁻. The stimulatory effect of extracellular glycine confirmed the involvement of transporters functioning in a reverse direction. A part of the release is mediated by Na⁺ and Cl⁻ channels, since it was inhibited by the inhibitors of these, riluzole and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonate, respectively. Glycine release was potentiated by the activation of protein kinase C and diminished by increasing cyclic guanosine monophosphate levels with a phosphodiesterase inhibitor, zaprinast. The release was also modulated by the phospholipase inhibitor quinacrine and the tyrosine kinase inhibitor genistein. Adenosine A₁ receptors likewise regulate glycine release, since it was enhanced by their agonist R(−)N⁶-(2-phenylisopropyl)adenosine, which effect was blocked by the antagonist 8-cyclopentyl-1,3-dipropylxanthine. The ionotropic glutamate receptor agonists N-methyl-d-aspartate, kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate failed to have any effects contrary to their effects in higher brain regions, e.g., in the hippocampus. The group I and III metabotropic glutamate receptor agonists (S)-3,5-dihydroxyphenylglycine and O-phospho-l-serine, respectively, increased the release in a receptor-mediated manner. Glycine release in the brain stem was also markedly enhanced by cell-damaging conditions, including hypoxia, hypoglycemia and ischemia.     

Ten-Year Mortality and Cardiovascular Morbidity in the Finnish Diabetes Prevention Study—Secondary Analysis of the Randomized Trial

Background

The Finnish Diabetes Prevention Study (DPS) was a randomized controlled trial, which showed that it is possible to prevent type 2 diabetes by lifestyle changes. The aim of the present study was to examine whether the lifestyle intervention had an effect on the ten-year mortality and cardiovascular morbidity in the DPS participants originally randomized either into an intervention or control group. Furthermore, we compared these results with a population-based cohort comprising individuals of varying glucose tolerance states.

Methods and Findings

Middle-aged, overweight people with IGT (n = 522) were randomized into intensive intervention (including physical activity, weight reduction and dietary counseling), or control “mini-intervention” group. Median length of the intervention period was 4 years and the mean follow-up was 10.6 years. The population-based reference study cohort included 1881 individuals (1570 with normal glucose tolerance, 183 with IGT, 59 with screen-detected type 2 diabetes, 69 with previously known type 2 diabetes) with the mean follow-up of 13.8 years. Mortality and cardiovascular morbidity data were collected from the national Hospital Discharge Register and Causes of Death Register. Among the DPS participants who consented for register linkage (n = 505), total mortality (2.2 vs. 3.8 per 1000 person years, hazard ratio HR = 0.57, 95% CI 0.21–1.58) and cardiovascular morbidity (22.9 vs. 22.0 per 1000 person years, HR = 1.04, 95% CI 0.72–1.51) did not differ significantly between the intervention and control groups. Compared with the population-based cohort with impaired glucose tolerance, adjusted HRs were 0.21 (95% CI 0.09–0.52) and 0.39 (95% CI 0.20–0.79) for total mortality, and 0.89 (95% CI 0.62–1.27) and 0.87 (0.60–1.27) for cardiovascular morbidity in the intervention and control groups of the DPS, respectively. The risk of death in DPS combined cohort was markedly lower than in FINRISK IGT cohort (adjusted HR 0.30, 95% CI 0.17–0.54), but there was no significant difference in the risk of CVD (adjusted HR 0.88, 95% CI 0.64–1.21).

Conclusions

Lifestyle intervention among persons with IGT did not decrease cardiovascular morbidity during the first 10 years of follow-up. However, the statistical power may not be sufficient to detect small differences between the intervention and control groups. Low total mortality among participants of the DPS compared with individuals with IGT in the general population could be ascribed to a lower cardiovascular risk profile at baseline and regular follow-up.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00518167","term_id":"NCT00518167"}}NCT00518167     

Synthesis and ocular effects of imidazole nitrolic acid and amidoxime esters

Esters of 1-(H)-imidazole-5-nitrolic acid and 1-methyl-imidazole-5-carboxamide oxime were prepared to study the effect of esterification on the ocular effects of these compounds. Esterifications were performed with acid chloride. Acid chloride also reacts with the ring nitrogen of 1-(H)-imidazole-5-nitrolic acid, but the desired esters could be selectively prepared by adjustment of the reaction conditions. Esterification led to loss of the ocular effects exhibited by the parent compounds.     

Higher Free Fatty Acid Uptake in Visceral Than in Abdominal Subcutaneous Fat Tissue in Men

Visceral adipose tissue has been shown to have high lipolytic activity. The aim of this study was to examine whether free fatty acid (FFA) uptake into visceral adipose tissue is enhanced compared to abdominal subcutaneous tissue in vivo. Abdominal adipose tissue FFA uptake was measured using positron emission tomography (PET) and [¹⁸F]‐labeled 6‐thia‐hepta‐decanoic acid ([¹⁸F]FTHA) and fat masses using magnetic resonance imaging (MRI) in 18 healthy young adult males. We found that FFA uptake was 30% higher in visceral compared to subcutaneous adipose tissue (0.0025 ± 0.0018 vs. 0.0020 ± 0.0016 µmol/g/min, P = 0.005). Visceral and subcutaneous adipose tissue FFA uptakes were strongly associated with each other (P < 0.001). When tissue FFA uptake per gram of fat was multiplied by the total tissue mass, total FFA uptake was almost 1.5 times higher in abdominal subcutaneous than in visceral adipose tissue. In conclusion, we observed enhanced FFA uptake in visceral compared to abdominal subcutaneous adipose tissue and, simultaneously, these metabolic rates were strongly associated with each other. The higher total tissue FFA uptake in subcutaneous than in visceral adipose tissue indicates that although visceral fat is active in extracting FFA, its overall contribution to systemic metabolism is limited in healthy lean males. Our results indicate that subcutaneous, rather than visceral fat storage plays a more direct role in systemic FFA availability. The recognized relationship between abdominal visceral fat mass and metabolic complications may be explained by direct effects of visceral fat on the liver.     

Caspase-8, c-FLIP, and caspase-9 in c-Myc-induced apoptosis of fibroblasts

c-Myc is known to induce or potentiate apoptotic processes predominantly by triggering or enhancing the activity of caspases, but the activation mechanisms of caspases by c-Myc remain still poorly understood. Here we found that in MycER™ rat fibroblasts the activation of c-Myc led to an early activation and cleavage of the initiator caspase-8, and concurrent processing and activation of the effector caspases 3 and 7. Interestingly, the expression of cellular FLICE inhibitory protein (c-FLIP) mRNA and the encoded protein, c-FLIP_L, a catalytically inactive homologue of caspase-8, were down-regulated prior to or coincidently with the activation of caspase-8. Of the other known initiators, caspase-9, involved in the mitochondrial pathway, was activated/processed surprisingly late, only after the effector caspases 3/7. Further, we studied the potential involvement of the Fas- and tumor necrosis factor receptor (TNFR)-mediated signaling in the activation of caspase-8 by c-Myc. Blocking of the function of these death receptors by neutralizing antibodies against Fas ligand and TNF-α did not prevent the processing of caspase-8 or cell death. c-Myc was neither found to induce any changes in the expression of TNF-related apoptosis inducing ligand (TRAIL) or its receptor. These data suggest that caspase-8 does not become activated through an extrinsic but an “intrinsic/intracellular” apoptotic pathway unleashed by the down-regulation of c-FLIP by c-Myc. Moreover, ectopic expression of c-FLIP_L inhibited the c-Myc-induced apoptosis.     

Emergent neutrality in adaptive asexual evolution

In nonrecombining genomes, genetic linkage can be an important evolutionary force. Linkage generates interference interactions, by which simultaneously occurring mutations affect each other's chance of fixation. Here, we develop a comprehensive model of adaptive evolution in linked genomes, which integrates interference interactions between multiple beneficial and deleterious mutations into a unified framework. By an approximate analytical solution, we predict the fixation rates of these mutations, as well as the probabilities of beneficial and deleterious alleles at fixed genomic sites. We find that interference interactions generate a regime of emergent neutrality: all genomic sites with selection coefficients smaller in magnitude than a characteristic threshold have nearly random fixed alleles, and both beneficial and deleterious mutations at these sites have nearly neutral fixation rates. We show that this dynamic limits not only the speed of adaptation, but also a population's degree of adaptation in its current environment. We apply the model to different scenarios: stationary adaptation in a time-dependent environment and approach to equilibrium in a fixed environment. In both cases, the analytical predictions are in good agreement with numerical simulations. Our results suggest that interference can severely compromise biological functions in an adapting population, which sets viability limits on adaptive evolution under linkage.     

Germline fitness-based scoring of cancer mutations

A key goal in cancer research is to find the genomic alterations that underlie malignant cells. Genomics has proved successful in identifying somatic variants at a large scale. However, it has become evident that a typical cancer exhibits a heterogenous mutation pattern across samples. Cases where the same alteration is observed repeatedly seem to be the exception rather than the norm. Thus, pinpointing the key alterations (driver mutations) from a background of variations with no direct causal link to cancer (passenger mutations) is difficult. Here we analyze somatic missense mutations from cancer samples and their healthy tissue counterparts (germline mutations) from the viewpoint of germline fitness. We calibrate a scoring system from protein domain alignments to score mutations and their target loci. We show first that this score predicts to a good degree the rate of polymorphism of the observed germline variation. The scoring is then applied to somatic mutations. We show that candidate cancer genes prone to copy number loss harbor mutations with germline fitness effects that are significantly more deleterious than expected by chance. This suggests that missense mutations play a driving role in tumor suppressor genes. Furthermore, these mutations fall preferably onto loci in sequence neighborhoods that are high scoring in terms of germline fitness. In contrast, for somatic mutations in candidate onco genes we do not observe a statistically significant effect. These results help to inform how to exploit germline fitness predictions in discovering new genes and mutations responsible for cancer.     

Distinguishing driver and passenger mutations in an evolutionary history categorized by interference

In many biological scenarios, from the development of drug resistance in pathogens to the progression of healthy cells toward cancer, quantifying the selection acting on observed mutations is a central question. One difficulty in answering this question is the complexity of the background upon which mutations can arise, with multiple potential interactions between genetic loci. We here present a method for discerning selection from a population history that accounts for interference between mutations. Given sequences sampled from multiple time points in the history of a population, we infer selection at each locus by maximizing a likelihood function derived from a multilocus evolution model. We apply the method to the question of distinguishing between loci where new mutations are under positive selection (drivers) and loci that emit neutral mutations (passengers) in a Wright-Fisher model of evolution. Relative to an otherwise equivalent method in which the genetic background of mutations was ignored, our method inferred selection coefficients more accurately for both driver mutations evolving under clonal interference and passenger mutations reaching fixation in the population through genetic drift or hitchhiking. In a population history recorded by 750 sets of sequences of 100 individuals taken at intervals of 100 generations, a set of 50 loci were divided into drivers and passengers with a mean accuracy of >0.95 across a range of numbers of driver loci. The potential application of our model, either in full or in part, to a range of biological systems, is discussed.     

Ectodysplasin,Edar and TNFRSF19 are expressed in complementary and overlapping patterns during mouse embryogenesis

Ectodysplasin (Eda), a member of the tumor necrosis factor (TNF) superfamily, and its receptor Edar are necessary components of ectodermal organ development. Analysis of their expression patterns and mutant phenotypes has shown that during mouse hair and tooth development they may be involved in signalling between separate epithelial compartments. Here we have analysed ectodysplasin and Edar expression in other embryonic mouse tissues, and show that Edar mRNA is confined to the epithelium. Ectodysplasin and Edar are expressed in separate epithelial compartments in the developing brain and the lacrimal gland. In the salivary gland ectodysplasin is expressed in the mesenchyme and Edar in the epithelium. This is the first indication of ectodysplasin-Edar signalling between the epithelium and the mesenchyme. We also studied the expression pattern of a related TNF receptor, TNFRSF19, and show that it is expressed in an overlapping domain with Edar in the tooth, mammary gland, whiskers, and limb bud suggesting a potentially redundant role.