Insulin sensitivity, body composition and adipose depots following 12 w combined endurance and strength training in dysglycemic and normoglycemic sedentary men.

CONTEXT: Insulin resistance and dysglycemia are associated with physical inactivity and adiposity, and may be improved by exercise. OBJECTIVE: Investigate the effect of exercise on insulin sensitivity, body composition and adipose depots in sedentary men with (n = 11) or without (n = 11) overweight and dysglycemia. MATERIAL AND METHODS: Euglycemic-hyperinsulinemic clamp, ankle-to-neck MRI, MRS, muscle and adipose tissue biopsies before and after 12 weeks combined strength and endurance exercise. RESULTS: Insulin sensitivity, VO2max, strength, whole-body and muscle fat content, and abdominal adipose depots were improved without obvious differences between normo- and dysglycemic men. Hepatic fat, waist circumference and subcutaneous adipose tissue were reduced in the dysglycemic group. For both groups plasma adiponectin was reduced, whereas IL-6 was unchanged. Visceral fat was preferentially lost compared with other adipose depots. DISCUSSION AND CONCLUSION: Body composition, fat distribution and insulin sensitivity improved following training in sedentary middle-aged men with and without dysglycemia.

Regulation of angiopoietin-like protein 4 production during and after exercise.

Angiopoietin-like protein 4 (ANGPTL4) may regulate lipoprotein lipase-dependent plasma clearance of triacylglycerol from skeletal muscle during exercise. The aim of this study was to examine the importance of muscle in regulating ANGPTL4 in response to exercise. We sampled muscle biopsies and serum before, immediately after, and 2 h after 45 min of ergometer cycling. Sampling was done before and after a 12-week training intervention in controls and dysglycemic subjects. Moreover, fat biopsies were taken before and after the training intervention. The regulation of ANGPTL4 was also investigated in several tissues of exercising mice, and in cultured myotubes. ANGPTL4 levels in serum and expression in muscle were highest 2 h after exercise in both groups. Whereas ANGPTL4 was higher in muscle of exercising controls as compared to dysglycemic subjects, the opposite was observed in serum. In exercising mice, Angptl4 mRNA showed both higher basal expression and induction in liver compared to muscle. Angptl4 mRNA was much higher in adipose tissue than muscle and was also induced by exercise. We observed two mRNA isoforms of ANGPTL4 in muscle and fat in humans. Both were induced by exercise in muscle; one isoform was expressed 5- to 10-fold higher than the other. Studies in mice and cultured myotubes showed that both fatty acids and cortisol have the potential to increase ANGPTL4 expression in muscle during exercise. In conclusion, ANGPTL4 is markedly induced in muscle in response to exercise. However, liver and adipose tissue may contribute more than muscle to the exercise-induced increase in circulating ANGPTL4.

The effects of acute and chronic exercise on PGC-1α, irisin and browning of subcutaneous adipose tissue in humans.

Irisin was first identified as a peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) dependent myokine with the potential to induce murine brown-fat-like development of white adipose tissue. In humans, the regulatory effect of training on muscle FNDC5 mRNA expression and subsequently irisin levels in plasma is more controversial. We recruited 26 inactive men (13 normoglycaemic and normal weight, controls; and 13 slightly hyperglycaemic and overweight, pre-diabetes group) aged 40-65 years for a 12-week intervention of combined endurance and strength training with four sessions of training per week. Before and after the 12-week intervention period, participants were exposed to an acute endurance workload of 45 min at 70% of VO(2max), and muscle biopsies were taken prior to and after exercise. Skeletal muscle mRNA for PGC1A and FNDC5 correlated and both PGC1A and FNDC5 mRNA levels increased after 12 weeks of training in both control and pre-diabetes subjects. Circulating irisin was reduced in response to 12 weeks of training, and was increased acutely (~1.2-fold) just after acute exercise. Plasma concentration of irisin was higher in pre-diabetes subjects compared with controls. There was little effect of 12 weeks of training on selected browning genes in subcutaneous adipose tissue. UCP1 mRNA did not correlate with FNDC5 expression in subcutaneous adipose tissue or skeletal muscle or with irisin levels in plasma. We observed no enhancing effect of long-term training on circulating irisin levels, and little or no effect of training on browning of subcutaneous white adipose tissue in humans.

Hyperoxic resuscitation after hypoxia-ischemia induces cerebral inflammation that is attenuated by tempol in a reporter mouse model with very young mice.

BACKGROUND: Oxygen supplementation is still part of international resuscitation protocols for premature children. Mechanisms for tissue damage by hypoxia/ischemia in the extreme premature involve inflammation. AIM AND METHOD: To study cerebral inflammation after hypoxia/ischemia and oxygen treatment in the premature, we measured NF-κB activity in 5-day-old transgenic reporter mice in response to experimental hypoxia/ischemia. results were correlated to cerebral histological evaluation and plasma cytokine levels. A treatment strategy with the antioxidant tempol was tested. RESULTS: One day after hypoxia/ischemia NF-κB activation was increased compared to controls [mean difference: 10.6±4.6% (P=0.03)]. Exposure to 100% oxygen after hypoxia/ischemia further increased NF-κB activation compared to hypoxia/ischemia alone [mean difference: 15.0±5.5% (P=0.01)]. Histological changes in the brain were positively correlated with NF-κB activity (P<0.001), but we found no significant difference in tissue damage between resuscitation with air and resuscitation with pure oxygen. Administration of tempol reduced NF-κB activation [mean difference: 14.6±5.0% (P=0.01)] and the plasma level of cytokines; however, the histological damage score was not affected. CONCLUSION: Cerebral inflammatory response after hypoxia/ischemia in a mouse model with immature brain development corresponding to human prematurity prior to 32 weeks' gestation was influenced by administration of oxygen. Tempol treatment attenuated inflammation but did not reduce the extent of histological cerebral damage.

Molecular nutrition research: the modern way of performing nutritional science.

In spite of amazing progress in food supply and nutritional science, and a striking increase in life expectancy of approximately 2.5 months per year in many countries during the previous 150 years, modern nutritional research has a great potential of still contributing to improved health for future generations, granted that the revolutions in molecular and systems technologies are applied to nutritional questions. Descriptive and mechanistic studies using state of the art epidemiology, food intake registration, genomics with single nucleotide polymorphisms (SNPs) and epigenomics, transcriptomics, proteomics, metabolomics, advanced biostatistics, imaging, calorimetry, cell biology, challenge tests (meals, exercise, etc.), and integration of all data by systems biology, will provide insight on a much higher level than today in a field we may name molecular nutrition research. To take advantage of all the new technologies scientists should develop international collaboration and gather data in large open access databases like the suggested Nutritional Phenotype database (dbNP). This collaboration will promote standardization of procedures (SOP), and provide a possibility to use collected data in future research projects. The ultimate goals of future nutritional research are to understand the detailed mechanisms of action for how nutrients/foods interact with the body and thereby enhance health and treat diet-related diseases.

NF-κB activity in perinatal brain during infectious and hypoxic-ischemic insults revealed by a reporter mouse.

Infants suffering from infection or hypoxia-ischemia around the time of birth can develop brain damage resulting in life-long impairment such as cerebral palsy, epilepsy and cognitive disability. Inflammation appears to be an important contributor irrespective of whether the primary event is infection or hypoxia-ischemia. Activation of the transcription factor NF-κB is a hallmark of inflammation. To study perinatal brain inflammation, we developed a transgenic reporter mouse for imaging NF-κB activity in live animals and tissue samples. The reporter genes firefly luciferase and a destabilized version of enhanced GFP (dEGFP) were regulated by common NF-κB sites using a bidirectional promoter. Luciferase activity was imaged in vivo, while dEGFP was detected at cellular level in tissue sections. In newborn mice subjected to experimental models of infections or hypoxia-ischemia; luciferase signal increased in brains of live animals. In brain sections dEGFP expression, revealing NF-κB activation was observed in the endothelial cells of the blood-brain barrier in all disease models. In meningitis and hypoxia-ischemia expression of dEGFP was also induced in perivascular astrocytes. In conclusion, by using this transgenic reporter mouse in experimental models of perinatal complications, we could assess NF-κB activity in vivo and subsequently determine the cellular origin in the tissues.

Adjuvant System AS03 containing α-tocopherol modulates innate immune response and leads to improved adaptive immunity.

AS03 is an Adjuvant System (AS) containing α-tocopherol and squalene in an oil-in-water (o/w) emulsion. AS03 has been considered for the development of pandemic and seasonal influenza vaccines. Key features of AS03's mode of action were investigated in vivo in mice and ex vivo in human cells. AS03's adjuvant activity was superior to that of aluminium hydroxide and required the spatio-temporal co-localisation of AS03 with the antigen. This requirement coincided with AS03 triggering a transient production of cytokines at the injection site and in the draining lymph nodes (dLNs). The nature of the cytokines produced was consistent with the enhanced recruitment of granulocytes and of antigen-loaded monocytes in the dLNs. The presence of α-tocopherol in AS03 was required for AS03 to achieve the highest antibody response. The presence of α-tocopherol also modulated the expression of some cytokines, including CCL2, CCL3, IL-6, CSF3 and CXCL1; increased the antigen loading in monocytes; and increased the recruitment of granulocytes in the dLNs. Hence, AS03's promotion of monocytes as the principal antigen-presenting cells, and its effects on granulocytes and cytokines, may all contribute to enhancing the antigen-specific adaptive immune response.

Molecular imaging of transcriptional regulation during inflammation.

Molecular imaging enables non-invasive visualization of the dynamics of molecular processes within living organisms in vivo. Different imaging modalities as MRI, SPECT, PET and optic imaging are used together with molecular probes specific for the biological process of interest. Molecular imaging of transcription factor activity is done in animal models and mostly in transgenic reporter mice, where the transgene essentially consists of a promoter that regulates a reporter gene. During inflammation, the transcription factor NF-kappaB is widely involved in orchestration and regulation of the immune system and almost all imaging studies in this field has revolved around the role and regulation of NF-kappaB. We here present a brief introduction to experimental use and design of transgenic reporter mice and a more extensive review of the various studies where molecular imaging of transcriptional regulation has been applied during inflammation.

AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity.

Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4'-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-kappaB activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4(+) T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.

In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012.

Production of reactive oxygen and nitrogen species (ROS/RNS) is an important part of the inflammatory response, but prolonged elevated levels of ROS/RNS as under chronic inflammation can contribute to the development of disease. Monitoring ROS/RNS in living animals is challenging due to the rapid turnover of ROS/RNS and the limited sensitivity and specificity of ROS/RNS probes. We have explored the use of the chemiluminescent probe L-012 for noninvasive imaging of ROS/RNS production during inflammation in living mice. Various inflammatory conditions were induced, and L-012-dependent luminescence was recorded with an ultrasensitive CCD camera. Strong luminescent signals were observed from different regions of the body corresponding to inflammation. The signal was reduced by administration of the SOD mimetic tempol, the NADPH oxidase inhibitor apocynin, and the inhibitor of nitric oxide synthesis L-NAME, signifying the requirement for the presence of ROS/RNS. Additionally, the L-012 signal was abolished in mice with a mutation in the Ncf1 gene, encoding a protein in the NADPH oxidase complex 2, which generates ROS/RNS during inflammation. In conclusion, L-012 is well distributed in the mouse body and mediates a strong ROS/RNS-dependent luminescent signal in vivo and is useful for monitoring the development and regulation of inflammation in living organisms.

Activity patterns govern synapse-specific AMPA receptor trafficking between deliverable and synaptic pools.

In single neurons, glutamatergic synapses receiving distinct afferent inputs may contain AMPA receptors (-Rs) with unique subunit compositions. However, the cellular mechanisms by which differential receptor transport achieves this synaptic diversity remain poorly understood. In lateral geniculate neurons, we show that retinogeniculate and corticogeniculate synapses have distinct AMPA-R subunit compositions. Under basal conditions at both synapses, GluR1-containing AMPA-Rs are transported from an anatomically defined reserve pool to a deliverable pool near the postsynaptic density (PSD), but further incorporate into the PSD or functional synaptic pool only at retinogeniculate synapses. Vision-dependent activity, stimulation mimicking retinal input, or activation of CaMKII or Ras signaling regulated forward GluR1 trafficking from the deliverable pool to the synaptic pool at both synapses, whereas Rap2 signals reverse GluR1 transport at retinogeniculate synapses. These findings suggest that synapse-specific AMPA-R delivery involves constitutive and activity-regulated transport steps between morphological pools, a mechanism that may extend to the site-specific delivery of other membrane protein complexes.

Tracking early autoimmune disease by bioluminescent imaging of NF-kappaB activation reveals pathology in multiple organ systems.

It is desirable to have an early and sensitive detection marker of autoimmune disease in intact animals. Nuclear factor (NF)-kappaB is a transcription factor that is associated with inflammatory responses and immune disorders. Previously, we demonstrated that so-called idiotypic-driven T-B cell collaboration in mice doubly transgenic for paired immunoglobulin and T cell receptor transgenes resulted in a systemic autoimmune disease with systemic lupus erythematosus-like features. Here, we investigated NF-kappaB activation by including an NF-kappaB-responsive luciferase reporter transgene in this animal model. Triply transgenic mice developed bioluminescence signals from diseased organs before onset of clinical symptoms and autoantibody production, and light emissions correlated with disease progression. Signals were obtained from secondary lymphoid organs, inflamed intestines, skin lesions, and arthritic joints. Moreover, bioluminescence imaging and immunohistochemistry demonstrated that a minority of mice suffered from an autoimmune disease of the small intestine, in which light emissions correlated with antibodies against tissue transglutaminase and gliadin. Detection of luciferase by immunohistochemistry revealed NF-kappaB activation in collaborating B and T cells, as well as in macrophages. These results demonstrate that bioluminescent in vivo imaging of NF-kappaB activation can be used for early and sensitive detection of autoimmune disease in an experimental mouse model, offering new possibilities for the evaluation of anti-inflammatory drugs.

Synapsin utilization differs among functional classes of synapses on thalamocortical cells.

Several proteins in nerve terminals participate in synaptic transmission between neurons. The synapsins, which are synaptic vesicle-associated proteins, have widespread distribution in the brain and are assumed essential for sustained recruitment of vesicles during high rates of synaptic transmission. We compared the role of synapsins in two types of glutamatergic synapses on thalamocortical cells in the dorsal lateral geniculate nucleus of mice: retinogeniculate synapses, which transmit primary afferent input at high frequencies and show synaptic depression, and corticogeniculate synapses, which provide modulatory feedback at lower frequencies and show synaptic facilitation. We used electrophysiological methods to determine effects of gene knock-out of synapsin I and II on short-term synaptic plasticity in paired-pulse, pulse-train, and posttetanic potentiation paradigms. The gene inactivation changed the plasticity properties in corticogeniculate, but not in retinogeniculate, synapses. Immunostaining with antibodies against synapsins in wild-type mice demonstrated that neither synapsin I nor II occurred in retinogeniculate terminals, whereas both occurred in corticogeniculate terminals. In GABAergic terminals, only synapsin I occurred. In corticogeniculate terminals of knock-out mice, the density of synaptic vesicles was reduced because of increased terminal size rather than reduced number of vesicles and the intervesicle distance was increased compared with wild-type mice. In the retinogeniculate terminals, no significant morphometric differences occurred between knock-out and wild-type mice. Together, this indicates that synapsin I and II are not present in the retinogeniculate terminals and therefore are not essential for sustained, high-rate synaptic transmission.

AMPA and NMDA currents show different short-term depression in the dorsal lateral geniculate nucleus of the rat.

Paired-pulse depression was studied at the glutamatergic synapse between retinal afferents and thalamocortical cells in the rat dorsal lateral geniculate nucleus. The main objective of this study was to examine the contributions of the pre- and postsynaptic sites to this depression by comparing AMPA- and NMDA-receptor-mediated responses. Equal depression of the two receptor components would indicate involvement of presynaptic mechanisms, while differences in depression would indicate involvement of postsynaptic mechanisms. Pharmacologically isolated AMPA- and NMDA-receptor-mediated currents were recorded using the whole-cell patch-clamp technique in acute thalamic slices. Both the AMPA and the NMDA components showed pronounced depression when retinal afferents were activated by paired pulses. The depression decayed within 5 s. The AMPA component was more strongly depressed than the NMDA component at paired-pulse intervals ranging from 20 to 200 ms, suggesting the involvement of postsynaptic mechanisms. For intervals of 500 ms and longer, the depression of the two components was identical, suggesting the involvement of purely presynaptic mechanisms. The degree of depression measured without the use of pharmacological tools produced similar results, thus excluding the involvement of presynaptic ionotropic glutamate receptors. Cyclothiazide, a blocker of AMPA-receptor desensitisation, reduced the difference in depression between the two components, suggesting that desensitisation of the AMPA receptors is a postsynaptic mechanism that contributes to the difference in depression between the AMPA and the NMDA components.