Maintenance of cardiorespiratory fitness, body composition, and a physically active lifestyle after structured exercise interventions in individuals with overweight and obesity: A mixed-method follow-up study.

Objectives: The aim of this mixed-method study was to explore maintenance of physical activity and health effects one year after completion of exercise interventions in transport and leisure-time domains of everyday life. We hypothesised that routinisation of active commuting would lead to better maintenance of physical activity and health effects compared with leisure-time exercise. Study design: Mixed-methods follow-up study. Methods: Individuals with overweight/obesity, who completed a 6-month exercise intervention (active commuting by bike (BIKE), moderate (MOD) or vigorous intensity leisure-time exercise (VIG)), were after one year invited to participate in a follow-up visit which included measurements of cardiorespiratory fitness during an incremental bicycle test and body composition using dual-energy X-ray absorptiometry. Variability in maintenance practices was assessed in a sub-sample of participants who experienced the greatest improvements ('VO2peak improvers') and reductions ('VO2peak reducers'), respectively, in cardiorespiratory fitness. Semi-structured interviews were conducted (15-30 min) and analysed using systematic text condensation to identify barriers and facilitators associated with maintenance of physical activity. Results: Out of the 74 participants completing an exercise intervention, 46 (62%) completed follow-up (BIKE: n = 14; MOD: n = 14; VIG: n = 18). Improvements in VO2peak and reductions in fat mass were maintained in BIKE and VIG. Body weight decreased in BIKE and fat free mass increased in VIG. Changes in VO2peak and anthropometry at follow-up did not differ between BIKE and MOD + VIG. Fat mass decreased and recreational physical activity increased in 'VO2peak improvers'. Findings from the interviews suggested that self-monitoring, collective exercising, and new personal exercise challenges facilitate maintenance of a physically active lifestyle. Conclusion: Completion of a structured exercise intervention consisting of 6 months of active commuting or vigorous intensity leisure-time exercise was associated with long-term maintenance of improvements in VO2peak and body composition, whereas moderate intensity leisure-time exercise was not. In contrast to our hypothesis, active commuting was not associated with better maintenance of physical activity and health effects after the intervention compared with leisure-time exercise.

EBI2 in splenic and local immune responses and in autoimmunity.

The seven transmembrane G protein-coupled receptor EBV-induced gene 2 (EBI2), also known as GPR183, is expressed in particular in immune cells. Activated by its endogenous ligands, which are a group of oxysterols, it functions as a chemo-attractant receptor, mediating cell migration. In coordination with other receptors, EBI2 plays important roles in controlling the migration of immune cells during the course of a T-dependent Ab response in the spleen. In recent years, it has become clear that EBI2 also has other roles to play in the immune system. Thus, EBI2 seems to be involved in innate immune responses, such as those mediated by TLR signaling, and it has been implicated in regional immune responses, including immune responses in the CNS. In this review, we describe the functions of EBI2 in B cells, T cells, and dendritic cells during the course of a T-dependent Ab response in the spleen. Furthermore, we review the existing evidence supporting a role for EBI2 in local immune responses and in autoimmune diseases, with a special focus on immune responses in the CNS. Finally, we discuss which type of role EBI2 may play in autoimmune diseases, and we give our opinion about the paths of future research in EBI2.

Effects of active commuting and leisure-time exercise on fat loss in women and men with overweight and obesity: a randomized controlled trial.

BACKGROUND: Aerobic exercise is recommended for weight management but energy balance is often less negative than predicted from exercise energy expenditure (ExEE). OBJECTIVE: To examine effects of active commuting and leisure-time exercise on fat loss in women and men with overweight and obesity. METHODS: We randomized 130 younger, physically inactive women and men with overweight and obesity (body mass index: 25-35 kg m-2) to 6 months of habitual lifestyle (control; CON, n=18), active commuting (BIKE, n=35) or leisure-time exercise of moderate (MOD, 50% VO2peak reserve, n=39) or vigorous intensity (VIG, 70% VO2peak reserve, n=38). The primary outcome was change in fat mass measured by dual-energy X-ray absorptiometry, which was analyzed intention-to-treat. Accumulated energy balance was calculated based on changes in body composition, and ExEE was calculated based on heart rate monitoring during exercise. RESULTS: Testing at 3 and 6 months was completed by 95 and 90 participants, respectively. Fat mass was reduced after 3 and 6 months in BIKE (3 months: -3.6 (-5.5; -1.7) kg (mean (95% CI)); 6 months: -4.2 (-6.6; -1.9) kg; both: P<0.001), MOD (3 months: -2.2 (-3.9; -0.4) kg; 6 months: -2.6 (-4.8; -0.5) kg, both: P<0.02) and VIG (3 months: -3.4 (-5.2; -1.7) kg; 6 months: -4.5 (-6.6; -2.3) kg; both: P<0.001) compared with CON. Furthermore, fat loss was greater in VIG compared with MOD (6 months: -1.8 (-3.6; -0.1) kg, P=0.043). Based on the ExEE and the accumulated energy balance MOD compensated for the ExEE (77 (48; 106) %) but not BIKE (38 (-18; 95) %) and VIG (21 (-14; 55) %). CONCLUSIONS: A meaningful fat loss was obtained by 6 months of active commuting and leisure-time exercise, but fat loss was greater with vigorous compared with moderate intensity exercise. Active commuting is an alternative to leisure-time exercise in the management of overweight and obesity. The trial was registered at clinicaltrials.gov as NCT01962259 (main trial) and NCT01973686 (energy metabolism sub-study).

Protein engineering of the chemokine CCL20 prevents psoriasiform dermatitis in an IL-23-dependent murine model.

Psoriasis is a chronic inflammatory skin disease characterized by the infiltration of T cell and other immune cells to the skin in response to injury or autoantigens. Conventional, as well as unconventional, γδ T cells are recruited to the dermis and epidermis by CCL20 and other chemokines. Together with its receptor CCR6, CCL20 plays a critical role in the development of psoriasiform dermatitis in mouse models. We screened a panel of CCL20 variants designed to form dimers stabilized by intermolecular disulfide bonds. A single-atom substitution yielded a CCL20 variant (CCL20 S64C) that acted as a partial agonist for the chemokine receptor CCR6. CCL20 S64C bound CCR6 and induced intracellular calcium release, consistent with G-protein activation, but exhibited minimal chemotactic activity. Instead, CCL20 S64C inhibited CCR6-mediated T cell migration with nominal impact on other chemokine receptor signaling. When given in an IL-23-dependent mouse model for psoriasis, CCL20 S64C prevented psoriatic inflammation and the up-regulation of IL-17A and IL-22. Our results validate CCR6 as a tractable therapeutic target for psoriasis and demonstrate the value of CCL20 S64C as a lead compound.

Radiological and pathological evaluation of the level of arterial division after colon cancer surgery.

AIM: In aiming to cure patients with colorectal cancer surgery, the surgeon must carefully dissect the mesocolon and mesorectum and divide the vascular pedicle near to its origin so as to include all local lymph nodes. This has been termed complete mesocolic excision. The distance from the distal vascular tie to the bowel wall in the fixed specimen is an indication as to the quality of surgery but this does not assess the length of the residual vascular pedicle and, by implication, residual lymph nodes. The aim of this study was to establish if our surgeons were carrying out complete mesocolic excision by assessing the length of the proximal arterial pedicle and relating this to arterial length in the fixed specimen. METHOD: This was a single centre prospective study of patients undergoing elective surgery for locally advanced colorectal cancer. An abdominal and pelvic CT scan was performed 2 days postoperatively and a radiologist blinded to the operative procedure measured the length of the residual arterial stump. Similarly, the length of the vessel in the fixed resected specimen and lymph node yield were also recorded. RESULTS: Fifty-two patients were recruited. The mean length of the residual arterial stump was 38 mm (95% CI: 33-43), which was significantly longer than the < 10 mm recommended in guidelines (P < 0.0001). The mean length was 31 mm (95% CI: 25-37) and 49 mm (95% CI: 40-57) for left and right sided resections respectively. There was no correlation between the residual arterial stump and the pathology. CONCLUSIONS: The residual arterial length was greater than suggested by guidelines and may indicate that our surgery is less radical than we planned. Caution should be taken when using pathological measurements of vascular ligation as it may not reflect the height of the pedicle division.

GIP(3-30)NH2 is a potent competitive antagonist of the GIP receptor and effectively inhibits GIP-mediated insulin, glucagon, and somatostatin release.

Alternative processing of the precursor protein pro-GIP results in endogenously produced GIP(1-30)NH2, that by DPP-4 cleavage in vivo results in the metabolite GIP(3-30)NH2. We showed previously that GIP(3-30)NH2 is a high affinity antagonist of the human GIPR in vitro. Here we determine whether it is suitable for studies of GIP physiology in rats since effects of GIP agonists and antagonists are strictly species-dependent. Transiently transfected COS-7 cells were assessed for cAMP accumulation upon ligand stimulation or assayed in competition binding using human 125I-GIP(1-42) as radioligand. In isolated perfused rat pancreata, insulin, glucagon, and somatostatin-releasing properties were evaluated. Competition binding demonstrated that on the rat GIP receptor (GIPR), rat GIP(3-30)NH2 bound with high affinity (Ki of 17nM), in contrast to human GIP(3-30)NH2 (Ki of 250nM). In cAMP studies, rat GIP(3-30)NH2 inhibited GIP(1-42)-induced rat GIPR activation and schild-plot analysis showed competitive antagonism with a pA2 of 13nM and a slope of 0.9±0.09. Alone, rat GIP(3-30)NH2 displayed weak, low-potent partial agonistic properties (EC50>1µM) with an efficacy of 9.4% at 0.32µM compared to GIP(1-42). In perfused rat pancreata, rat GIP(3-30)NH2 efficiently antagonized rat GIP(1-42)-induced insulin, somatostatin, and glucagon secretion. In summary, rat GIP(3-30)NH2 is a high affinity competitive GIPR antagonist and effectively antagonizes GIP-mediated G protein-signaling as well as pancreatic hormone release, while human GIP(3-30)NH2, despite a difference of only one amino acid between the two (arginine in position 18 in rat GIP(3-30)NH2; histidine in human), is unsuitable in the rat system. This underlines the importance of species differences in the GIP system, and the limitations of testing human peptides in rodent systems.

Targeting the latent cytomegalovirus reservoir with an antiviral fusion toxin protein.

Reactivation of human cytomegalovirus (HCMV) in transplant recipients can cause life-threatening disease. Consequently, for transplant recipients, killing latently infected cells could have far-reaching clinical benefits. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, and one viral gene expressed by latently infected myeloid cells is US28. This viral gene encodes a cell surface G protein-coupled receptor (GPCR) that binds chemokines, triggering its endocytosis. We show that the expression of US28 on the surface of latently infected cells allows monocytes and their progenitor CD34+ cells to be targeted and killed by F49A-FTP, a highly specific fusion toxin protein that binds this viral GPCR. As expected, this specific targeting of latently infected cells by F49A-FTP also robustly reduces virus reactivation in vitro. Consequently, such specific fusion toxin proteins could form the basis of a therapeutic strategy for eliminating latently infected cells before haematopoietic stem cell transplantation.

Double-blind randomized controlled trial of collagen mesh for the prevention of abdominal incisional hernia in patients having a vertical rectus abdominis myocutaneus flap during surgery for advanced pelvic malignancy.

AIM: The study investigated the effect of collagen mesh-assisted closure at the donor site in preventing the formation of incisional hernia following construction of a vertical rectus abdominis myocutaneus (VRAM) flap as part of pelvic surgery for recurrent colorectal cancer. METHOD: The study was a double-blinded randomized controlled superiority trial that was designed and performed according to the Consolidated Standards of Reporting Trials (CONSORT) Statement. Eligible patients undergoing surgery that included a VRAM flap for advanced colorectal pelvic malignancy were prospectively randomized to conventional abdominal wound closure or collagen mesh-assisted closure. The primary end-point was incisional herniation at 1 year confirmed by CT. Secondary end-points were CT-verified incisional herniation at 3 and 36 months, clinically recognizable incisional herniation, donor-site and reconstructive-site complications, surgical mortality, postoperative morbidity, postoperative recovery and survival. RESULTS: In total, 58 (29 conventional closure; 29 mesh-assisted closure) patients were included. At 1 year, incisional herniation on the CT scan was found in 12 (50%) of 24 patients in the conventional closure group, and in 8 (33%) of 24 in the mesh-assisted closure group (P = 0.38). No significant difference between the groups was found in surgical mortality, early or late complications or survival. Donor-site morbidity was comparable between the two groups. CONCLUSION: No preventative effect of collagen mesh-assisted closure was observed following VRAM flap reconstruction.

Oral 2-oleyl glyceryl ether improves glucose tolerance in mice through the GPR119 receptor.

The intestinal G protein-coupled receptor GPR119 is a novel metabolic target involving glucagon-like peptide-1 (GLP-1)-derived insulin-regulated glucose homeostasis. Endogenous and diet-derived lipids, including N-acylethanolamines and 2-monoacylglycerols (2-MAG) activate GPR119. The purpose of this work is to evaluate whether 2-oleoyl glycerol (2-OG) improves glucose tolerance through GPR119, using wild type (WT) and GPR 119 knock out (KO) mice. We here show that GPR119 is essential for 2-OG-mediated release of GLP-1 and CCK from GLUTag cells, since a GPR119 specific antagonist completely abolished the hormone release. Similarly, in isolated primary colonic crypt cultures from WT mice, GPR119 was required for 2-OG-stimulated GLP-1 release while there was no response in crypts from KO mice. In vivo, gavage with 2-oleyl glyceryl ether ((2-OG ether), a stable 2-OG analog with a potency of 5.3 µM for GPR119 with respect to cAMP formation as compared to 2.3 µM for 2-OG), significantly (P < 0.05) improved glucose clearance in WT littermates, but not in GPR119 KO mice. Finally, deletion of GPR119 in mice resulted in lower glucagon levels, whereas the levels of insulin and GIP were unchanged. In the present study we show that 2-OG stimulates GLP-1 secretion through GPR119 activation in vitro, and that fat-derived 2-MAGs are potent candidates for mediating fat-induced GLP-1 release through GPR119 in vivo. © 2016 BioFactors, 42(6):665-673, 2016.

Maintenance of improvements in fitness and fatness 1 year after a 3-month lifestyle intervention in overweight men.

Sustained improvements in cardiovascular fitness and body composition after lifestyle interventions are challenging. The present study investigated whether changes in cardiovascular fitness and body composition were maintained for up to 1 year after similar exercise-induced (T) or diet-induced weight loss (D) or exercise without weight loss (T-iD) in overweight sedentary men. Six and 12 months after the interventions, we measured cardiovascular fitness and body composition. Cardiovascular fitness was higher at both 6- (3.2±1.5 ml O2/kg/min, P=0.053) and 12-month follow-up (3.9±1.4 ml O2/kg/min, P=0.049) compared with pre-intervention in T. Fat mass (-3.0±1.2 kg, P=0.04) and abdominal fat (-3.6±1.5%, P=0.04) were lower within T at 12-month follow-up compared with pre-intervention. This did not occur in D (P>0.13) or T-iD (P>0.14), although body weight was lower in D (-2.5±2.2 kg, P=0.09). This study showed that fitness and fatness were not returned to pre-intervention levels 1 year after a 3-month exercise-induced weight-loss intervention.

N-terminally and C-terminally truncated forms of glucose-dependent insulinotropic polypeptide are high-affinity competitive antagonists of the human GIP receptor.

BACKGROUND AND PURPOSE: Glucose-dependent insulinotropic polypeptide (GIP) affects lipid, bone and glucose homeostasis. High-affinity ligands for the GIP receptor are needed to elucidate the physiological functions and pharmacological potential of GIP in vivo. GIP(1-30)NH2 is a naturally occurring truncation of GIP(1-42). Here, we have characterized eight N-terminal truncations of human GIP(1-30)NH2 . EXPERIMENTAL APPROACH: COS-7 cells were transiently transfected with human GIP receptors and assessed for cAMP accumulation upon ligand stimulation or competition binding with (125) I-labelled GIP(1-42), GIP(1-30)NH2 , GIP(2-30)NH2 or GIP(3-30)NH2 . KEY RESULTS: GIP(1-30)NH2 displaced (125) I-GIP(1-42) as effectively as GIP(1-42) (Ki 0.75 nM), whereas the eight truncations displayed lower affinities (Ki 2.3-347 nM) with highest affinities for GIP(3-30)NH2 and GIP(5-30)NH2 (5-30)NH2 . Only GIP(1-30)NH2 (Emax 100% of GIP(1-42)) and GIP(2-30)NH2 (Emax 20%) were agonists. GIP(2- to 9-30)NH2 displayed antagonism (IC50 12-450 nM) and Schild plot analyses identified GIP(3-30)NH2 and GIP(5-30)NH2 as competitive antagonists (Ki 15 nM). GIP(3-30) NH2 was a 26-fold more potent antagonist than GIP(3-42). Binding studies with agonist ((125) I-GIP(1-30)NH2 ), partial agonist ((125) I-GIP(2-30)NH2 ) and competitive antagonist ((125) I-GIP(3-30)NH2 ) revealed distinct receptor conformations for these three ligand classes. CONCLUSIONS AND IMPLICATIONS: The N-terminus is crucial for GIP agonist activity. Removal of the C-terminus of the endogenous GIP(3-42) creates another naturally occurring, more potent, antagonist GIP(3-30)NH2 , which like GIP(5-30)NH2 , was a high-affinity competitive antagonist. These peptides may be suitable tools for basic GIP research and future pharmacological interventions.

Species-specific action of (Pro3)GIP - a full agonist at human GIP receptors, but a partial agonist and competitive antagonist at rat and mouse GIP receptors.

BACKGROUND AND PURPOSE: Specific, high potency receptor antagonists are valuable tools when evaluating animal and human physiology. Within the glucose-dependent, insulinotropic polypeptide (GIP) system, considerable attention has been given to the presumed GIP receptor antagonist, (Pro3)GIP, and its effect in murine studies. We conducted a pharmacological analysis of this ligand including interspecies differences between the rodent and human GIP system. EXPERIMENTAL APPROACH: Transiently transfected COS-7 cells were assessed for cAMP accumulation upon ligand stimulation and assayed in competition binding using (125) I-human GIP. Using isolated perfused pancreata both from wild type and GIP receptor-deficient rodents, insulin-releasing, glucagon-releasing and somatostatin-releasing properties in response to species-specific GIP and (Pro3)GIP analogues were evaluated. KEY RESULTS: Human (Pro3)GIP is a full agonist at human GIP receptors with similar efficacy (Emax ) for cAMP production as human GIP, while both rat and mouse(Pro3)GIP were partial agonists on their corresponding receptors. Rodent GIPs are more potent and efficacious at their receptors than human GIP. In perfused pancreata in the presence of 7 mM glucose, both rodent (Pro3)GIP analogues induced modest insulin, glucagon and somatostatin secretion, corresponding to the partial agonist activities observed in cAMP production. CONCLUSIONS AND IMPLICATIONS: When evaluating new compounds, it is important to consider interspecies differences both at the receptor and ligand level. Thus, in rodent models, human GIP is a comparatively weak partial agonist. Human (Pro3)GIP was not an antagonist at human GIP receptors, so there is still a need for a potent antagonist in order to elucidate the physiology of human GIP.

Transfer of liraglutide from blood to cerebrospinal fluid is minimal in patients with type 2 diabetes.

Treatment with liraglutide leads to weight loss. We investigated whether blood-to-cerebrospinal fluid (CSF) transfer of liraglutide occurs, and if so, whether it associates with clinical weight loss following liraglutide treatment in humans. We performed lumbar puncture and blood sampling in eight patients with type 2 diabetes (mean (range)): age 63 (54-79) years; actual body weight: 90 (75-118) kg treated with 1.8 mg liraglutide for 14 (5-22) months and with a treatment-induced weight loss of 8.4 (7-11) kg. We measured liraglutide in plasma and CSF with a radioimmunoassay specific for the N-terminus of the GLP-1 moiety of liraglutide. Mean plasma liraglutide was 31 (range: 21-63) nmol l(-1). The mean CSF-liraglutide concentration was 6.5 (range: 0.9-13.9) pmol l(-1). Ratio of CSF: plasma-liraglutide concentrations was 0.02 (range: 0.07-0.002)% and plasma liraglutide did not correlate with CSF-liraglutide levels (P=0.67). Body weight loss tended to correlate with plasma-liraglutide levels (P=0.06), but not with CSF-liraglutide levels (P=0.69). In conclusion, we measured very low concentrations of liraglutide in CSF, and the levels of CSF liraglutide did not correlate with the actual clinical weight loss in these patients. The amount of liraglutide in plasma tended to correlate with the clinical weight loss.

Changes in peak fat oxidation in response to different doses of endurance training.

The effect of different doses of endurance training on the capacity to oxidize fat during exercise in sedentary, overweight men and assessment of variables associated with changes in peak fat oxidation (PFO) were evaluated. Young, sedentary, overweight men were randomized to either the high-dose (HIGH, 600 kcal/day, n = 17) or moderate-dose (MOD, 300 kcal/day, n = 18) endurance training groups or controls (CON, n = 15). PFO and peak oxygen uptake (VO2 peak) were measured using indirect calorimetry, body composition using dual-energy x-ray absorptiometry, and protein levels of mitochondrial enzymes determined by Western blotting. PFO increased in both MOD [1.2 mg/kg fat-free mass (FFM)/min, 95% confidence interval (CI): 0.08:2.3, P = 0.03] and HIGH (1.8 mg/kg FFM/min, CI: 0.6:2.9, P < 0.001) compared with CON. Skeletal muscle expression of citrate synthase, ß-hydroxyacyl-CoA dehydrogenase, and mitochondrial oxphos complexes II-V increased similarly in MOD and HIGH. Stepwise multiple linear regression analysis with backward elimination of individual variables correlated with changes in PFO revealed increases in cycling efficiency, FFM, and VO2 peak as the remaining associated variables. In conclusion, PFO during exercise increased with both moderate- and high-dose endurance training. Increases in PFO were mainly predicted by changes in VO2 peak, FFM, and cycling efficiency, and less with skeletal muscle mitochondrial enzymes.

Determination of the binding mode for the cyclopentapeptide CXCR4 antagonist FC131 using a dual approach of ligand modifications and receptor mutagenesis.

BACKGROUND AND PURPOSE: The cyclopentapeptide FC131 (cyclo(-L-Arg(1) -L-Arg(2) -L-2-Nal(3) -Gly(4) -D-Tyr(5) -)) is an antagonist at the CXC chemokine receptor CXCR4, which plays a role in human immunodeficiency virus infection, cancer and stem cell recruitment. Binding modes for FC131 in CXCR4 have previously been suggested based on molecular docking guided by structure-activity relationship (SAR) data; however, none of these have been verified by in vitro experiments. EXPERIMENTAL APPROACH: Heterologous (125) I-12G5-competition binding and functional assays (inhibition of CXCL12-mediated activation) of FC131 and three analogues were performed on wild-type CXCR4 and 25 receptor mutants. Computational modelling was used to rationalize the experimental data. KEY RESULTS: The Arg(2) and 2-Nal(3) side chains of FC131 interact with residues in TM-3 (His(113) , Asp(171) ) and TM-5 (hydrophobic pocket) respectively. Arg(1) forms charge-charge interactions with Asp(187) in ECL-2, while D-Tyr(5) points to the extracellular side of CXCR4. Furthermore, the backbone of FC131 interacts with the chemokine receptor-conserved Glu(288) via two water molecules. Intriguingly, Tyr(116) and Glu(288) form a H-bond in CXCR4 crystal structures and mutation of either residue to Ala abolishes CXCR4 activity. CONCLUSIONS AND IMPLICATIONS: Ligand modification, receptor mutagenesis and computational modelling approaches were used to identify the binding mode of FC131 in CXCR4, which was in agreement with binding modes suggested from previous SAR studies. Furthermore, insights into the mechanism for CXCR4 activation by CXCL12 were gained. The combined findings will facilitate future design of novel CXCR4 antagonists.

Gating function of isoleucine-116 in TM-3 (position III:16/3.40) for the activity state of the CC-chemokine receptor 5 (CCR5).

BACKGROUND AND PURPOSE: A conserved amino acid within a protein family indicates a significance of the residue. In the centre of transmembrane helix (TM)-5, position V:13/5.47, an aromatic amino acid is conserved among class A 7TM receptors. However, in 37% of chemokine receptors - a subgroup of 7TM receptors - it is a leucine indicating an altered function. Here, we describe the significance of this position and its possible interaction with TM-3 for CCR5 activity. EXPERIMENTAL APPROACH: The effects of [L203F]-CCR5 in TM-5 (position V:13/5.47), [I116A]-CCR5 in TM-3 (III:16/3.40) and [L203F;G286F]-CCR5 (V:13/5.47;VII:09/7.42) were determined in G-protein- and ß-arrestin-coupled signalling. Computational modelling monitored changes in amino acid conformation. KEY RESULTS: [L203F]-CCR5 increased the basal level of G-protein coupling (20-70% of Emax ) and ß-arrestin recruitment (50% of Emax ) with a threefold increase in agonist potency. In silico, [I116A]-CCR5 switched χ1-angle in [L203F]-CCR5. Furthermore, [I116A]-CCR5 was constitutively active to a similar degree as [L203F]-CCR5. Tyr(244) in TM-6 (VI:09/6.44) moved towards TM-5 in silico, consistent with its previously shown function for CCR5 activation. On [L203F;G286F]-CCR5 the antagonist aplaviroc was converted to a superagonist. CONCLUSIONS AND IMPLICATIONS: The results imply that an aromatic amino acid in the centre of TM-5 controls the level of receptor activity. Furthermore, Ile(116) acts as a gate for the movement of Tyr(244) towards TM-5 in the active state, a mechanism proposed previously for the ß2 -adrenoceptor. The results provide an understanding of chemokine receptor function and thereby information for the development of biased and non-biased antagonists and inverse agonists.

Only minor additional metabolic health benefits of high as opposed to moderate dose physical exercise in young, moderately overweight men.

OBJECTIVE: The dose-response effects of exercise training on insulin sensitivity, metabolic risk, and quality of life were examined. METHODS: Sixty-one healthy, sedentary (VO2max: 35 ± 5 ml/kg/min), moderately overweight (BMI: 27.9 ± 1.8), young (age: 29 ± 6 years) men were randomized to sedentary living (sedentary control group; n = 18), moderate (moderate dose training group [MOD]: 300 kcal/day, n = 21), or high (high dose training group [HIGH]: 600 kcal/day, n = 22) dose physical exercise for 11 weeks. RESULTS: The return rate for post-intervention testing was 82-94% across groups. Weekly exercise amounted to 2,004 ± 24 and 3,774 ± 68 kcal, respectively, in MOD and HIGH. Cardiorespiratory fitness increased (P < 0.001) 18 ± 3% in MOD and 17 ± 3% in HIGH, and fat percentage decreased (P < 0.001) similarly in both exercise groups (MOD: 32 ± 1 to 29 ± 1%; HIGH: 30 ± 1 to 27 ± 1%). Peripheral insulin sensitivity increased (P < 0.01) (MOD: 28 ± 7%; HIGH: 36 ± 8%) and the homeostasis model assessment of insulin resistance decreased (P < 0.05) (MOD: -17 ± 7%; HIGH: -18 ± 10%). The number of subjects meeting the criteria of the metabolic syndrome decreased by 78% in MOD (P < 0.01) and by 80% in HIGH (P < 0.05). General health assessed by questionnaire increased similarly in MOD (P < 0.05) and HIGH (P < 0.01). CONCLUSIONS: Only minor additional health benefits were found when exercising ∼3,800 as opposed to ∼2,000 kcal/week in young moderately overweight men. This finding may have important public health implications.

Exercise training favors increased insulin-stimulated glucose uptake in skeletal muscle in contrast to adipose tissue: a randomized study using FDG PET imaging.

Physical exercise increases peripheral insulin sensitivity, but regional differences are poorly elucidated in humans. We investigated the effect of aerobic exercise training on insulin-stimulated glucose uptake in five individual femoral muscle groups and four different adipose tissue regions, using dynamic (femoral region) and static (abdominal region) 2-deoxy-2-[¹8F]fluoro-d-glucose (FDG) PET/CT methodology during steady-state insulin infusion (40 mU·m⁻²·min⁻¹). Body composition was measured by dual X-ray absorptiometry and MRI. Sixty-one healthy, sedentary [V(O2max) 36(5) ml·kg⁻¹·min⁻¹; mean(SD)], moderately overweight [BMI 28.1(1.8) kg/m²], young [age: 30(6) yr] men were randomized to sedentary living (CON; n = 17 completers) or moderate (MOD; 300 kcal/day, n = 18) or high (HIGH; 600 kcal/day, n = 18) dose physical exercise for 11 wk. At baseline, insulin-stimulated glucose uptake was highest in femoral skeletal muscle followed by intraperitoneal visceral adipose tissue (VAT), retroperitoneal VAT, abdominal (anterior + posterior) subcutaneous adipose tissue (SAT), and femoral SAT (P < 0.0001 between tissues). Metabolic rate of glucose increased similarly (~30%) in the two exercise groups in femoral skeletal muscle (MOD 24[9, 39] µmol·kg⁻¹·min⁻¹, P = 0.004; HIGH 22[9, 35] µmol·kg⁻¹·min⁻¹, P = 0.003) (mean[95% CI]) and in five individual femoral muscle groups but not in femoral SAT. Standardized uptake value of FDG decreased ~24% in anterior abdominal SAT and ~20% in posterior abdominal SAT compared with CON but not in either intra- or retroperitoneal VAT. Total adipose tissue mass decreased in both exercise groups, and the decrease was distributed equally among subcutaneous and intra-abdominal depots. In conclusion, aerobic exercise training increases insulin-stimulated glucose uptake in skeletal muscle but not in adipose tissue, which demonstrates some interregional differences.

Molecular requirements for inhibition of the chemokine receptor CCR8--probe-dependent allosteric interactions.

BACKGROUND AND PURPOSE: Here we present a novel series of CCR8 antagonists based on a naphthalene-sulfonamide structure. This structure differs from the predominant pharmacophore for most small-molecule CC-chemokine receptor antagonists, which in fact activate CCR8, suggesting that CCR8 inhibition requires alternative structural probes. EXPERIMENTAL APPROACH: The compounds were tested as inverse agonists and as antagonists against CCL1-induced activity in Gα(i) signalling and chemotaxis. Furthermore, they were assessed by heterologous competition binding against two radiolabelled receptor ligands: the endogenous agonist CCL1 and the virus-encoded antagonist MC148. KEY RESULTS: All compounds were highly potent inverse agonists with EC(50) values from 1.7 to 23 nM. Their potencies as antagonists were more widely spread (EC(50) values from 5.9 to 1572 nM). Some compounds were balanced antagonists/inverse agonists whereas others were predominantly inverse agonists with >100-fold lower potency as antagonists. A correspondingly broad range of affinities, which followed the antagonist potencies, was disclosed by competition with [(125)I]-CCL1 (K(i) 3.4-842 nM), whereas the affinities measured against [(125)I]-MC148 were less widely spread (K(i) 0.37-27 nM), and matched the inverse agonist potencies. CONCLUSION AND IMPLICATIONS: Despite highly potent and direct effects as inverse agonists, competition-binding experiments against radiolabelled agonist and tests for antagonism revealed a probe-dependent allosteric effect of these compounds. Thus, minor chemical changes affected the ability to modify chemokine binding and action, and divided the compounds into two groups: predominantly inverse agonists and balanced antagonists/inverse agonists. These studies have important implications for the design of new inverse agonists with or without antagonist properties.

Reversed binding of a small molecule ligand in homologous chemokine receptors - differential role of extracellular loop 2.

BACKGROUND AND PURPOSE: The majority of small molecule compounds targeting chemokine receptors share a similar pharmacophore with a centrally located aliphatic positive charge and flanking aromatic moieties. Here we describe a novel piperidine-based compound with structural similarity to previously described CCR8-specific agonists, but containing a unique phenyl-tetrazol moiety which, in addition to activity at CCR8 was also active at CCR1. EXPERIMENTAL APPROACH: Single point mutations were introduced in CCR1 and CCR8, and their effect on small molecule ligand-induced receptor activation was examined through inositol trisphosphate (IP(3) ) accumulation. The molecular interaction profile of the agonist was verified by molecular modeling. KEY RESULTS: The chemokine receptor conserved glutamic acid in TM-VII served as a common anchor for the positively charged amine in the piperidine ring. However, whereas the phenyl-tetrazol group interacted with TyrIV:24 (Tyr(172) ) and TyrIII:09 (Tyr(114) ) in the major binding pocket (delimited by TM-III to VII) of CCR8, it also interacted with TrpII:20 (Trp(90) ) and LysII:24 (Lys(94) ) in the minor counterpart (delimited TM-I to III, plus TM-VII) in CCR1. A straightening of TM-II by Ala-substitution of ProII:18 confirmed its unique role in CCR1. The extracellular loop 2 (ECL-2) contributed directly to the small molecule binding site in CCR1, whereas it contributed to efficacy, but not potency in CCR8. CONCLUSION AND IMPLICATIONS: Despite high ligand potency and efficacy and receptor similarity, this dual-active and bitopic compound binds oppositely in CCR1 and CCR8 with different roles of ECL-2, thereby expanding and diversifying the influence of extracellular receptor regions in drug action.