Lipolytic Markers, Insulin and Resting Fat Oxidation are Associated with Maximal Fat Oxidation.

The maximal capacity to oxidize fat during exercise (MFO) is associated with 24-h fat balance and insulin sensitivity. Understanding factors that influence MFO could have implications for metabolic health. We investigated relationships between selected plasma metabolites, hormones and overnight-fasted resting fat oxidation rates (Resting), with MFO. Resting fat oxidation and MFO was measured in 57 men with blood collected at rest and during exercise. Plasma glycerol (R=0.39, P=0.033), non-esterified fatty acids (NEFA: R=0.27, P=0.030) and insulin (R=- 0.36, P=0.007) measured at MFO correlated with MFO; only glycerol remained correlated when controlled for resting concentrations (R=0.36, P=0.008). The change in glycerol from rest to MFO correlated with exercise-induced fat oxidation (R=0.32, P=0.012). V˙O 2max correlated with resting fat oxidation (R=0.44, P=0.001) and MFO (R=0.52, P<0.001). Resting fat oxidation correlated with MFO (R=0.55, P<0.001); this remained when controlled for V˙O 2max (R=0.41, P=0.001). This study reports weak-to-moderate, albeit significant, relationships between plasma lipolytic markers, insulin and resting overnight-fasted fat oxidation with MFO and shows the plasma glycerol response to uniquely reflect exercise-induced fat oxidation. V˙O 2max correlates with fat oxidation but the relationship can be dissociated. Interventions to increase fat oxidation for optimal metabolic health would benefit from, but are not reliant on, increases in V˙O 2max.

Development of a fully automatic shape model matching (FASMM) system to derive statistical shape models from radiographs: application to the accurate capture and global representation of proximal femur shape.

OBJECTIVE: To evaluate the accuracy and sensitivity of a fully automatic shape model matching (FASMM) system to derive statistical shape models (SSMs) of the proximal femur from non-standardised anteroposterior (AP) pelvic radiographs. DESIGN: AP pelvic radiographs obtained with informed consent and appropriate ethical approval were available for 1105 subjects with unilateral hip osteoarthritis (OA) who had been recruited previously for The arcOGEN Study. The FASMM system was applied to capture the shape of the unaffected (i.e., without signs of radiographic OA) proximal femur from these radiographs. The accuracy and sensitivity of the FASMM system in calculating geometric measurements of the proximal femur and in shape representation were evaluated relative to validated manual methods. RESULTS: De novo application of the FASMM system had a mean point-to-curve error of less than 0.9 mm in 99% of images (n = 266). Geometric measurements generated by the FASMM system were as accurate as those obtained manually. The analysis of the SSMs generated by the FASMM system for male and female subject groups identified more significant differences (in five of 17 SSM modes after Bonferroni adjustment) in their global proximal femur shape than those obtained from the analysis of conventional geometric measurements. Multivariate gender-classification accuracy was higher when using SSM mode values (76.3%) than when using conventional hip geometric measurements (71.8%). CONCLUSIONS: The FASMM system rapidly and accurately generates a global SSM of the proximal femur from radiographs of varying quality and resolution. This system will facilitate complex morphometric analysis of global shape variation across large datasets. The FASMM system could be adapted to generate SSMs from the radiographs of other skeletal structures such as the hand, knee or pelvis.

Post-exercise dietary macronutrient composition modulates components of energy balance in young, physically active adults.

The effectiveness of exercise to reduce body mass is typically modest, partially due to energy compensation responses which may be linked to energy substrate availability around exercise. The present study aimed to investigate the effect of manipulating post-exercise energy substrate availability (high carbohydrate/low fat [HCLF] or low carbohydrate/high fat [LCHF] energy replacement) on energy balance components in the short-term (i.e., appetite, energy intake (EI) and energy expenditure (EE)). METHODS: Appetite, EI, activity- and total- EE were measured in twelve healthy, young (21.0 ± 2.3 years) physically active participants (10 men, 2 women) on two occasions across 4 days after a 75-min run and an isocaloric energy replacement drink (HCLF and LCHF). Appetite was measured daily by visual analogue scales, EI was calculated by subtracting the energy content of food leftovers from a provided food package, activity- and total- EE determined by heart-rate accelerometery. RESULTS: Composite appetite ratings between days were lower in HCLF (62.4 ± 12) compared to LCHF (68.3 ± 8.9 mm; p = 0.048). No differences between conditions were detected for EI. Cumulative activity-EE (HCLF=  20.9 ± 3.7, LCHF=  16.9 ± 3.1 MJ; p = 0.037), but not total-EE (HCLF=  44.6 ± 7.7, LCHF=  39.9 ± 4.7 MJ; p = 0.060), was higher for the HCLF condition than the LCHF across the measurement period. CONCLUSION: Compared with low carbohydrate/high fat, immediate post-exercise energy replacement with a high carbohydrate/low fat drink resulted in higher short-term activity energy expenditure and lower appetite ratings.

Insights into the genetic architecture of osteoarthritis from stage 1 of the arcOGEN study.

OBJECTIVES: The genetic aetiology of osteoarthritis has not yet been elucidated. To enable a well-powered genome-wide association study (GWAS) for osteoarthritis, the authors have formed the arcOGEN Consortium, a UK-wide collaborative effort aiming to scan genome-wide over 7500 osteoarthritis cases in a two-stage genome-wide association scan. Here the authors report the findings of the stage 1 interim analysis. METHODS: The authors have performed a genome-wide association scan for knee and hip osteoarthritis in 3177 cases and 4894 population-based controls from the UK. Replication of promising signals was carried out in silico in five further scans (44,449 individuals), and de novo in 14 534 independent samples, all of European descent. RESULTS: None of the association signals the authors identified reach genome-wide levels of statistical significance, therefore stressing the need for corroboration in sample sets of a larger size. Application of analytical approaches to examine the allelic architecture of disease to the stage 1 genome-wide association scan data suggests that osteoarthritis is a highly polygenic disease with multiple risk variants conferring small effects. CONCLUSIONS: Identifying loci conferring susceptibility to osteoarthritis will require large-scale sample sizes and well-defined phenotypes to minimise heterogeneity.

Pre-race dietary carbohydrate intake can independently influence sub-elite marathon running performance.

We examined whether selected anthropometric and nutritional factors influenced field-based marathon running performance. An internet-based data collection tool allowed competitors in the 2009 London Marathon (n=257, mean ± SD age: 39 ± 8 years, finish time: 273.8 ± 59.5 min) to record a range of anthropometric, training and nutritional predictors. Multivariate statistical methods were used to quantify the change in running speed mediated by a unit change in each predictor via the 95% confidence interval for each covariate-controlled regression slope ( B). Gender ( B=1.22 to 1.95 km/h), body mass index ( B=-0.14 to -0.27 km/h), training distance ( B=0.01 to 0.04 km/h) and the amount of carbohydrate consumed the day before the race ( B=0.08 to 0.26 km/h) were significant predictors, collectively accounting for 56% of the inter-individual variability in running speed (P<0.0005). Further covariate-adjusted analysis revealed that those competitors who consumed carbohydrate the day before the race at a quantity of >7 g/kg body mass had significantly faster overall race speeds (P=0.01) and maintained their running speed during the race to a greater extent than with those who consumed <7 g/kg body mass (P=0.02). We conclude that, in addition to gender, body size and training, pre-race day carbohydrate intake can significantly and independently influence marathon running performance.

Coordinated expression of matrix Gla protein is required during endochondral ossification for chondrocyte survival.

Matrix Gla protein (MGP) is a 14-kD extracellular matrix protein of the mineral-binding Gla protein family. Studies of MGP-deficient mice suggest that MGP is an inhibitor of extracellular matrix calcification in arteries and the epiphyseal growth plate. In the mammalian growth plate, MGP is expressed by proliferative and late hypertrophic chondrocytes, but not by the intervening chondrocytes. To investigate the functional significance of this biphasic expression pattern, we used the ATDC5 mouse chondrogenic cell line. We found that after induction of the cell line with insulin, the differentiating chondrocytes express MGP in a stage-specific biphasic manner as in vivo. Treatment of the ATDC5 cultures with MGP antiserum during the proliferative phase leads to their apoptosis before maturation, whereas treatment during the hypertrophic phase has no effect on chondrocyte viability or mineralization. After stable transfection of ATDC5 cells with inducible sense or antisense MGP cDNA constructs, we found that overexpression of MGP in maturing chondrocytes and underexpression of MGP in proliferative and hypertrophic chondrocytes induced apoptosis. However, overexpression of MGP during the hypertrophic phase has no effect on chondrocyte viability, but it does reduce mineralization. This work suggests that coordinated levels of MGP are required for chondrocyte differentiation and matrix mineralization.

Bone growth: coordinating chondrocyte differentiation.

Two signalling molecules-Indian hedgehog and parathyroid hormone-related peptide-have been found to function in a negative feedback loop that is crucial for the coordinated regulation of the rate and extent of endochondral bone growth.

Cartilage disorders. The importance of being sulphated.

Mutations within a gene encoding a novel sulphate transporter cause diastrophic dysplasia. This finding has implications for the management of the disorder and for understanding the structure and function of cartilage.

The ribosomal protein QM is expressed differentially during vertebrate endochondral bone development.

Endochondral ossification is a carefully coordinated developmental process that converts the cartilaginous model of the embryonic skeleton to bone with accompanying long bone growth. To identify genes that regulate this process we performed a complementary DNA (cDNA) subtractive hybridization of fetal bovine proliferative chondrocyte cDNA from epiphyseal cartilage cDNA. The subtracted product was used to screen a fetal bovine cartilage cDNA library. Ten percent of the clones identified encoded the bovine orthologue of the human ribosomal protein "QM." Northern and western blot analysis confirmed that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In contrast, no detectable difference in the expression of mRNA for the ribosomal protein S11 was detected. Immunohistochemical analysis of fetal bovine limb sections revealed that QM was not expressed by the majority of the epiphyseal chondrocytes but only by chondrocytes in close proximity to capillaries that had invaded the epiphyseal cartilage. Strongest QM expression was seen in osteoblasts in the diaphyseal region of the bone adjoining the growth plate, within the periosteum covering the growth plate and within secondary centers of ossification. Hypertrophic chondrocytes within the growth plate adjoining the periosteum also were positive for QM as were chondrocytes in the perichondrium adjoining the periosteum. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. The in vivo and in vitro expression pattern of QM suggests that this protein may have a role in cell differentiation before mineralization.

A novel cell culture model of chondrocyte differentiation during mammalian endochondral ossification.

Endochondral ossification (EO) occurs in the growth plate where chondrocytes pass through discrete stages of proliferation, maturation, hypertrophy, and calcification. We have developed and characterized a novel bovine cell culture model of EO that mirrors these events and will facilitate in vitro studies on factors controlling chondrocyte differentiation. Chondrocytes derived from the epiphyses of long bones of fetal calves were treated with 5-azacytidine (aza-C) for 48 h. Cultures were maintained subsequently without aza-C and harvested at selected time points for analyses of growth and differentiation status. A chondrocytic phenotype associated with an extensive extracellular matrix rich in proteoglycans and collagen types II and VI was observed in aza-C-treated and -untreated cultures. aza-C-treated cultures were characterized by studying the expression of several markers of chondrocyte differentiation. Parathyroid hormone-related protein (PTHrP) and its receptor, both markers of maturation, were expressed at days 5-9. Type X collagen, which is restricted to the stage of hypertrophy, was expressed from day 11 onward. Hypertrophy was confirmed by a 14-fold increase in cell size by day 15 and an increased synthesis of alkaline phosphatase during the hypertrophic period (days 14-28). The addition of PTHrP to aza-C-treated cultures at day 14 led to the down-regulation of type X collagen by 6-fold, showing type X collagen expression is under the control of PTHrP as in vivo. These findings show that aza-C can induce fetal bovine epiphyseal chondrocytes to differentiate in culture in a manner consistent with that which occurs during the EO process in vivo.

Sequence comparison of three mammalian type-X collagen promoters and preliminary functional analysis of the human promoter.

The mechanism(s) controlling the specific expression of the type-X collagen (COL10A1)-encoding gene in the growth plate of developing long bones is not known. In preparation for identifying and characterizing the 5'-regulatory sequences and transcription factors which control mammalian Col10a1 gene expression, we have isolated and sequenced the first exon and 5' flanking promoter regions of bovine Col10a1. Sequence comparisons, including those previously published for mouse Col10a1, highlighted a number of conserved domains within the promoter and upstream elements. Reporter cat gene (encoding chloramphenicol acetyltransferase, CAT) constructs containing 5'-regulatory sequences of human COL10a1 (hCOL10a1) were transfected into primary cultures of foetal bovine growth plate chondrocytes producing COL10A1 and non-producing epiphyseal cartilage chondrocytes. Constructs containing up to 900 bp of promoter sequence exhibited low levels of CAT production in expressing cells and non-expressing cells. Addition of a further 1.5 kb of upstream sequence resulted in a dramatic increase in CAT production in expressing cells only. The results demonstrate the presence of enhancer-like elements between 900 bp and 2.4 kb upstream of the transcription start point(s) of hCOL10a1, which is distinctly different from that reported for the chick.

Tracing the pathway between mutation and phenotype in osteogenesis imperfecta: isolation of mineralization-specific genes.

The brittleness of bone in people with lethal (type II) osteogenesis imperfecta, a heritable disorder caused by mutations in the type I collagen genes, arises from the deposition of abnormal collagen in the bone matrix. The inability of the abnormal collagen to participate in mineralization may be caused by its failure to interact with other bone proteins. Here, we have designed a strategy to isolate the genes important for mineralization of collagen during bone formation. Cells isolated from 16-day embryonic chick calvaria and seeded post-confluence in culture deposited a mineralized matrix over a period of 2 weeks. Chick skin fibroblasts seeded and cultured under the same conditions did not mineralize. Using RT-PCR, we prepared short cDNAs (approximately 300 bp) corresponding to the 3' ends of mRNA from fibroblasts and separately from the mineralizing calvarial cells. Subtractive cDNA hybridization generated a pool of cDNAs that were specific to mineralizing calvarial cells but not to fibroblasts. Screening of 100,000 plaques of a chick bone ZAP Express cDNA library with this pool of mineralizing-specific cDNAs identified ten clones which comprised full-length cDNAs for the bone proteins osteopontin (eight of the ten positives), bone sialoprotein II (one of the ten positives), and cystatin (one of the ten positives). cDNAs for type I collagen, fibronectin, alkaline phosphatase, house-keeping genes, and other genes expressed in fibroblasts were not identified in this preliminary screen. The pool of short cDNAs is likely to comprise cDNAs for further bone-specific genes and will be used to screen the entire bone cDNA library of 4.2 million clones.

Increasing shape modelling accuracy by adjusting for subject positioning: an application to the analysis of radiographic proximal femur symmetry using data from the Osteoarthritis Initiative.

In total hip arthroplasty, the shape of the contra-lateral femur frequently serves as a template for preoperative planning. Previous research on contra-lateral femoral symmetry has been based on conventional hip geometric measurements (which reduce shape to a series of linear measurements) and did not take the effect of subject positioning on radiographic femur shape into account. The aim of this study was to analyse proximal femur symmetry based on statistical shape models (SSMs) which quantify global femoral shape while also adjusting for differences in subject positioning during image acquisition. We applied our recently developed fully automatic shape model matching (FASMM) system to automatically segment the proximal femur from AP pelvic radiographs to generate SSMs of the proximal femurs of 1258 Caucasian females (mean age: 61.3 SD=9.0). We used a combined SSM (capturing the left and right femurs) to identify and adjust for shape variation attributable to subject positioning as well as a single SSM (including all femurs as left femurs) to analyse proximal femur symmetry. We also calculated conventional hip geometric measurements (head diameter, neck width, shaft width and neck-shaft angle) using the output of the FASMM system. The combined SSM revealed two modes that were clearly attributable to subject positioning. The average difference (mean point-to-curve distance) between left and right femur shape was 1.0mm before and 0.8mm after adjusting for these two modes. The automatic calculation of conventional hip geometric measurements after adjustment gave an average absolute percent asymmetry of within 3.1% and an average absolute difference of within 1.1mm or 2.9° for all measurements. We conclude that (i) for Caucasian females the global shape of the right and left proximal femurs is symmetric without isolated locations of asymmetry; (ii) a combined left-right SSM can be used to adjust for radiographic shape variation due to subject positioning; and (iii) adjusting for subject positioning increases the accuracy of predicting the shape of the contra-lateral hip.

Fully automatic segmentation of the proximal femur using random forest regression voting.

Extraction of bone contours from radiographs plays an important role in disease diagnosis, preoperative planning, and treatment analysis. We present a fully automatic method to accurately segment the proximal femur in anteroposterior pelvic radiographs. A number of candidate positions are produced by a global search with a detector. Each is then refined using a statistical shape model together with local detectors for each model point. Both global and local models use Random Forest regression to vote for the optimal positions, leading to robust and accurate results. The performance of the system is evaluated using a set of 839 images of mixed quality. We show that the local search significantly outperforms a range of alternative matching techniques, and that the fully automated system is able to achieve a mean point-to-curve error of less than 0.9 mm for 99% of all 839 images. To the best of our knowledge, this is the most accurate automatic method for segmenting the proximal femur in radiographs yet reported.

Accurate fully automatic femur segmentation in pelvic radiographs using regression voting.

Extraction of bone contours from radiographs plays an important role in disease diagnosis, pre-operative planning, and treatment analysis. We present a fully automatic method to accurately segment the proximal femur in anteroposterior pelvic radiographs. A number of candidate positions are produced by a global search with a detector. Each is then refined using a statistical shape model together with local detectors for each model point. Both global and local models use Random Forest regression to vote for the optimal positions, leading to robust and accurate results. The performance of the system is evaluated using a set of 519 images. We show that the fully automated system is able to achieve a mean point-to-curve error of less than 1 mm for 98% of all 519 images. To the best of our knowledge, this is the most accurate automatic method for segmenting the proximal femur in radiographs yet reported.

Measurement of substrate oxidation during exercise by means of gas exchange measurements.

Measures of substrate oxidation have traditionally been calculated from indirect calorimetry measurements using stoichiometric equations. Although this has proven to be a solid technique and it has become one of the standard techniques to measure whole body substrate metabolism, there are also several limitations that have to be considered. When indirect calorimetry is used during exercise most of the assumptions on which the method is based hold true although changes in the size of the bicarbonate pool at higher exercise intensities may invalidate the calculations of carbohydrate and fat oxidation. Most of the existing equations are based on stoichiometric equations of glucose oxidation and the oxidation of a triacylglycerol that is representative of human adipose tissue. However, in many exercise conditions, glycogen and not glucose is the predominant carbohydrate substrate. Therefore we propose slightly modified equations for the calculation of carbohydrate and fat oxidation for use during low to high intensity exercise. Studies that investigated fat oxidation over a wide range of intensities and that determined the exercise intensity at which fat oxidation is maximal have provided useful insights in the variation in fat oxidation between individuals and in the factors that affect fat oxidation. Fat oxidation during exercise can be influenced by exercise intensity and duration, diet, exercise training, exercise mode and gender. Although a number of important factors regulating fat oxidation have been identified, it is apparent that a considerable degree of inter-subject variability in substrate utilization persists and cannot be explained by the aforementioned factors. Future research should investigate the causes of the large inter-individual differences in fat metabolism between individuals and their links with various disease states.