Identification of brown adipose tissue in mice with fat-water IDEAL-MRI

Purpose: To investigate the feasibility of using IDEAL (Iterative Decomposition with Echo Asymmetry and Least squares estimation) fat-water imaging and the resultant fat fraction metric in detecting brown adipose tissue (BAT) in mice, and in differentiating BAT from white adipose tissue (WAT). Materials and Methods: Excised WAT and BAT samples and whole-mice carcasses were imaged with a rapid three-dimensional fat-water IDEAL-SPGR sequence on a 3 Tesla scanner using a single-channel wrist coil. An isotropic voxel size of 0.6 mm was used. Excised samples were also scanned with single-voxel proton spectroscopy. Fat fraction images from IDEAL were reconstructed online using research software, and regions of WAT and BAT were quantified. Results: A broad fat fraction range for BAT was observed (40-80%), in comparison to a tighter and higher WAT range of 90-93%, in both excised tissue samples and in situ. Using the fat fraction metric, the interscapular BAT depot in each carcass could be clearly identified, as well as peri-renal and inguinal depots that exhibited a mixed BAT and WAT phenotype appearance. Conclusion: Due to BAT's multi-locular fat distribution and extensive mitochondrial, cytoplasm, and vascular supply, its fat content is significantly less than that of WAT. We have demonstrated that the fat fraction metric from IDEAL-MRI is a sensitive and quantitative approach to noninvasively characterize BAT

Hemoglobin A1c above Threshold Level is Associated with Decreased b-Cell Function in Overweight Latino Youth

Objective To examine whether a hemoglobin A1c (HbA1c)-identified prediabetic state (HbA1c $6.0%-6.4%) is associated with decreased insulin sensitivity (SI) and b-cell dysfunction, known factors in the pathogenesis of type 2 diabetes, in an overweight pediatric population. Study design A total of 206 healthy overweight Latino adolescents (124 males and 82 females; mean age, 13.1 AE 2.0 years) were divided into 2 groups: lower risk (n = 179), with HbA1c <6.0%, and higher risk (n = 27), with HbA1c 6.0%-6.4%. Measurements included HbA1c, oral glucose tolerance testing, fasting and 2-hour glucose and insulin, SI, acute insulin response, and disposition index (an index of b-cell function) by the frequently sampled intravenous glucose tolerance test with minimal modeling. Body fat was determined by dual-energy X-ray absorptiometry. Results Compared with the lower risk group, the higher risk group had 21% lower SI (1.21 AE 0.06 vs 1.54 AE 0.13; P < .05), 30% lower acute insulin response (928 AE 102 vs 1342 AE 56; P < .01), and a 31% lower disposition index (1390 AE 146 vs 2023 AE 83; P = .001) after adjusting for age and total percent body fat. Conclusion These data provide clear evidence of greater impairment of b-cell function in overweight Latino children with HbA1c 6.0%-6.4%, and thereby support the adoption of the International Expert Committee's HbA1c-determined definition of high-risk state for overweight children at risk for type 2 diabetes. (J Pediatr 2012;160:751-6). M ore than 40% of the US population suffers from diabetes or prediabetes

Measurement issues related to studies of childhood obesity: assessment of body composition, body fat distribution, physical activity, and food intake

ABSTRACT. This article reviews the current status of various methodologies used in obesity and nutrition research in children, with particular emphasis on identifying priorities for research needs. The focus of the article is 1) to review methodologic aspects involved with measurement of body composition, body-fat distribution, energy expenditure and substrate use, physical activity, and food intake in children; and 2) to present an inventory of research priorities. Pediatrics 1998;101:505-518; obesity, body composition, energy expenditure, physical activity, diet, methodology. ABBREVIATIONS. DXA, dual energy x-ray absorptiometry; SEE, standard error of the estimate; MRI, magnetic resonance imaging; CT, computed tomography; RQ, respiratory quotient; METs, metabolic equivalents. BODY COMPOSITION A ccurate assessment of body composition is important in many areas of obesity and nutrition-related research. In addition to providing fundamental whole-body descriptive characteristics, accurate measures of body composition often are required as scaling factors to normalize physiologic variables (eg, metabolic rate, physical activity, physical fitness, etc). As described by Wang et al, 1 the composition of the human body can be thought of in terms of an atomic model (ie, oxygen, carbon, hydrogen, etc), a molecular model (ie, water, lipid, protein, minerals, and glycogen), a cellular model (ie, cell mass, extracellular fluid, etc), or a tissue model (ie, skeletal muscle, adipose tissue, bone, etc). In terms of obesity research, the most useful model is probably the molecular model, in which the body's composition is broken down into its main molecular components: lipids (includes essential and nonessential lipids), water, protein, minerals, and glycogen. Fat and fat-free mass are terms used frequently that refer to the classic two-component body composition model in which body mass is broken down into fat and nonfat tissue masses. Measurement of the masses of the individual compartments of body mass is extremely challenging, because no direct method exists other than in vivo neutron activation analysis (very limited availability) and chemical analysis of the cadaver (useful for animal studies only). The lack of direct methods has led to development of various models and indirect methods for estimation of fat and fat-free mass, all of which are imperfect and require a number of assumptions, many of which require age-specific considerations, because the usual assumptions in multicompartmental models (eg, hydration of fat-free mass, density of fat-free mass) are known to be influenced by age and state of maturation. 2, Densitometry Densitometry is based on estimating body composition from measurement of total body density. The most widely used approach is to measure body volume by underwater weight and determine density by dividing body mass by body volume. The technique is a two-compartment model and is based on the different tissue densities of the fat and fat-free compartments of the body. If total body density and the specific densities of fat and fat-free mass are known, an equation can be generated for converting total body density to percentage of body fat based on the Archimedes principle. 9 Generally, at least in adults, the densities of fat and fat-free mass are assumed to be 0.9 g/mL and 1.1 g/mL; however, the density of fat-free mass is known to be influenced by factors such as age, gender, and ethnicity, Current limitations for applying densitometry to the pediatric population include practical problems and theoretic considerations. The technique requires climbing into a large tank of water, emptying the lungs by maximal exhalation, and sitting still underwater for several seconds. Thus, from a practical standpoint, testing adherence is extremely difficult for young children and impossible for infants. Recent developments using air rather than water displacement 10 for measurement of volume may be more practical for pediatric populations. Such a device, called the BodPod, 10 has been developed, and it produces an alternative method for measuring body volume that is simpler, quicker, and more practical than hydrostatic weighing. From a theoretic standpoint, the limitations of successfully applying densitometry requires additional knowledge of the specific densities of fat and fat-free mass in children of different states of maturity, gender, and ethnicity. One previous study suggests that between birth and 22 years of age, the density of the fat-free mass increases from 1.063 to 1.102 g/mL in boys and from 1.064 to 1.096 g/mL in girls. The density of fat mass probably does not change, because it is more or less fixed by the biophysical properties of fat in vivo at a density of 0.9 g/mL. 11 Thus, as described by Lohman, 3 application of densitometry and development of accurate age-, gender-, and ethnic-specific equations requires knowledge of the density of fat-free mass for each subgroup being investigated. DXA Recent advances in techniques to measure body composition have provided DXA for assessment of whole-body as well as regional measurements of bone mass, lean mass, and fat mass. 12 DXA is based on the exponential attenuation resulting from absorption by body tissues of photons emitted at two energy levels to resolve body weight into bone mineral and lean and fat soft tissue masses; the theoretic considerations are reviewed elsewhere. Since the introduction of DXA, numerous studies have compared this technique with other researchbased methods. 18 Collectively, the validation studies of DXA suggest that the relationship between actual chemical content of the carcass and DXA estimates may be affected by factors such as the size of the animal, the equipment used, and the operation mode. These validation studies and generation of new calibration equations are important steps in development of standardized techniques to measure body composition in children. 20 Skinfolds and Anthropometry Estimation of fat mass from anthropometry involves development of prediction models in which anthropometric measures (eg, sum of skinfolds) are related to body-fat mass. Thus, the use of an accurate criterion method is important for development of these equations. Several prediction equations have been developed for children based on use of a multicompartmental model 2 or DXA 21 as criterion method. Slaughter and colleagues 2 developed body composition prediction equations from data on 310 subjects (8 to 29 years of age), including 66 prepubescent children (50 boys and 16 girls). A multicompartmental model of body composition was used as a criterion method by combining measures of total body density (from underwater weight), total body water (from deuterium dilution), and bone mineral density (from photon absorptiometry) on the right and left radius and ulna. This study led to development of gender-, race-, and maturation-specific equations for estimating body fat based on measurement of either triceps plus calf (two gender-specific equations) or triceps plus subscapular skinfolds (nine equations recommended depending on gender, race, maturation state, and sum of skinfold thickness). Several studies have examined the accuracy of the Slaughter equations in cross-validation studies. Janz and coworkers 22 were unable to cross-validate the Slaughter equation based on triceps and calf skinfolds in girls and boys. In 98 white children (6.6 Ϯ 1.4 years old; 24.1 Ϯ 5.9 kg), 21 fat mass by DXA (4.8 Ϯ 3.0 kg) was significantly lower than fat mass by skinfolds (5.0 Ϯ 3.1 kg) with the Slaughter equation, 2 although fat masses by these two techniques were strongly related (R 2 ϭ 0.87; standard error of the estimate [SEE] ϭ 1.1 kg). A possible explanation for the differences between equations for predicting fat 506 SUPPLEMENT at USC Norris Medical Library on July 15, 2008 www.pediatrics.org Downloaded from mass may be the small sample size of young girls (n ϭ 16) in the original study by Slaughter et al. 2 We recently developed a series of new anthropometric prediction equations based on data from 98 white children. 24 The cross-validation and testing of anthropometric prediction equations in independent groups is of particular importance for several reasons. First, although there are standardized methods, 25 the measurement of skinfolds is by nature sensitive to interuser variability. Second, because of potential gender-, ethnic-, and maturation-related changes in body composition, the relationship between skinfold measures and body fat may vary between subgroups of the population. Thus, it is important to identify anthropometric measures that are robust to interuser variability and equally reflective of body composition in all subgroups of the population. Bioelectrical Impedance Analysis Bioelectrical resistance is an alternative technique for assessing body composition in clinical and population-based studies. The technique is based on measurement of electrical resistance in the body to a tiny imperceptible current. The electrical resistance is a function of body shape and the volume of conductive tissues in the body. 4 Thus, the techniques appear to be reliable as well as robust to interlaboratory variation, and the Kushner equation is generally recommended in young children, although this approach has not been validated in children of different ethnic groups. In adolescents, the equations of Houtkooper 30 are recommended, although these equations have not been cross-validated widely or examined in other ethnic groups. One of the limitations of bioelectrical resistance is that this approach provides an estimate of total body water, which then must be transformed into fat-free mass. This requires knowledge of the hydration of fat-free mass, which generally is thought to be constant in adults at 73.2%, 31 but known to vary in children. 7 Fomon and colleagues 7 published age-and gender-specific hydration constants, although the original constants may have to be modified slightly. Body Fat Distribution In adults, intraabdominal adipose tissue (body fat around the visceral organs) is related to negative health outcome independent of total body fat. 42 CT and MRI are accurate imaging techniques for assessing body-fat distribution, but the disadvantages are cost, radiation exposure, and limited use to a research setting. Thus, other indirect indicators of body fat distribution have been used. For example, in adults, the waist-to-hip ratio or the waist circumference are used often as markers of intraabdominal adipose tissue. However, in children 34 and adolescents, The use of DXA to measure total abdominal fat may provide a stronger index, but this technique cannot resolve subcutaneous from intraabdominal adipose tissue. The combination of total abdominal fat by DXA and skinfold and anthropometry data (as an index of subcutaneous fat) has been used in adults to estimate intraabdominal adipose tissue with reasonable accuracy. 14,45 Preliminary equations have been developed and cross-validated in children with this approach (Goran MI, unpublished observations). The combination of trunk fat by DXA, total fat by DXA, and abdominal skinfold thickness predicted intraabdominal adipose tissue as measured with CT scanning with a model R 2 of 0.85 and an SEE of Ϯ 9 cm 2 . In fact, the incorporation of DXA was not essential, because abdominal skinfold thickness, ethnicity (white versus black), and subscapular skinfold predicted intraabdominal adipose tissue with a model R 2 of 0.82 and an SEE of Ϯ 10 cm 2 . ASSESSMENT OF ENERGY EXPENDITURE AND SUBSTRATE USE Energy expenditure is typically measured in humans by either direct or indirect calorimetry. Direct calorimetry involves measurement of heat production directly. This approach is technically demanding, especially in human studies, and is now infrequently used. Indirect calorimetry measures energy production by respiratory gas analysis. This approach is based on measurement of oxygen consumption and carbon dioxide production that occurs during the combustion (or oxidation) of protein, carbohydrate, fat, and alcohol. Respiratory gas analysis can easily be achieved in humans either over short measurement periods at rest or during exercise with a face mask, mouthpiece, or canopy system used for gas collection, and over longer periods of 24 hours or more by having subjects live in a metabolic chamber. Indirect calorimetry has the added advantage that the ratio of carbon dioxide production to oxygen consumption (the respiratory quotient [RQ]) is indicative of the type of substrate (ie, fat versus carbohydrate) being oxidized; for example, carbohydrate oxidation has an RQ of 1.0, and fat oxidation has an RQ close to 0.7. Finally, carbon dioxide production rates can be measured directly and used to estimate energy expenditure over extended free-living periods of 7 to 14 days with stable isotope methodology and double-labeled water. up to 2 weeks can be measured by the combination of double-labeled water to measure total energy expenditure and indirect calorimetry to measure resting energy expenditure and the thermic effect of a meal. Indirect Calorimetry Indirect calorimetry can be performed easily in children by using standard commercial equipment as performed in adults. There are no special assumptions or theoretic considerations in children. However, there may be important considerations for measurement conditions before and during measurement of metabolic rate to achieve the need for having subjects lie still and quiet for 30 minutes. For example, basal metabolic rate is often understood to encompass measurements performed after a 10-to 12-hour fast in a nonaroused state, with minimal muscular movement. These conditions are often met in adults when measurements can be performed immediately on awakening after the subject has slept in the laboratory or clinical research center. 48 This approach is viable and reproducible in young children, 49 but may not always be possible. The terms basal metabolic rate and resting metabolic rate are often used interchangeably. Resting metabolic rate implies a resting but nonbasal state and may include the increase in metabolic rate associated with arousal. In children, a relaxed and quiet state can be achieved in younger children by allowing them to watch television or standardized cartoons One of the limitations of using indirect calorimetry to measure resting metabolic rate is that measurements can be performed over only a very short time (usually 30 minutes). Measurements over 24 hours can be achieved by having subjects live in a metabolic chamber. This approach has been used successfully in children as young as 4 years of age (FigueroaColon R, unpublished observations). An additional advantage of this approach is that activity and food intake can be monitored and controlled. The disadvantage is that the chamber environment is not habitual, especially because movement and physical activity may be restricted. Double-labeled Water The double-labeled water technique is the first truly noninvasive means to measure total daily energy expenditure accurately in free-living humans. The technique was first introduced by Lifson and colleagues 51 in the 1950s as an isotopic technique for measuring carbon dioxide production rate in small animals. Unfortunately, it was not possible to apply the technique to humans, because the dose required was cost-prohibitive given the relatively poor sensitivity of isotope ratio mass spectrometry at that time. It was not for another 20 years that Lifson and coworkers 52 described the feasibility of applying the technique to measure free-living energy expenditure in humans, an application that was recognized later by Schoeller et al. The major advantages of the double-labeled water method are that 1) the technique is noninvasive and unobtrusive; 2) measurements are performed under free-living conditions over extended periods (7 to 14 days); and 3) the technique can be used to estimate activity energy expenditure when combined with measurement of resting metabolic rate. The major disadvantages include the following: 1) the cost of 18 O (ϳ$200 for a 30-kg child); 2) the need for an isotope ratio mass spectrometer for sample analysis; 3) the method provides a direct measure of CO 2 production and not energy expenditure (the food quotient of the diet is required for generating estimates of energy expenditure); 4) the method gives an integrated measure of activity energy expenditure over 14 days and does not provide any qualitative information on physical activity pattern; and 5) the technique is not really suitable for large-scale epidemiologic studies. The double-labeled water technique has been validated in humans in several laboratories around the world by comparison with indirect calorimetry in both adults and infants, as described previously. 56 These studies generally show the technique to be accurate to within 5% to 10% relative to data derived by indirect calorimetry for subjects living in metabolic chambers. There are no special considerations or limitations in applying the technique to children. The theoretic precision of the double-labeled water technique is 3% to 5%, 57 although the experimental variability is Ϯ12% in free-living adults because of fluctuations in physical activity levels 58 and Ϯ 8% under more controlled sedentary living conditions. 59 The accuracy and reasonable precision of the technique therefore allow the double-labeled water method to be used as a standard measure of freeliving energy expenditure in humans with which other methods can be compared. ASSESSMENT OF PHYSICAL ACTIVITY Physical activity is a broadly used term, and its heterogeneous nature makes it extremely difficult to characterize and quantify. Physical activity can be defined by any physical movement that is a result of skeletal muscle contraction. Physical activity is often measured in terms of caloric cost, but this may not be appropriate because the benefits and health effect of physical activities using a high-energy expenditure (eg, running at a certain intensity) versus a lowenergy expenditure (eg, strength training) may not be related to the caloric cost of the physical activity. Thus, quantification and description of physical activity should probably consider all aspects, including the following: 1) type and purpose of physical activity (eg, recreational or obligatory, aerobic or anaerobic, occupational); 2) intensity (strenuousness); 3) efficiency; 4) duration (ie, time); 5) frequency (ie, times per week); and 6) specific energy cost of the activity performed. It is also important to consider that physical activity and exercise may not be synonymous. Exercise typically refers to structured activities that are performed for the purpose of improving physical SUPPLEMENT 509 at USC Norris Medical Library on July 15, 2008 www.pediatrics.org Downloaded from fitness and well-being. This distinction is of particular relevance in children. An additional difficulty with the development of rigorous techniques for measuring physical activity is the lack of an ideal standard with which to validate the data, thus making it difficult to truly validate any given technique. Standard techniques that are available include measurement of specific energy costs of different activities in a laboratory setting by indirect calorimetry and measurement of free-living physical activity-related energy expenditure with doublelabeled water. Various methods are available for assessing physical activity in children, including questionnaires, accelerometry or pedometry, doublelabeled water for assessment of free-living physical activity-related energy expenditure, and heart-rate monitoring. Use of these techniques in children has been reviewed previously 60 -62 and is briefly described below. Activity Energy Expenditure With Double-labeled Water As described above in the section on energy expenditure, the combination of double-labeled water (to measure total energy expenditure) and indirect calorimetry (to measure resting energy expenditure) can be used to estimate physical activity-related energy expenditure by difference. Thus, the major advantage of this approach is the quantification of physical activity-related energy expenditure under free-living conditions over extended periods. However, the technique does not describe physical activity patterns, nor does it differentiate between different types of physical activity (eg, playing outside at a low intensity for 2 hours vs riding a bike for 30 minutes). In addition, although the 14-day period is generally considered long-term relative to other metabolic studies, this window of time actually might be quite short when considered relative to the time scale for development of obesity. Several studies have used this approach to measure physical activity-related energy expenditure in children under free-living conditions. It is important to note that activity energy expenditure estimated from the difference between total and resting energy expenditure may be an imprecise measure. There are no test/retest studies of physical activity energy expenditure in children, but precision can be estimated by a propagation-of-error approach. Questionnaires Questionnaires may be useful for large-scale epidemiologic studies. However, this approach is fraught with many difficulties. The major difficulty with the questionnaire approach is that it relies on the ability of the subject (or the parent) to recall behavioral information accurately. Also, it is difficult to translate qualitative information on physical activity (eg, playing for 30 minutes) to quantitative data (ie, kcal per exercise session). Although many different types

Editor-in-Chief Editor-in-Chief JEPonline Increasing Physical Activity Decreases Hepatic Fat and Metabolic Risk Factors

ABSTRACT Alderete TL, Gyllenhammer LE, Byrd-Williams CE, Spruijt-Metz D, Goran MI, Davis JN. Increasing Physical Activity Decreases Hepatic Fat and Metabolic Risk Factors. JEPonline 2012;15(2):40-54. This study assessed the changes in time spent in moderate to vigorous physical activity (MVPA) on fat depots, insulin action, and inflammation. Longitudinal data were generated from 66 Hispanic adolescents (15.6±1.1 yr; BMI percentile 97.1±3.0) who participated in a 16-wk nutrition or nutrition+exercise intervention. There were no effects of the intervention on PA, but there were inter-individual changes in PA. For purposes of this analysis, all intervention groups were combined to assess how changes in PA during 16 wk affected changes in adiposity, insulin action, and markers of inflammation. MVPA was assessed by 7-day accelerometry, total body fat via DXA, liver fat by MRI, and insulin, glucose and HOMA-IR via a fasting blood draw. A repeated measures ANCOVA was used to assess the effect of MVPA on fat depots, insulin action, and inflammatory markers. Sixty-two percent of participants increased MVPA (mean increase, 19.7±16.5 min/day) and 38% decreased MVPA (mean decrease, 10.7±10.1 min/day). Those who increased MVPA by as little as 20 min per day over 16 wk, compared to those who decreased MVPA, had significant reductions in liver fat (-13% vs. +3%; P=0.01), leptin levels (-18% vs. +4%; P=0.02), and fasting insulin (-23% vs. +5%; P=0.05). These findings indicate that a modest increase in MVPA can improve metabolic health in sedentary overweight Hispanic adolescents