Approximate testing in two-stage nonlinear mixed models.

We investigate here small sample properties of approximate F-tests about fixed effects parameters in nonlinear mixed models. For estimation of population fixed effects parameters as well as variance components, we apply the two-stage approach. This method is useful and popular when the number of observations per sampling unit is large enough. The approximate F-test is constructed based on large sample approximation to the distribution of nonlinear least squares estimates of subject-specific parameters. We recommend a modified test statistic that takes into consideration approximation to the large sample Fisher information matrix (See [1]). Our main focus is on comparing finite sample properties of broadly used approximate tests (Wald test and likelihood ratio test) and the modified F-test under the null hypothesis, especially accuracy of p-values (See [2]). For that purpose two extensive simulation studies are conducted based on pharmacokinetic models (See [3, 4]).

Intrahepatic and intramyocellular lipids are determinants of insulin resistance in prepubertal children.

AIMS/HYPOTHESIS: We hypothesised that ectopic fat deposition is present in liver and skeletal muscle before puberty and that both are potentially important factors in the early pathogenesis of insulin resistance. METHODS: Proton magnetic resonance spectroscopy was used to evaluate intramyocellular and intrahepatic lipids in 50 male and 42 female multi-ethnic, prepubertal (Tanner < 2) children (8.1 ± 0.8 years; 35.4 ± 10.7 kg; 27.9 ± 8.3% body fat; means ± SD). Intramyocellular lipid was measured in soleus muscle and intrahepatic lipid in the middle right lobe. Abdominal fat was measured by magnetic resonance imaging, body fat by dual energy X-ray absorptiometry, and insulin resistance using homeostatic model assessment. RESULTS: Intrahepatic lipid ranged from 0.11% to 4.6% relative to the liver water signal (mean 0.79 ± 0.79%) whereas intramyocellular lipid ranged from 0.13% to 1.86% relative to the muscle water signal (mean 0.51 ± 0.28%). Intramyocellular and intrahepatic lipids were significantly correlated with total adiposity (r = 0.49 and 0.59), abdominal adiposity (r = 0.44 and 0.54), and each other (r = 0.39, p < 0.05, Spearman). Both intramyocellular and intrahepatic lipid were positively correlated with fasting insulin (r = 0.37 and 0.38 respectively) and insulin resistance (r = 0.37 and 0.37; p < 0.01). After adjustment for race and sex, the relations between ectopic fat and insulin resistance remained, whereas both disappeared when further adjusted for body fat or BMI z scores. CONCLUSIONS/INTERPRETATIONS: These results suggest that typical relations between body composition, ectopic fat and insulin resistance are present in children before puberty. Thus, interventions aimed at reducing adiposity have the potential to decrease ectopic fat accumulation, delay the onset of insulin resistance and decrease the risk for development of type 2 diabetes in children.

Pre-school obesity is inversely associated with vegetable intake, grocery stores and outdoor play.

The study determined the association between body mass index (BMI) z score and fruit and vegetable intake, frequency and ratio of fast food outlets and grocery stores in concentric areas around the child's residence, outdoor play and total crime index. Data from 78 Louisiana pre-school children were analyzed using Pearson's correlation and multiple regression analysis. Parental-reported fruit intake was linearly associated with increased number of grocery store counts in concentric areas around the child's residence (P = 0.0406, P = 0.0281). Vegetable intake was inversely (P = 0.04) and the ratio of fast food outlets to grocery stores in a 2-mile concentric area around the child's residence was positively (P = 0.05) associated to BMI z score after applying Best Model regression analysis (F = 3.06, P = 0.0346). Children residing in neighbourhoods with greater access to fast foods and lower access to fruits and vegetables may be at higher risk for developing obesity during pre-school years.

The gene structure and minimal promoter of the human agouti related protein.

The murine agouti related protein (mAGRP) is upregulated in obese and diabetic mice and stimulates hyperphagia when administered intracerebroventricularly (i.c.v.) or when overexpressed in transgenic mice. The human ortholog, hAGRP, has been isolated and has similar molecular and physiological properties. Here, we report the complete gene structure of the human AGRP gene and upstream regions with differential promoter activity. A polymorphism, A67T, in the third exon was identified but was not associated with obesity- or type 2 diabetes-related phenotypes. Putative binding sites for transcription factors were identified in the promoter of the gene including recognition sites for the signal transducers and activators of transcription (STATs) that may potentially mediate leptin's action in the hypothalamus. The upstream non-coding exon had significant promoter activity in a periphery- but not so in a hypothalamus-derived cell line, suggesting that it might contain the minimal promoter required for expression of the short transcript of hAGRP in the periphery.

Evaluation of body fat in fatter and leaner 10-y-old African American and white children: the Baton Rouge Children's Study.

BACKGROUND: Only a few published studies in children used several methods to compare body fat in large groups of fatter and leaner multiethnic children. We hypothesized that the preferred methods of determining body fat may differ in children with larger compared with smaller amounts of body fat, in boys compared with girls, and in African Americans compared with whites. OBJECTIVE: Our objective was to evaluate several methods of predicting body fat in 10-12-y-old white and African American boys and girls. DESIGN: The body fat of 129 African American and white boys and girls aged 10-12 y, distributed equally by sex and race, was measured with use of dual-energy X-ray absorptiometry (DXA), underwater weighing (densitometry), isotope dilution (H(2)18O), bioelectrical impedance, skinfold thicknesses, corporal diameters, and circumferences. RESULTS: With use of DXA as the criterion variable, body fat was bimodally distributed in the boys and skewed to higher values in the girls. Biceps skinfold thickness had the highest predictive value of any single skinfold thickness compared with DXA fat. All formulas for estimating body fat from skinfold thicknesses, body density, or impedance performed better in the children in the upper one-half of the fat distribution (the fatter children) than in those in the lower one-half (the leaner children). Body mass index was highly correlated with body fat (R2 = 0.77); there was a good correlation for the fatter children (R2 = 0.66) and no correlation for the leaner children (R2 = 0.09). The hydration of the fat-free mass was significantly higher in the fatter children than in the leaner ones (79.2% compared with 76.7%). CONCLUSIONS: These data are consistent with the hypothesis that all methods of estimating body fat work better in children with larger amounts of body fat. The best formulas use skinfold thicknesses, bioelectrical impedance, and a 4-compartment model.

Concurrent physical activity increases fat oxidation during the shift to a high-fat diet.

BACKGROUND: It takes several days to adapt to a high-fat diet. In an earlier study, we observed a large degree of interindividual variation in the capacity to adapt to a high-fat diet. We hypothesized that concurrent physical activity would accelerate fat oxidation during an isoenergetic high-fat diet. OBJECTIVE: The objective of this study was to determine the effect of increased physical activity on the ability of young healthy men to increase fat oxidation during the shift to a high-fat diet. DESIGN: Six young healthy men participated in a randomized, single-blind crossover study. The volunteers consumed a diet contributing 37% of energy as fat, 14% as protein, and 49% as carbohydrate for 4 d. Energy expenditure and macronutrient balance were then measured in a respiration chamber as the energy content of the isoenergetic diet was changed to 50% fat, 14% protein, and 36% carbohydrate. Treadmill walking, as the physical activity, was used to increase total daily energy expenditure to 1.8 times the resting metabolic rate during 1 of 2 stays in the metabolic chamber. Total daily energy expenditure was maintained at 1.4 times the resting metabolic rate for the other stay. RESULTS: Energy balance was not significantly different between the 2 conditions. The 24-h respiratory quotient decreased more rapidly and to a greater extent under conditions of increased energy expenditure. Further, there was a decrease in the interindividual variability in the response of the respiratory quotient to a high-fat diet with increased energy expenditure (physical activity). Cumulative carbohydrate and protein balances were greater under conditions of increased physical activity. Conversely, cumulative fat balance was greater under sedentary conditions. CONCLUSION: Concurrent physical activity increases fat oxidation during the shift to a high-fat diet.

Video-based spatio-temporal maps for analysis of gastric motility in vitro: effects of vagal stimulation in guinea-pigs.

Our aim was to evaluate topographically specific gastric motility changes induced by graded vagal activation. A recently developed method of constructing spatio-temporal maps of motility from video movies was adapted to the in vitro perfused guinea-pig stomach with an intact vagal nerve supply. In the unstimulated preparation, spontaneous activity was low or absent. Bilateral vagal stimulation with frequencies as low as 0.2 Hz triggered weak anally, and in some cases orally, propagating antral contractions at rates of about 5-6 min-1. Upon stimulation with higher frequencies, antral contractions increased significantly in length (starting more proximally) and amplitude, and produced large pressure peaks of up to 25 hPa, with maximal effects at 2-4 Hz. In contrast, the speed of propagation and the interval between peristaltic waves did not change with vagal stimulation at any frequency. Vagal stimulation also produced a significant and frequency-dependent enlargement of the fundus with a maximal effect at 4 Hz. It is concluded that a very low tonic vagal activity is apparently necessary and sufficient to express basic antral motility, while more sustained vagal activity is necessary for high-amplitude gastric contractions and significant sustained fundic relaxation. The constant interval between propagating contractions supports the concept that vagal input impinges on intrinsic enteric neural circuits that have a modulatory role in the myogenic mechanism underlying slow-wave peristalsis, rather than directly on gastric musculature.

Increased flavor preference and lick activity for sucrose and corn oil in SWR/J vs. AKR/J mice.

Nutrient preferences and orosensory responses were characterized in two mouse inbred strains. In two-bottle solution tests (tastant vs. vehicle; ascending concentrations), the effects of strain and chow type (12 or 26% fat) on preference thresholds for sucrose and corn oil were compared in AKR/J and SWR/J mice. SWR/J mice displayed lower preference thresholds and ingested more sucrose than AKR/J mice did. SWR/J mice also showed lower preference thresholds and consumed more corn oil than AKR/J mice did; corn oil preference was suppressed 3.5-fold in AKR/J mice compared with SWR/J mice when fed 26% fat chow. Next, licking was recorded during 30-s access to sucrose or corn oil across a range of concentrations. SWR/J mice licked the tastants more than AKR/J mice did. Analysis of modal interlick intervals during lick training revealed that SWR/J mice licked water faster than AKR/J mice when water deprived, suggesting that motor as well as sensory factors may determine lick responses to tastants in brief-access tests. Finally, in two-bottle tests pitting maximally preferred concentrations of sucrose (8 or 16%) against corn oil (20%), SWR/J mice highly preferred sucrose over corn oil at either sucrose concentration. AKR/J mice preferred corn oil over 8% sucrose but reversed their preference when 16% sucrose was offered. These results support a primary role of flavor in the nutrient preferences of SWR/J mice. In AKR/J mice, the low lick activity for sucrose and corn oil and greater suppression of corn oil preference by the high-fat chow suggest that their preferences depend more on postingestive factors than on flavor.

Body composition of African American and white children: a 2-year follow-up of the BAROC study.

OBJECTIVE: To evaluate the 2-year changes in body composition of white and African American boys and girls. RESEARCH METHODS AND PROCEDURES: A total of 114 boys and girls ages 12 to 14 years with equal sex and ethnic distribution between African American and white races participated in measurements of body composition using DXA, underwater weighing (densitometry), skinfold thickness, corporal diameters, circumferences, isotope dilution (H(2)(18)O), and bioelectric impedance. RESULTS: Sixty-eight of the 114 children advanced from Tanner Stages 1 and 2 to Tanner Stages 3 to 5 over a 2-year period. More than 50% of the children were in the top 15th percentile according to normative data for body mass index but not for triceps skinfold. All measures except for percentage of fat, density, and four of the six skinfolds increased significantly during the 2 years, with no differences between races, genders, or fat group. The boys who advanced in Tanner Stage reduced their percentage of fat and a number of skinfolds and increased their lean body mass, but the girls did not. The percentage of water was significantly higher in the fatter children and declined significantly over 2 years. Most children remained in the same quartile of body fat, lean body mass, and bone mineral content over 2 years. DISCUSSION: The data are consistent with the hypothesis that over 2 years, growth is the major determinant of changing body composition, with body-fat group and sexual maturation being additional variables.

Changes in skeletal muscle vascular resistance with weight gain: associations with insulin and sympathetic activity.

OBJECTIVE: This study was designed to characterize changes in peripheral vascular resistance with weight gain, and whether these changes are correlated with insulin and/or sympathetic activity. RESEARCH METHODS AND PROCEDURES: Femoral vascular resistance (FVR), mean arterial pressure, heart rate, and plasma insulin were measured before and during overfeeding in seven dogs with unilateral lumbar ganglionectomy (L3 to L6). Measurements were taken standing and while walking on a treadmill. RESULTS: There was a significant main effect of weight gain to increase mean arterial pressure (16.5+/-8.4 mmHg and 12.5+/-6.8 mmHg increase for standing and walking baseline, respectively) and heart rate (increase from week 1 of 31.6+/-10.6 beats/minute standing and 38.3+/-9.1 walking beat/minute). FVR increased immediately with overfeeding/ weight gain [standing: denervated (DNX):1.32+/-0.3 to 2.34+/-0.5; intact: 0.88+/-0.17 to 1.9+/-0.33 mmHg/mL.min(-1)], but returned to baseline with continued weight gain. Return of FVR to baseline occurred between weeks 2 and 3 of overfeeding in the DNX limb, but did not return to baseline until week 6 in the innervated limb. These changes were not correlated with plasma insulin levels. DISCUSSION: These data suggest that vascular resistance may be normal in the obese, but increases in vascular resistance occur early with weight gain (before changes in arterial pressure). This initial increase in vascular resistance could initiate the series of events leading to obesity-associated hypertension. Additionally, changing vascular resistance during weight gain may be influenced by sympathetic activity, because DNX limb FVR returned to baseline approximately 3 weeks earlier than the innervated limb.

Effect of restraint stress on food intake and body weight is determined by time of day.

Three experiments were conducted to investigate the effect of restraint stress applied at different times of the light-dark cycle on feeding behavior and body weight of rats. Sprague-Dawley rats were restrained for 3 h in restraining tubes either at the start or the end of the light cycle. There was a significant reduction in food intake on the day of restraint and no change in food intake during a 10-day recovery period in either experiment. Reductions of food intake on the day of restraint were about the same for both restrained groups compared with their controls. When stress was applied in the evening, eating was inhibited during the first 2 h after restraint, whereas in rats restrained in the morning, feeding was suppressed twice: during the 4 h after restraint and during the first 2 h of the dark cycle. Restraint induced a significant weight loss that was greater in the rats stressed in the morning. Neuropeptide Y (NPY) levels determined at the time of food suppression for both experiments (beginning of the dark cycle) revealed an elevation of NPY in the paraventricular nucleus of rats stressed in the morning compared with other groups, but no difference in hypothalamic NPY mRNA expression. Expression of uncoupling protein mRNA in brown adipose tissue and leptin mRNA in epididymal fat, measured at the start of the dark period, was not altered by stress. There was an elevation of dopamine turnover in the hypothalami of rats restrained at the end of light cycle, but not those restrained in the morning. These results show that restraint stress has a greater effect on metabolism and energy balance when it is applied in the morning. Additional studies are needed to elucidate mechanisms involved in the suppression of food intake 9 h after restraint.

Serial echocardiographic and clinical evaluation of valvular regurgitation before, during, and after treatment with fenfluramine or dexfenfluramine and mazindol or phentermine.

OBJECTIVE: The prevalence of cardiac valvular regurgitation demonstrated by echocardiography in patients who took appetite-suppressant medication for weight loss has been assessed at 5%-30%. We studied 86 patients who had echocardiograms before treatment with appetite suppressants to determine the incidence of new cases and to evaluate the clinical implication of the echocardiographic findings. RESEARCH METHODS AND PROCEDURES: We studied 69 men [Mean+/-Standard Deviation (S) age 49+/-8] and 17 women (mean+/-S age 50+/-7) who had 233 echocardiograms before, during, and after a weight-loss program that used predominantly fenfluramine (or dexfenfluramine) with mazindol (or phentermine). Mean drug exposure was 17 months. Blinded echocardiographic readings were performed to identify and grade aortic regurgitation (AR) or mitral regurgitation (MR). RESULTS: Seven of 86 patients (8%) had pre-existing regurgitation with five (6%) meeting our case definition. Thirteen (16.5%) of initially normal patients developed valvular regurgitation and were new cases. Of the new cases, 12 were grade I/IV AR and one was both grade II/III MR and II/IV AR. All 13 patients were asymptomatic, and only two aortic insufficiency murmurs could be auscultated. There was significantly greater risk for developing valvulopathy for those who took medications longer than 6 months (p = 0.03), and no new cases were observed in patients exposed for less than 8 months. No increased risk associated with age, presence of hypertension, or exposure to fenfluramine-phentermine combination was demonstrated. Although there was a higher incidence of new regurgitation in women (31% vs. 13% for men), this was not statistically significant (p = 0.093). DISCUSSION: Some patients who had normal echocardiograms at baseline developed cardiac valvular regurgitation after exposure to fenfluramine or dexfenfluramine with mazindol or phentermine. The development of valvulopathy was significantly correlated with duration of exposure. The clinical implications of echocardiographically demonstrated regurgitation are uncertain, since there were only two audible murmurs and no other clinically relevant signs or symptoms among the patients.

Consumption of a controlled low-fat diet containing olestra for 9 months improves health risk factors in conjunction with weight loss in obese men: the Ole' Study.

OBJECTIVE: To compare the effects of a standard American diet, a traditional low-fat diet, and a low-fat diet containing the fat substitute olestra on risk factors for heart disease and diabetes. DESIGN: A 9-month, double-blind, randomized, parallel-arm, feeding study comparing three diets: (1). control (33% fat), (2). fat-reduced (FR; 25% fat), and (3). fat-substituted (FS) where olestra replaced 1/3 of dietary fat (33% lipid and 25% digestible fat). Subjects were allowed to adjust their total energy intake as desired, allowing weight to fluctuate. SUBJECTS: A total of 37 healthy, obese men (age 36.7+/-1.3 y; body mass index 30.8+/-0.4 kg/m(2)). MEASUREMENTS: Body weight and composition by dual-energy X-ray absorptiometry, blood pressure, serum lipids, lipoproteins, hemostatic factors, glucose, insulin, and leptin at baseline and every 3 months. RESULTS: The FS group lost 6.27 kg of body weight by 9 months vs 4.0 kg in the control and 1.79 kg in the FR groups. There was a significant diet main effect on cholesterol (P=0.002), low-density lipoprotein cholesterol (P=0.003), and triglycerides (P=0.01), all of which decreased in the FS group but not the other groups by 9 months. Apolipoprotein B (ApoB) increased in the FR and control groups but was unchanged in the FS group (diet main effect P=0.04). High-density lipoprotein (HDL) cholesterol increased in all groups over 9 months (time main effect P=0.0001). Time main effects were also observed for cholesterol, ApoA1, ApoB, Factor VII, diastolic blood pressure, and glucose. After adjustment for % fat loss at 9 months, the effects of diet on change in risk factors remained significant only for triglycerides. DISCUSSION: Consumption of a low-fat diet containing olestra for 9 months produced significant improvement in cardiovascular risk factors, an effect largely explained by weight loss. Long-term low-fat diet consumption with or without olestra does not decrease HDL cholesterol.