Geminin Is Essential for Pluripotent Cell Viability During Teratoma Formation, but Not for Differentiated Cell Viability During Teratoma Expansion.

Pluripotent embryonic stem cells (ESCs) are unusual in that geminin has been reported to be essential either to prevent differentiation by maintaining expression of pluripotency genes or to prevent DNA rereplication-dependent apoptosis. To distinguish between these two incompatible hypotheses, immune-compromised mice were inoculated subcutaneously with ESCs harboring conditional Gmnn alleles alone or together with a tamoxifen-dependent Cre recombinase gene. Mice were then injected with tamoxifen at various times during which the ESCs proliferated and differentiated into a teratoma. For comparison, the same ESCs were cultured in vitro in the presence of monohydroxytamoxifen. The results revealed that geminin is a haplosufficient gene that is essential for ESC viability before they differentiate into a teratoma, but once a teratoma is established, the differentiated cells can continue to proliferate in the absence of Gmnn alleles, geminin protein, and pluripotent stem cells. Thus, differentiated cells did not require geminin for efficient proliferation within the context of a solid tissue, although they did when teratoma cells were cultured in vitro. These results provide proof-of-principle that preventing geminin function could prevent malignancy in tumors derived from pluripotent cells by selectively eliminating the progenitor cells with little harm to normal cells.

A mouse model for a partially inactive obesity-associated human MC3R variant.

We previously reported children homozygous for two MC3R sequence variants (C17A+G241A) have greater fat mass than controls. Here we show, using homozygous knock-in mouse models in which we replace murine Mc3r with wild-type human (MC3R(hWT/hWT)) and double-mutant (C17A+G241A) human (MC3R(hDM/hDM)) MC3R, that MC3R(hDM/hDM) have greater weight and fat mass, increased energy intake and feeding efficiency, but reduced length and fat-free mass compared with MC3R(hWT/hWT). MC3R(hDM/hDM) mice do not have increased adipose tissue inflammatory cell infiltration or greater expression of inflammatory markers despite their greater fat mass. Serum adiponectin levels are increased in MC3R(hDM/hDM) mice and MC3R(hDM/hDM) human subjects. MC3R(hDM/hDM) bone- and adipose tissue-derived mesenchymal stem cells (MSCs) differentiate into adipocytes that accumulate more triglyceride than MC3R(hWT/hWT) MSCs. MC3R(hDM/hDM) impacts nutrient partitioning to generate increased adipose tissue that appears metabolically healthy. These data confirm the importance of MC3R signalling in human metabolism and suggest a previously-unrecognized role for the MC3R in adipose tissue development.

Sex-associated differences in free fatty acid flux of obese adolescents.

CONTEXT: In obesity, increases in free fatty acid (FFA) flux can predict development of insulin resistance. Adult women release more FFA relative to resting energy expenditure (REE) and have greater FFA clearance rates than men. In adolescents, it is unknown whether sex differences in FFA flux occur. OBJECTIVE: Our objective was to determine the associations of sex, REE, and body composition with FFA kinetics in obese adolescents. PARTICIPANTS: Participants were from a convenience sample of 112 non-Hispanic white and black adolescents (31% male; age range, 12-18 years; body mass index SD score range, 1.6-3.1) studied before initiating obesity treatment. MAIN OUTCOME MEASURES: Glucose, insulin, and FFA were measured during insulin-modified frequently sampled iv glucose tolerance tests. Minimal models for glucose and FFA calculated insulin sensitivity index (SI) and FFA kinetics, including maximum (l0 + l2) and insulin-suppressed (l2) lipolysis rates, clearance rate constant (cf), and insulin concentration for 50% lipolysis suppression (ED50). Relationships of FFA measures to sex, REE, fat mass (FM), lean body mass (LBM) and visceral adipose tissue (VAT) were examined. RESULTS: In models accounting for age, race, pubertal status, height, FM, and LBM, we found sex, pubertal status, age, and REE independently contributed to the prediction of l2 and l0 + l2 (P < .05). Sex and REE independently predicted ED50 (P < .05). Sex, FM/VAT, and LBM were independent predictors of cf. Girls had greater l2, l0 + l2 and ED50 (P < .05, adjusted for REE) and greater cf (P < .05, adjusted for FM or VAT) than boys. CONCLUSION: Independent of the effects of REE and FM, FFA kinetics differ significantly in obese adolescent girls and boys, suggesting greater FFA flux among girls.

Long-term ritonavir exposure increases fatty acid and glycerol recycling in 3T3-L1 adipocytes as compensatory mechanisms for increased triacylglycerol hydrolysis.

Lipodystrophy with high nonesterified fatty acid (FA) efflux is reported in humans receiving highly active antiretroviral therapy (HAART) to treat HIV infection. Ritonavir, a common component of HAART, alters adipocyte FA efflux, but the mechanism for this effect is not established. To investigate ritonavir-induced changes in FA flux and recycling through acylglycerols, we exposed differentiated murine 3T3-L1 adipocytes to ritonavir for 14 d. FA efflux, uptake, and incorporation into acylglycerols were measured. To identify a mediator of FA efflux, we measured adipocyte triacylglycerol lipase (ATGL) transcript and protein. To determine whether ritonavir-treated adipocytes increased glycerol backbone synthesis for FA reesterification, we measured labeled glycerol and pyruvate incorporation into triacylglycerol (TAG). Ritonavir-treated cells had increased FA efflux, uptake, and incorporation into TAG (all P < 0.01). Ritonavir increased FA efflux without consistently increasing glycerol release or changing TAG mass, suggesting increased partial TAG hydrolysis. Ritonavir-treated adipocytes expressed significantly more ATGL mRNA (P < 0.05) and protein (P < 0.05). Ritonavir increased glycerol (P < 0.01) but not pyruvate (P = 0.41), utilization for TAG backbone synthesis. Consistent with this substrate utilization, glycerol kinase transcript (required for glycerol incorporation into TAG backbone) was up-regulated (P < 0.01), whereas phosphoenolpyruvate carboxykinase transcript (required for pyruvate utilization) was down-regulated (P < 0.001). In 3T3-L1 adipocytes, long-term ritonavir exposure perturbs FA metabolism by increasing ATGL-mediated partial TAG hydrolysis, thus increasing FA efflux, and leads to compensatory increases in FA reesterification with glycerol and acylglycerols. These changes in FA metabolism may, in part, explain the increased FA efflux observed in ritonavir-associated lipodystrophy.

Brain-derived neurotrophic factor and obesity in the WAGR syndrome.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) has been found to be important in energy homeostasis in animal models, but little is known about its role in energy balance in humans. Heterozygous, variably sized, contiguous gene deletions causing haploinsufficiency of the WT1 and PAX6 genes on chromosome 11p13, approximately 4 Mb centromeric to BDNF (11p14.1), result in the Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome. Hyperphagia and obesity were observed in a subgroup of patients with the WAGR syndrome. We hypothesized that the subphenotype of obesity in the WAGR syndrome is attributable to deletions that induce haploinsufficiency of BDNF. METHODS: We studied the relationship between genotype and body-mass index (BMI) in 33 patients with the WAGR syndrome who were recruited through the International WAGR Syndrome Association. The extent of each deletion was determined with the use of oligonucleotide comparative genomic hybridization. RESULTS: Deletions of chromosome 11p in the patients studied ranged from 1.0 to 26.5 Mb; 58% of the patients had heterozygous BDNF deletions. These patients had significantly higher BMI z scores throughout childhood than did patients with intact BDNF (mean [+/-SD] z score at 8 to 10 years of age, 2.08+/-0.45 in patients with heterozygous BDNF deletions vs. 0.88+/-1.28 in patients without BDNF deletions; P=0.03). By 10 years of age, 100% of the patients with heterozygous BDNF deletions (95% confidence interval [CI], 77 to 100) were obese (BMI > or = 95th percentile for age and sex) as compared with 20% of persons without BDNF deletions (95% CI, 3 to 56; P<0.001). The critical region for childhood-onset obesity in the WAGR syndrome was located within 80 kb of exon 1 of BDNF. Serum BDNF concentrations were approximately 50% lower among the patients with heterozygous BDNF deletions (P=0.001). CONCLUSIONS: Among persons with the WAGR syndrome, BDNF haploinsufficiency is associated with lower levels of serum BDNF and with childhood-onset obesity; thus, BDNF may be important for energy homeostasis in humans.

Effects of ritonavir on adipocyte gene expression: evidence for a stress-related response.

To understand the molecular mechanisms underlying the development of dyslipidemia and lipodystrophy that occurs after administration of aspartic acid protease inhibitors, we examined transcriptional profiles using cDNA microarrays in 3T3-L1 adipocytes exposed to 10 micromol/l ritonavir for 2-21 days. The expression levels of approximately 12,000 transcripts were assessed using the MgU74Av2 mouse microarray chip. Ritonavir altered gene expression of inflammatory cytokines, stress response genes localized to endoplasmic reticulum, oxidative stress genes, apoptosis-related genes, and expression of genes involved in cell adhesion and extracellular matrix remodeling. Microarray analysis also identified a novel gene downregulated by ritonavir, Cidea, whose expression levels may affect free-fatty acid metabolism. These changes suggest a unique, stress-related pattern in adipocytes induced by chronic exposure to the protease inhibitor, ritonavir.

Orthopedic complications of overweight in children and adolescents.

OBJECTIVE: Few studies have quantified the prevalence of weight-related orthopedic conditions in otherwise healthy overweight children. The goal of the present investigation was to describe the musculoskeletal consequences of pediatric overweight in a large pediatric cohort of children that included severely overweight children. METHODS: Medical charts from 227 overweight and 128 nonoverweight children and adolescents who were enrolled in pediatric clinical studies at the National Institutes of Health from 1996 to 2004 were reviewed to record pertinent orthopedic medical history and musculoskeletal complaints. Questionnaire data from 183 enrollees (146 overweight) documented difficulties with mobility. In 250, lower extremity alignment was determined by bilateral metaphyseal-diaphyseal and anatomic tibiofemoral angle measurements made from whole-body dual-energy x-ray absorptiometry scans. RESULTS: Compared with nonoverweight children, overweight children reported a greater prevalence of fractures and musculoskeletal discomfort. The most common self-reported joint complaint among those who were questioned directly was knee pain (21.4% overweight vs 16.7% nonoverweight). Overweight children reported greater impairment in mobility than did nonoverweight children (mobility score: 17.0 +/- 6.8 vs 11.6 +/- 2.8). Both metaphyseal-diaphyseal and anatomic tibiofemoral angle measurements showed greater malalignment in overweight compared with nonoverweight children. CONCLUSIONS: Reported fractures, musculoskeletal discomfort, impaired mobility, and lower extremity malalignment are more prevalent in overweight than nonoverweight children and adolescents. Because they affect the likelihood that children will engage in physical activity, orthopedic difficulties may be part of the cycle that perpetuates the accumulation of excess weight in children.

Serum brain-derived neurotrophic factor concentrations in lean and overweight children and adolescents.

CONTEXT: Brain-derived neurotrophic factor (BDNF) and its receptor appear to be important components of the leptin-signaling cascade involved in energy homeostasis, and mice with BDNF or TrkB gene haploinsufficiency have excessive adiposity. Little is known about the relationship between adiposity and BDNF, particularly in children. OBJECTIVE: The objective of the study was to study the association of serum BDNF with measures of adiposity in children. DESIGN/SETTING/PATIENTS: BDNF was determined by a sandwich-type ELISA after an overnight fast in convenience sample of 328 subjects, aged 3-19 yr enriched for extreme obesity. In 43, BDNF was also measured before, and again 1 h after, consuming a high-energy content (787 kcal) milkshake. MAIN OUTCOME MEASURES: Measures included associations between BDNF and measures of adiposity. RESULTS: There were no significant univariate associations between log BDNF and adiposity measured by body mass index (BMI), BMI-Z score, or fat mass. However, in an analysis of covariance accounting for age, sex, race, pubertal status, and platelet count, BDNF was lower in overweight children (mean +/- sd, 39.8 +/- 24.8 vs. 47.0 +/- 25.4 ng/dl, P = 0.03); in multiple regression analyses with log BDNF as the dependent variable, BMI (P = 0.03), BMI-Z (P = 0.01), and body fat (P < 0.02) were all negatively associated with BDNF once age, pubertal status, and platelet count were included in the model. Ingestion of a meal did not significantly alter serum BDNF 1 h later (P = 0.26). CONCLUSIONS: Serum BDNF is lower in extremely overweight children and adolescents than those of normal weight. It remains to be determined whether obese individuals with low serum BDNF for age and platelet count have mutations that alter BDNF function.

Co-occurrence of two partially inactivating polymorphisms of MC3R is associated with pediatric-onset obesity.

Both human linkage studies and MC3R knockout mouse models suggest that the MC3R may play an important role in energy homeostasis. Here we show that among 355 overweight and nonoverweight children, 8.2% were double homozygous for a pair of missense MC3R sequence variants (Thr6Lys and Val81Ile). Such children were significantly heavier (BMI and BMI SD score: P < 0.0001), had more body fat (body fat mass and percentage fat mass: P < 0.001), and had greater plasma leptin (P < 0.0001) and insulin concentrations (P < 0.001) and greater insulin resistance (P < 0.008) than wild-type or heterozygous children. Both sequence variants were more common in African-American than Caucasian children. In vitro expression studies found the double mutant MC3R was partially inactive, with significantly fewer receptor binding sites, decreased signal transduction, and less protein expression. We conclude that diminished MC3R expression in this double MC3R variant may be a predisposing factor for excessive body weight gain in children.

Effects of the human immunodeficiency virus-protease inhibitor, ritonavir, on basal and catecholamine-stimulated lipolysis.

Several of the aspartic acid protease inhibitors used to treat HIV infection increase basal lipolysis in adipocytes, but the cellular mechanisms leading to this augmentation are not well understood. We therefore studied the effects of chronic exposure to the HIV protease inhibitor, ritonavir, on the lipolytic cascade in 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with ritonavir for 14 d (during and after differentiation) enhanced basal, isoproterenol (Iso)-stimulated, and cAMP analog-stimulated lipolysis. Enhancement of lipolysis was observed after Iso at concentrations between 0.1 and 10 mum. Despite a significant decrease in cyclic nucleotide phosphodiesterase (PDE)3B activity and protein levels, there were no changes in Iso-stimulated intracellular cAMP, protein kinase A (PKA) expression, or PKA activity. Ritonavir-augmented lipolysis was also observed under conditions that reversed the effect on PDE3B activity via preincubation with 1 mum (-)-N(6)-(2-phenylisopropyl)adenosine. In ritonavir-treated cells, protein expression of the lipid droplet-protective protein, perilipin, was significantly decreased, whereas there was no change in hormone-sensitive lipase. Activation of ERK1/2 by Iso did not play a role in the augmentation. We conclude that ritonavir decreases PDE3B and perilipin protein expression and affects both basal and catecholamine-stimulated lipolysis in 3T3-L1 adipocytes primarily through actions at sites downstream of PKA.

Prediction equations for resting energy expenditure in overweight and normal-weight black and white children.

BACKGROUND: Accurate estimation of children's resting energy expenditure (REE) is important for planning dietary therapy. OBJECTIVE: Our objective was to compare the utility of 5 REE prediction equations in a diverse sample of young children. DESIGN: REE was obtained in 502 black and white girls and boys aged 6-11 y by using indirect calorimetry at 4 US sites. Measured REE and REE predicted from the equations were compared. RESULTS: None of the equations provided both accurate and unbiased estimates of REE. Two new sets of sex-specific equations including race as a factor were generated and evaluated. One set used easily measured variables-females: REE = 0.046 x weight - 4.492 x 1/height(2) - 0.151 x race + 5.841; males: REE = 0.037 x weight - 4.67 x 1/height(2) - 0.159 x race + 6.792-and accounted for 72% and 69%, respectively, of REE variance. The other set used body-composition variables-females: REE = 0.101 x fat-free mass + 0.025 x fat mass + 0.293 x height(3) - 0.185 x race + 1.643; males: REE = 0.078 x fat-free mass + 0.026 x fat mass - 2.646 x 1/height(2) - 0.244 x race + 4.8-and accounted for 75% and 71%, respectively, of REE variance. When split by race and adiposity, the small bias generated could be corrected to within 0.25 MJ (60 kcal) of the mean measured value. CONCLUSION: Sex-specific equations must take race into account to predict REE adequately in children.