High energy expenditure is not protective against increased adiposity in children.

BACKGROUND: Low levels of energy expenditure (TEE) may contribute to excess weight during childhood, but limited longitudinal data exist. OBJECTIVES: This is to test whether low TEE during the first 6 years of life could predict excess weight status at 8 years. METHODS: Total energy expenditure from doubly labelled water, weight, stature, waist circumference and fat mass and fat-free mass (FFM) in children at 0.25, 2, 4 and 6 years of age. This cohort includes individuals at high (n = 27) and low risk (n = 26) for childhood obesity, based upon whether pre-pregnant maternal obesity. A linear mixed effects model was fit to TEE. Individual variation was accounted for as a random effect. Residual TEE was calculated for age and individually averaged across time. RESULTS: Fat-free mass (kg) was highly correlated (R2 = 0.91) with TEE (kcal/day), and waist circumference and sex were also significant predictors of TEE. TEE residual tracked within individuals. TEE residuals did not correlate with either BMI or %fat at age 8 years. CONCLUSION: Using the residual TEE approach to identify high and low TEE during the first 6 years of life did not explain excess weight at 8 years of life in this cohort of children at high and low risk of obesity based upon maternal obesity status.

Comparison of total energy expenditure between school and summer months.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Childhood obesity has increased 3 to 4 fold. Some children gain excess weight in summer. WHAT THIS STUDY ADDS: Total energy expenditure increases almost linearly with fat-free mass. A lower total energy expenditure was not detected in summer. OBJECTIVE: Recent data report that the youth experience greater weight gain during summer than during school months. We tested the hypothesis that a difference in total energy expenditure (TEE) between school and summer months exists and may contribute to summer weight gain. SUBJECTS AND METHODS: A secondary analysis was performed on cross-sectional TEE data from school-age, sedentary African-American and Caucasian youth based in or near the District of Columbia who were at-risk for adult obesity because they had body mass index (BMI) ≥ 85th percentile or had overweight parents. TEE was estimated from 18-O and deuterium measurements during 1-week intervals using urine samples collected after ingestion of doubly labelled water. Differences in summer- and school-time TEE were assessed using analysis of covariance. The data were adjusted for fat-free mass (FFM) as determined by deuterium dilution to adjust for the effect of body size on TEE. RESULTS: Data were collected from 162 youth (average age 10 ± 2 years, BMI 28 ± 8 kg m(-2) and BMI z-score 1.96 + 0.96). Of these, 96 youth had TEE measured during the school year (September-June); 66 different youths had TEE measured during summer months (June-August). After adjustment for FFM, average summertime TEE was 2450 ± 270 kcal d(-1) and average school-time TEE was 2510 ± 350 kcal d(-1) (P = 0.26). CONCLUSION: No difference in TEE was detected between the school year and the summer months. These data suggest that seasonal differences in youth weight gain are not necessarily due to differences in energy expenditures.

The loss of the BH3-only Bcl-2 family member Bid delays T-cell leukemogenesis in Atm-/- mice.

Multicellular organisms maintain genomic integrity and resist tumorigenesis through a tightly regulated DNA damage response (DDR) that prevents propagation of deleterious mutations either through DNA repair or programmed cell death. An impaired DDR leads to tumorigenesis that is accelerated when programmed cell death is prevented. Loss of the ATM (ataxia telangiectasia mutated)-mediated DDR in mice results in T-cell leukemia driven by accumulation of DNA damage accrued during normal T-cell development. Pro-apoptotic BH3-only Bid is a substrate of Atm, and Bid phosphorylation is required for proper cell cycle checkpoint control and regulation of hematopoietic function. In this report, we demonstrate that, surprisingly, loss of Bid increases the latency of leukemogenesis in Atm-/- mice. Bid-/-Atm-/- mice display impaired checkpoint control and increased cell death of DN3 thymocytes. Loss of Bid thus inhibits T-cell tumorigenesis by increasing clearance of damaged cells, and preventing propagation of deleterious mutations.

Rejuvenating Bi(d)ology.

The BH3-only Bid protein is a critical sentinel of cellular stress in the liver and the hematopoietic system. Bid's initial 'claim to fame' came from its ability-as a caspase-truncated product-to trigger the mitochondrial apoptotic program following death receptor activation. Today we know that Bid can response to multiple types of proteases, which are activated under different conditions such as T-cell activation, ischemical reperfusion injury and lysosomal injury. Activation of the mitochondrial apoptotic program by Bid-via its recently identified receptor mitochondrial carrier homolog 2-involves multiple mechanisms, including release of cytochrome c and second mitochondria-derived activator of caspase (Smac), alteration of mitochondrial cristae organization, generation of reactive oxygen species and engagement of the permeability transition pore. Bid is also emerging-in its full-length form-as a pivotal sentinel of DNA damage in the bone marrow regulated by the ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR) kinases. The ATM/ATR-Bid pathway is critically involved in preserving the quiescence and survival of hematopoietic stem cells both in the absence and presence of external stress, and a large part of this review will be dedicated to recent advances in this area of research.

Bid protects the mouse hematopoietic system following hydroxyurea-induced replicative stress.

Hematopoietic stem cells (HSCs) possess long-term self-renewal capacity and multipotent differentiative capacity, to maintain the hematopoietic system. Long-term hematopoietic homeostasis requires effective control of genotoxic damage to maintain HSC function and prevent propagation of deleterious mutations. Here we investigate the role of the BH3-only Bcl-2 family member Bid in the response of murine hematopoietic cells to long-term replicative stress induced by hydroxyurea (HU). The PI3-like serine/threonine kinase, ATR, initiates the DNA damage response (DDR) to replicative stress. The pro-apoptotic Bcl-2 family member, Bid, facilitates this response to replicative stress in hematopoietic cells, but the in vivo role of this DDR function of Bid has not been defined. Surprisingly, we demonstrate that long-term HU treatment expands wild-type myeloid progenitor cells (MPCs) and HSC-enriched Lin(-)Sca1(+)Kit(+) (LSK) cells to maintain bone marrow function as measured by long-term competitive repopulating ability. Bid-/- MPCs demonstrate increased sensitivity to HU and are depleted. Bid-/- LSK cells demonstrate increased mobilization manifest by increased Bromodeoxyuridine (BrdU) incorporation. Bid-/- MPCs and LSK cells are relatively depleted, however, and bone marrow from Bid-/- mice demonstrates decreased long-term competitive repopulating ability in both primary and secondary transplants. We thus describe a survival function of Bid in hematopoiesis in the setting of chronic replicative stress.

Proapoptotic Bid mediates the Atr-directed DNA damage response to replicative stress.

Proapoptotic BH3 interacting domain death agonist (Bid), a BH3-only Bcl-2 family member, is situated at the interface between the DNA damage response and apoptosis, with roles in death receptor-induced apoptosis as well as cell cycle checkpoints following DNA damage.(1, 2, 3) In this study, we demonstrate that Bid functions at the level of the sensor complex in the Atm and Rad3-related (Atr)-directed DNA damage response. Bid is found with replication protein A (RPA) in nuclear foci and associates with the Atr/Atr-interacting protein (Atrip)/RPA complex following replicative stress. Furthermore, Bid-deficient cells show an impaired response to replicative stress manifest by reduced accumulation of Atr and Atrip on chromatin and at DNA damage foci, reduced recovery of DNA synthesis following replicative stress, and decreased checkpoint kinase 1 activation and RPA phosphorylation. These results establish a direct role for the BH3-only Bcl-2 family member, Bid, acting at the level of the damage sensor complex to amplify the Atr-directed cellular response to replicative DNA damage.

BCL2 family in DNA damage and cell cycle control.

Individual BCL2 family members couple apoptosis regulation and cell cycle control in unique ways. Antiapoptotic BCL2 and BCL-x(L) are antiproliferative by facilitating G0. BAX is proapoptotic and accelerates S-phase progression. The dual functions in apoptosis and cell cycle are coordinately regulated by the multi-domain BCL2 family members (MCL-1) and suggest that survival is maintained at the expense of proliferation. The role of BH3-only molecules in cell cycle is more variable. BAD antagonizes both the cell cycle and antiapoptotic functions of BCL2 and BCL-x(L) through BH3 binding. BID has biochemically separable functions in apoptosis and S-phase checkpoint, determined by post-translational modification. p53-induced PUMA is known only to have apoptotic function. Inhibition of apoptosis is oncogenic, whereas promotion of cell cycle arrest is tumor suppressive. Paradoxically, selected BCL2 family members can be both oncogenic and tumor suppressive. Which of the dual functions predominates is lineage specific and context dependent.

BID mediates neuronal cell death after oxygen/ glucose deprivation and focal cerebral ischemia.

Mitochondria and cytochrome c release play a role in the death of neurons and glia after cerebral ischemia. In the present study, we investigated whether BID, a proapoptotic promoter of cytochrome c release and caspase 8 substrate, was expressed in brain, activated after an ischemic insult in vivo and in vitro, and contributed to ischemic cell death. We detected BID in the cytosol of mouse brain and primary cultured mouse neurons and demonstrated, by using recombinant caspase 8, that neuronal BID also is a caspase 8 substrate. After 2 h of oxygen/glucose deprivation, BID cleavage was detected in neurons concurrent with caspase 8 activation but before caspase 3 cleavage. Bid(-/-) neurons were resistant to death after oxygen/glucose deprivation, and caspase 3 cleavage was significantly reduced; however, caspase 8 cleavage did not differ from wild type. In vivo, BID was cleaved 4 h after transient middle cerebral artery occlusion. Infarct volumes and cytochrome c release also were less in Bid(-/-) mice (-67% and -41%, respectively) after mild focal ischemia. These findings suggest that BID and the mitochondrial-amplification pathway promoting caspase activation contributes importantly to neuronal cell death after ischemic insult.

Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis.

The protein Bid is a participant in the pathway that leads to cell death (apoptosis), mediating the release of cytochrome c from mitochondria in response to signals from 'death' receptors known as TNFR1/Fas on the cell surface. It is a member of the proapoptotic Bcd-2 family and is activated as a result of its cleavage by caspase 8, one of a family of proteolytic cell-death proteins. To investigate the role of Bid in vivo, we have generated mice deficient for Bid. We find that when these mice are injected with an antibody directed against Fas, they nearly all survive, whereas wild-type mice die from hepatocellular apoptosis and haemorrhagic necrosis. About half of the Bid-deficient animals had no apparent liver injury and showed no evidence of activation of the effector caspases 3 and 7, although the initiator caspase 8 had been activated. Other Bid-deficient mice survived with only moderate damage: all three caspases (8 and 37) were activated but their cell nuclei were intact and no mitochondrial cytochrome c was released. We also investigated the effects of Bid deficiency in cultured cells treated with anti-Fas antibody (hepatocytes and thymocytes) or with TNFalpha. (fibroblasts). In these Bid-/- cells, mitochondrial dysfunction was delayed, cytochrome c was not released, effector caspase activity was reduced and the cleavage of apoptosis substrates was altered. This loss-of-function model indicates that Bid is a critical substrate in vivo for signalling by death-receptor agonists, which mediates a mitochondrial amplification loop that is essential for the apoptosis of selected cells.

Transgenic studies with a keratin promoter-driven growth hormone transgene: prospects for gene therapy.

Keratinocytes are potentially appealing vehicles for the delivery of secreted gene products because they can be transferred to human skin by the relatively simple procedure of grafting. Adult human keratinocytes can be efficiently propagated in culture with sufficient proliferative capacity to produce enough epidermis to cover the body surface of an average adult. However, the feasibility of delivering secreted proteins through skin grafting rests upon (i) the strength of the promoter in keratinocytes and (ii) the efficiency of protein transport through the basement membrane of the stratified epithelium and into the bloodstream. In this paper, we use transgenic technology to demonstrate that the activity of the human keratin 14 promoter remains high in adult skin and that keratinocyte-derived human growth hormone (hGH) can be produced, secreted, and transported to the bloodstream of mice with efficiency that is sufficient to exceed by an order of magnitude the circulating hGH concentration in growing children. Transgenic skin grafts from these adults continue to produce and secrete hGH stably, at approximately 1/10 physiological levels in the bloodstream of nontransgenic recipient mice. These studies underscore the utility of the keratin 14 promoter for expressing foreign transgenes in keratinocytes and demonstrate that keratinocytes can be used as effective vehicles for transporting factors to the bloodstream and for eliciting metabolic changes. These findings have important implications for considering the keratinocyte as a possible vehicle for gene therapy.

Skin cancer and transgenic mice.

At the outer surface of the body, the epidermis of skin forms a protective barrier to keep microorganisms out and essential body fluids in. This barrier is composed of one main cell type, the keratinocyte, which undergoes a precisely defined program of differentiation to produce a durable and renewable integument. The delicate balance between differentiation and proliferation is maintained by regulatory molecules and growth factors. In basal and squamous cell carcinomas of the skin, the balance between growth and differentiation goes awry, although the detailed pathways underlying these cancers remain to be elucidated. Transgenic mice provide the opportunity to precisely perturb growth factors in the skin in the context of a whole organism, leading to valuable insight into both appropriate and deranged growth. This article will review the biology and biochemistry of epidermal growth and differentiation, with emphasis on basal and squamous cell carcinomas and hyperproliferative diseases of the skin, and how recent transgenic research has been utilized to take a penetrating look into these issues.

Identification of a negative regulatory element that inhibits c-mos transcription in somatic cells.

We have used transient expression assays to identify a cis-acting region in the 5' flanking sequence of murine c-mos which, when deleted, allows expression from the c-mos promoter in NIH 3T3 cells. This negative regulatory sequence, located 400 to 500 nucleotides upstream of the c-mos ATG, also inhibited expression from a heterologous promoter. In addition to NIH 3T3 cells, the c-mos negative regulatory sequence was active in BALB/3T3 cells, PC12 rat pheochromocytoma cells, and A549 human lung carcinoma cells. Site-specific mutagenesis identified three possibly interacting regions that were involved in negative regulatory activity, located around -460, -425, and -405 with respect to the ATG. RNase protection analysis indicated that once the negative regulatory sequences were deleted, transcription in NIH 3T3 cells initiated from the same transcription initiation sites normally utilized in spermatocytes, approximately 280 nucleotides upstream of the ATG. Deletions beyond the spermatocyte promoter, however, allowed transcription initiation from progressively downstream c-mos sequences. Deletion or mutation of sequences surrounding the oocyte promoter at -53 also had little effect on expression of c-mos constructs in NIH 3T3 cells. Therefore, the major determinant of c-mos expression in NIH 3T3 cells was removal of the negative regulatory sequence rather than the utilization of a unique promoter. The c-mos negative regulatory sequences thus appear to play a significant role in tissue-specific c-mos expression by inhibiting transcription in somatic cells.

c-mos expression in mouse oocytes is controlled by initiator-related sequences immediately downstream of the transcription initiation site.

We have employed transient expression assays to analyze the sequences that direct c-mos transcription in mouse oocytes. Plasmids containing the chloramphenicol acetyltransferase (CAT) gene fused to either a 2.4-kb or a 731-bp fragment from the 5'-flanking region of c-mos produced similar levels of CAT activity when injected into nuclei of growing oocytes. BAL 31 deletions revealed that sequences up to 20 bp upstream of the major transcription start site could be removed without any significant loss of CAT activity. Promoter activity only decreased when these deletions closely approached the transcription start site, which was mapped at 53 nucleotides upstream of the first ATG in the c-mos open reading frame. On the other hand, deletion of sequences within 20 nucleotides downstream of the transcription initiation site resulted in a 10-fold reduction in CAT expression. A similar decrease in promoter activity was observed as a result of point mutations in these 5' untranslated sequences. Thus, sequences immediately downstream of the transcription start site, including a consensus sequence (PyPyCAPyPyPyPyPy) present in the initiator elements of several genes, appear to regulate c-mos expression in mouse oocytes. Reverse transcription-polymerase chain reaction analysis of RNA from injected oocytes showed that this regulation is manifest at the transcriptional level. Expression of c-mos in mouse oocytes thus appears to be directed by a simple promoter consisting only of sequences immediately surrounding the transcription start site, including an initiator element in the untranslated leader.

Catabolite activator protein-induced DNA bending in transcription initiation.

We describe experiments that enable us to track the presence and direction of the DNA bend induced by Escherichia coli catabolite activator protein (CAP) through the intermediate stages of transcription initiation at the lac promoter. Transcriptional complexes examined were formed on superhelical templates to enhance specific complex formation, and detected by electrophoretic analysis after restriction digestion. We found that the bend is maintained and even increased upon formation of closed and open complexes. Our results exclude the hypothesis that the energy of the CAP-induced bend is used to promote open complex formation. We now suggest a new model, in which DNA wraps around the CAP-polymerase complex to form a writhing structure equivalent to that at the end of an interwound superhelical domain. Formation of this structure may facilitate open complex formation. We further propose that the stored bend energy may be used to help counteract strong protein-protein or protein-DNA interactions, thus assisting the process of RNA polymerase escape from the promoter.

Comparative gel electrophoresis measurement of the DNA bend angle induced by the catabolite activator protein.

We describe a method to determine the magnitude of protein-induced DNA bends relative to a set of standard A tract bends using comparative gel electrophoresis. The DNA bend of interest was that induced by the catabolite activator protein (CAP), the transcriptional activator protein of the lac operon. The set of comparison molecules contained both bends of known magnitude and a bound CAP. The electrophoretic influence of the bound protein was accounted for by placing its binding site at the end of the molecule where its induced bend has little influence. Standard bends at the DNA center were introduced by incorporating 3-9 A6 tracts at approximately 10.5 base-pair phasing. The mobility of these control molecules was compared to the mobility of a test molecule of comparable length containing a central CAP-induced DNA bend. The CAP bend angle was found to be 5.6 +/- 0.3 A tract equivalents, or approximately 100 degrees, independent of the concentration of the gel used within the range tested. The dependence of gel retardation on DNA end-to-end distance was found to break down for A tract bend angles above 120 degrees, corresponding roughly to the angle beyond which the long axis of the molecule is no longer parallel to the end-to-end vector. We speculate that this may reflect a switch in the mode of migration of molecules through the gel.

DNA bend direction by phase sensitive detection.

Gel electrophoresis of DNA and protein-DNA complexes has been a key method used in studies of sequence-directed and protein-induced DNA bending. Natural DNA sequences can have protein binding sites adjacent to A-tract bending sites, resulting in the potential for the formation of topologically complex shapes in a localized DNA regulatory domain. An essential first step in deducing the structure and functional significance of such domains is elucidation of the relative direction of bending, which can be determined from the electrophoretic mobilities of isomers having varied helical phasing between the bends. Taking DNA bent around CAP protein as a standard, we conclude that the junction bending model correctly predicts the direction of bending at A tracts in kinetoplast DNA. The overall direction of the bend is towards the minor groove at the centre of the A tract.