Investigating the effects of Orexin-A on thermogenesis in human deep neck brown adipose tissue.

BACKGROUND: Despite successful preclinical testing, 85% of early clinical trials for novel drugs fail. Most futilities originate from molecular mechanisms of the drug(s) tested. It is critically important to develop validated human cell-based model systems in which animal-based research can be translated in order to complement the preclinical in vivo findings prior to implementation of a clinical trial. Obesity is associated with reduced circulating levels of Orexin-A (OX-A) in humans. OX-A increases thermogenesis in rodent brown adipose tissue (AT), yet this phenomenon has not been explored in humans. METHODS: We established a cell-based model system of human brown and white adipocytes and tested the effects of OX-A on thermogenesis. RESULTS: Contrary to published in vivo and in vitro reports in rodents, OX-A treatment alone or in combination with an adrenergic stimulus did neither enhance thermogenesis nor its related transcriptional program in a human in vitro model of brown adipocytes or AT explants. CONCLUSIONS: Translating preclinical findings in human model systems poses a challenge that must be overcome for the development of effective therapeutic compounds and targets.

Impact of a targeted direct marketing price promotion intervention (Buywell) on food-purchasing behaviour by low income consumers: a randomised controlled trial.

BACKGROUND: Price promotions are a promising intervention for encouraging healthier food purchasing. We aimed to assess the impact of a targeted direct marketing price promotion combined with healthy eating advice and recipe suggestions on the purchase of selected healthier foods by low income consumers. METHODS: We conducted a randomised controlled trial (n = 53 367) of a direct marketing price promotion (Buywell) combined with healthy eating advice and recipe suggestions for low income consumers identified as 'less healthy' shoppers. Impact was assessed using electronic point of sale data for UK low income shoppers before, during and after the promotion. RESULTS: The proportion of customers buying promoted products in the intervention month increased by between 1.4% and 2.8% for four of the five products. There was significantly higher uptake in the promotion month (P < 0.001) for the intervention group than would have been expected on the basis of average uptake in the other months. When product switching was examined for semi-skimmed/skimmed milk, a modest increase (1%) was found in the intervention month of customers switching from full-fat to low-fat milk. This represented 8% of customers who previously bought only full-fat milk. The effects were generally not sustained after the promotion period. CONCLUSIONS: Short-term direct marketing price promotions combined with healthy eating advice and recipe suggestions targeted at low income consumers are feasible and can have a modest impact on short-term food-purchasing behaviour, although further approaches are needed to help sustain these changes.

Intermuscular and perimuscular fat expansion in obesity correlates with skeletal muscle T cell and macrophage infiltration and insulin resistance.

BACKGROUND/OBJECTIVES: Limited numbers of studies demonstrated obesity-induced macrophage infiltration in skeletal muscle (SM), but dynamics of immune cell accumulation and contribution of T cells to SM insulin resistance are understudied. SUBJECTS/METHODS: T cells and macrophage markers were examined in SM of obese humans by reverse transcription-PCR (RT-PCR). Mice were fed high-fat diet (HFD) for 2-24 weeks, and time course of macrophage and T-cell accumulation was assessed by flow cytometry and quantitative RT-PCR. Extramyocellular adipose tissue (EMAT) was quantified by high-resolution micro-computed tomography (CT), and correlation to T-cell number in SM was examined. CD11a-/- mice and C57BL/6 mice were treated with CD11a-neutralizing antibody to determine the role of CD11a in T-cell accumulation in SM. To investigate the involvement of Janus kinase/signal transducer and activator of transcription (JAK/STAT), the major pathway for T helper I (TH1) cytokine interferon-γ, in SM and adipose tissue inflammation and insulin resistance, mice were treated with a JAK1/JAK2 inhibitor, baricitinib. RESULTS: Macrophage and T-cell markers were upregulated in SM of obese compared with lean humans. SM of obese mice had higher expression of inflammatory cytokines, with macrophages increasing by 2 weeks on HFD and T cells increasing by 8 weeks. The immune cells were localized in EMAT. Micro-CT revealed that EMAT expansion in obese mice correlated with T-cell infiltration and insulin resistance. Deficiency or neutralization of CD11a reduced T-cell accumulation in SM of obese mice. T cells polarized into a proinflammatory TH1 phenotype, with increased STAT1 phosphorylation in SM of obese mice. In vivo inhibition of JAK/STAT pathway with baricitinib reduced T-cell numbers and activation markers in SM and adipose tissue and improved insulin resistance in obese mice. CONCLUSIONS: Obesity-induced expansion of EMAT in SM was associated with accumulation and proinflammatory polarization of T cells, which may regulate SM metabolic functions through paracrine mechanisms. Obesity-associated SM 'adiposopathy' may thus have an important role in the development of insulin resistance and inflammation.

Clitoral anesthesia disrupts paced copulation in the female rat.

Clitoral stimulation produced by sexual contact with a partner or during manual stimulation is associated with pleasure in humans, and produces conditioned place preference in rats. The present experiment investigated the effect of blocking genitosensory stimulation of the clitoris with lidocaine during copulation in female rats on a measure of female sexual motivation: pacing behavior. Sexually naïve, ovariectomized female rats were treated with 10µg estradiol benzoate 48h and 500µg progesterone 4h prior to a 30-min copulatory trial with a sexually vigorous stimulus male scheduled every 4days. A total of 10 copulatory sessions were divided into two phases of 5 trails each. In the first phase, females received an injection (0.05ml) of either 2% lidocaine, saline, or no injection to the clitoral sheath under isoflurane anesthesia immediately prior to the start of a copulatory session, and were then placed on one side of a paced mating chamber and allowed to copulate for 30min. In the second phase, females previously injected with lidocaine were switched to saline and vice versa, and the no injection group remained the same. Variables measured included overall time spent with the males, number of solicitations, contact-return latencies following male mounts, intromissions, and ejaculations; the frequency of entrances and exits from the male chamber, and frequency of mounts, intromissions, ejaculations. Sexual behavior was examined at session 1, session 5, and session 10. At test 5, females that received LID had a greater number of entrances/exits but spent significantly less time in the presence of the male during the copulatory bout than CNTL animals. These females also displayed a trend for longer contact return latencies s after ejaculations than VEH and CNTL groups. On session 10, females that received LID and subsequently switched to VEH treatment no longer differed from controls in entrance/exit numbers, time spent with males or ejaculation contact return latency. They did however, receive a greater number of intromissions and displayed shorter inter intromission intervals compared to CNTLs. We suggest that clitoral stimulation in the rat serves as both a reward signal and may contribute to the detection of differences in copulatory stimuli that are critical to pacing and potentially, the initiation of pregnancy.

Comparisons of milk quality on North Carolina organic and conventional dairies.

The organic dairy industry is growing rapidly across the United States and has recently expanded into the southeastern states. To date, no published comparisons of milk quality exist between organic and conventional dairies in the Southeastern United States. Maintaining high milk quality is challenging in this region due to the longer periods of high heat and humidity. The objective of this observational study was to compare milk quality on organic and conventional dairies in North Carolina during the warm summer months of the year. Data were compared from 7 organically and 7 conventionally managed herds in North Carolina. To assess milk quality, milk samples were aseptically collected from each functional quarter of each cow in the milking herds at the time of sampling and linear somatic cell scores (SCS) were obtained for individual cows. A total of 4,793 quarter milk samples (2,526 conventional and 2,267 organic) were collected from 1,247 cows (652 conventional and 595 organic). Milk samples were cultured and bacterial growth was identified using protocols consistent with those of the National Mastitis Council (Verona, WI). Subclinical mastitis was defined as the presence of SCS ≥ 4 and also a microbiological infection in at least 1 quarter. The proportion of cows with subclinical mastitis did not differ between conventional (20.8%) and organic (23.3%) herds. No significant difference was observed between herd management types in the proportion of cows without microbiological growth in milk samples. Also, no significant differences were observed between organic and conventional herds for cow-level prevalence of Staphylococcus aureus, coagulase-negative Staphylococcus spp., Streptococcus spp., or Corynebacterium spp. Two of the organic herds had a notably higher prevalence of Corynebacterium spp. and higher SCS. Coliforms were found in 5 of 7 conventional herds and in only 1 of 7 organic herds. Mean SCS did not differ between conventional (3.3±0.2) and organic (3.5±0.2) herds. Despite differences in herd management, milk quality was remarkably similar between the organic and conventional dairies compared for this study.

Overexpression of PLIN5 in skeletal muscle promotes oxidative gene expression and intramyocellular lipid content without compromising insulin sensitivity.

AIMS/HYPOTHESIS: While lipid deposition in the skeletal muscle is considered to be involved in obesity-associated insulin resistance, neutral intramyocellular lipid (IMCL) accumulation per se does not necessarily induce insulin resistance. We previously demonstrated that overexpression of the lipid droplet coat protein perilipin 2 augments intramyocellular lipid content while improving insulin sensitivity. Another member of the perilipin family, perilipin 5 (PLIN5), is predominantly expressed in oxidative tissues like the skeletal muscle. Here we investigated the effects of PLIN5 overexpression - in comparison with the effects of PLIN2 - on skeletal muscle lipid levels, gene expression profiles and insulin sensitivity. METHODS: Gene electroporation was used to overexpress PLIN5 in tibialis anterior muscle of rats fed a high fat diet. Eight days after electroporation, insulin-mediated glucose uptake in the skeletal muscle was measured by means of a hyperinsulinemic euglycemic clamp. Electron microscopy, fluorescence microscopy and lipid extractions were performed to investigate IMCL accumulation. Gene expression profiles were obtained using microarrays. RESULTS: TAG storage and lipid droplet size increased upon PLIN5 overexpression. Despite the higher IMCL content, insulin sensitivity was not impaired and DAG and acylcarnitine levels were unaffected. In contrast to the effects of PLIN2 overexpression, microarray data analysis revealed a gene expression profile favoring FA oxidation and improved mitochondrial function. CONCLUSIONS/INTERPRETATION: Both PLIN2 and PLIN5 increase neutral IMCL content without impeding insulin-mediated glucose uptake. As opposed to the effects of PLIN2 overexpression, overexpression of PLIN5 in the skeletal muscle promoted expression of a cluster of genes under control of PPARα and PGC1α involved in FA catabolism and mitochondrial oxidation.

Neighbourhood deprivation and the price and availability of fruit and vegetables in Scotland.

BACKGROUND: Previous research has suggested that fruits and vegetables are more expensive and less readily available in more deprived communities. However, this evidence is mainly based on small samples drawn from specific communities often located in urban settings and thus is not generalisable to national contexts. The present study explores the influence of neighbourhood deprivation and local retail structure on the price and availability of fruit and vegetables in a sample of areas representing the diversity of urban-rural environments across Scotland, UK. METHODS: A sample of 310 stores located in 10 diverse areas of Scotland was surveyed and data on the price and availability of a basket of 15 fruit and vegetable items were collected. The data were analysed to identify the influence of store type and neighbourhood deprivation on the price and availability of fruits and vegetables. RESULTS: Neighbourhood deprivation and store type did not significantly predict the price of a basket of fruit and vegetables within the sample, although baskets did decrease in price as store size increased. The highest prices were found in the smallest stores located in the most deprived areas. Availability of fruit and vegetables is lower in small shops located within deprived neighbourhoods compared to similar shops in affluent areas. Overall, availability increases with increasing store size. CONCLUSIONS: Availability of fruit and vegetables significantly varies by neighbourhood deprivation in small stores. Policies aimed at promoting sales of fruit and vegetable in these outlets may benefit residents in deprived areas.

Subtyping obesity with microarrays: implications for the diagnosis and treatment of obesity.

OBJECTIVE: Obese patients respond differently to weight loss interventions. No efficient diagnostic tool exists to separate obese patients into subtypes as a means to improve prediction of response to interventions. We aimed to separate obese subjects into distinct subgroups using microarray technology to identify gene expression-based subgroups to predict weight loss. DESIGN: A total of 72 obese men and women without family history of diabetes were enrolled in the study; 52 were treated with ephedra and caffeine (E+C) and 20 with placebo for 8 weeks. Adipose and skeletal muscle tissue biopsies were performed at baseline. RNA sample pairs were labeled and hybridized to oligonucleotide microarrays. Quantile normalization and gene shaving were performed, and a clustering algorithm was then applied to cluster subjects based on their gene expression profile. Clusters were visualized using heat maps and related to weight changes. RESULTS: Cluster analysis of gene expression data revealed two distinct subgroups of obesity and predicted weight loss in response to the treatment with E+C. One cluster ('red') decreased to 96.87+/-2.35% body weight, and the second cluster ('green') decreased to 95.59+/-2.75% body weight (P<0.05). 'Red' cluster had less visceral adipose tissue mass (2.77+/-1.08 vs 3.43+/-1.49 kg; P<0.05) and decreased size of the very large fat cells (1.45+/-0.61 vs 2.16+/-1.74 microl; P<0.05) compared to 'green' cluster. Gene expression for both skeletal muscle and adipose tissue was also different between clusters. CONCLUSIONS: Our study provides the first evidence that the combined approach of gene expression profiling and cluster analysis can identify discrete subtypes of obesity, these subtypes have different physiological characteristics and respond differently to an adrenergic weight loss therapy. This brings us that into an era of personalized treatment in the obesity clinic.

Strong association between mitochondrial DNA copy number and lipogenesis in human white adipose tissue.

AIMS/HYPOTHESIS: Recent studies suggest a link between insulin resistance and mitochondrial function in white fat cells. The aim of this study was to evaluate adipocyte mitochondrial DNA (mtDNA) copy number in relation to adipocyte and clinical variables that are related to insulin sensitivity. METHODS: We studied a group of 148 healthy volunteers with a large inter-individual variation in BMI. Relative amounts of mtDNA and nuclear DNA were determined by quantitative RT-PCR. The mtDNA:nuclear DNA ratio reflects the tissue concentration of mtDNA per cell. RESULTS: The mtDNA copy number was enriched in adipocytes of adipose tissue and decreased slightly by ageing (p = 0.015) and increasing BMI (p = 0.004); however, it was not influenced by sex, energy-restricted diets or marked long-term weight reduction. Adipose mtDNA copy number was not independently related to resting energy expenditure, overall insulin sensitivity or adipocyte lipolysis. However, it showed a strong positive correlation with basal (p = 0.0012) and insulin-stimulated lipogenesis (p < 0.0001) in fat cells, independently of age and BMI, and a weak positive correlation with levels of mRNA from several genes involved in mitochondrial oxidative capacity (r = 0.2-0.3). CONCLUSIONS/INTERPRETATION: The mtDNA copy number in human white fat cells is fairly stable within healthy individuals. It is not influenced by sex or weight loss and is not important for overall insulin sensitivity or energy expenditure at rest. However, it is strongly related to adipocyte lipogenesis and weakly to mitochondrial oxidative capacity, suggesting that adipocyte mitochondria are, above all, local regulators.

The development of a healthy eating indicator shopping basket tool (HEISB) for use in food access studies-identification of key food items.

OBJECTIVES: To develop an objective, nutrient-based, healthy eating indicator shopping basket (HEISB) tool for use in studies of access to healthy food. DESIGN: Tool development used a literature search to identify previous practice, web information on current definition of healthy foods by the UK Food Standards Agency, and population-based dietary surveys to identify culturally acceptable foods. These findings were then appraised with respect to practical fieldwork considerations. SETTING: The review took account of surveys undertaken in a range of geographical areas. RESULTS: Previous tools have varied in the foods selected and the rationale for inclusion. Most have considered nutritional composition but no systematic definition has been used and foods have been subjectively classified as 'less healthy' or 'more healthy'. Recent UK work on nutrient profiling enabled individual food items to be objectively assessed for inclusion. Data from national food surveys enabled commonly consumed and culturally acceptable foods to be identified. Practical considerations included item use in meals, convenience, price, and fieldwork constraints. Other issues including health and price discriminators as well as regional preferences were considered. The final HEISB tool comprised 35 items within the following categories - 17 from fruit and vegetables, nine from potatoes, bread and cereal, five from fish/meats, three from dairy, and one from fatty and sugary foods. CONCLUSIONS: The tool provides a rational basis for examining access and availability of healthy foods in cross-sectional and longitudinal retail and consumer studies.

Natural experiments: an underused tool for public health?

Policymakers and public health researchers alike have demanded better evidence of the effects of interventions on health inequalities. These calls have been repeated most recently in the UK in the final Wanless report, which spoke of the "almost complete lack of an evidence base on the cost-effectiveness of public health interventions", and pointed more generally to the limited evidence base for public health policy and practice. Wanless and others have suggested that the gaps may be partially filled by exploiting the opportunities offered by "natural experiments", such as changes in employment opportunities, housing provision, or cigarette pricing. Natural experiments have an important contributions to make within the health inequalities agenda. First, they can play an important role in investigating the determinants of health inequalities. Second, they can assist in the identification of effective interventions, an area where it is widely acknowledged that the evidence-base is currently sparsely populated. This paper discusses some of the benefits and limitations of using this type of evidence, drawing on two ongoing quasi-experimental studies as examples.

Taking the "ouch" out of injections for children. Using distraction to decrease pain.

PURPOSE: This research compared the effect of two forms of distraction on injection pain in a convenience sample of preschool children. DESIGN: A quasi-experimental study of 105 children (53 girls and 52 boys) ages 4 to 6 years needing DPT immunizations. Data were collected at three sites: two school-based immunization clinics and one public health center with a walk-in immunization program. METHODS: Study children were randomly assigned to receive one of three treatments with their DTP injection: touch, bubble-blowing, or standard care. Prior to injection, a measure of medical fear was obtained (Child Medical Fear Scale) and pain was measured through use of the Oucher Scale. DATA ANALYSIS: Planned comparisons within analysis of variance (ANOVA) tested the differences in pain scores by treatment. Factorial ANOVA was used to determine the influence of age or gender on treatment, and the effect of medical fear on pain was analyzed using correlational statistics and factorial ANOVA. RESULTS: Both forms of distraction, touch and bubble-blowing, significantly reduced pain perception. There were no interaction effects of either age or gender. Fear was a significant covariate, but distraction was effective even when fear was not held constant. CLINICAL IMPLICATIONS: Distraction appears to be an effective method for decreasing injection pain in young children. It is an easy, practical nursing intervention to help children cope with this common, painful experience.

Allograft aortic root replacement in 418 patients over a span of 15 years: 1985 to 2000.

Allograft aortic root replacement for primary aortic valve or ascending aortic root pathology is the favored method of technical implantation. Results in 418 patients over 15 years demonstrate exceedingly low early mortality (<1%), complete eradication of preoperative endocarditis but poor long-term durability in the young age group of 20 years or less.

LICOS, a primordial costimulatory ligand?

In mammals, the classical B7 molecules expressed on antigen-presenting cells, B7-1 (CD80) and B7-2 (CD86), bind the structurally related glycoproteins CD28 and CTLA-4 (CD152), generating costimulatory signals that regulate the activation state of T cells. A recently identified human CD28-like protein, ICOS, also induces costimulatory signals in T cells when crosslinked with antibodies, but it is unclear whether ICOS is part of a B7-mediated regulatory pathway of previously unsuspected complexity, or whether it functions independently and in parallel. Here, we report that, rather than binding B7-1 or B7-2, ICOS binds a new B7-related molecule of previously unknown function that we call LICOS (for ligand of ICOS). At 37 degrees C, LICOS binds only to ICOS but, at lower, non-physiological temperatures, it also binds weakly to CD28 and CTLA-4. Sequence comparisons suggest that LICOS is the homologue of a molecule expressed by avian macrophages and of a murine protein whose expression is induced in non-lymphoid organs by tumour necrosis factor alpha (TNFalpha). Our results define the components of a distinct and novel costimulatory pathway and raise the possibility that LICOS, rather than B7-1 or B7-2, is the contemporary homologue of a primordial vertebrate costimulatory ligand.