Cloning and expression of the large zebrafish protocadherin gene, Fat

The cadherin superfamily members play an important role in mediating cell-cell contact and adhesion (Takeichi, M., 1991. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251, 1451-1455). A distinct subfamily, neither belonging to the classical or protocadherins includes Fat, the largest member of the cadherin super-family. Fat was originally identified in Drosophila. Subsequently, orthologues of Fat have been described in man (Dunne, J., Hanby, A. M., Poulsom, R., Jones, T. A., Sheer, D., Chin, W. G., Da, S. M., Zhao, Q., Beverley, P. C., Owen, M. J., 1995. Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule. Genomics 30, 207-223), rat (Ponassi, M., Jacques, T. S., Ciani, L., ffrench, C. C., 1999. Expression of the rat homologue of the Drosophila fat tumour suppressor gene. Mech. Dev. 80, 207-212) and mouse (Cox, B., Hadjantonakis, A. K., Collins, J. E., Magee, A. I., 2000. Cloning and expression throughout mouse development of mfat 1, a homologue of the Drosophila tumour suppressor gene fat [In Process Citation]. Dev. Dyn. 217, 233-240). In Drosophila, Fat has been shown to play an important role in both planar cell polarity and cell boundary formation during development. In this study we describe the characterization of zebrafish Fat, the first non-mammalian, vertebrate Fat homologue to be identified. The Fat protein has 64% amino acid identity and 80% similarity to human FAT and an identical domain structure to other vertebrate Fat proteins. During embryogenesis fat mRNA is expressed in the developing brain, specialised epithelial surfaces the notochord, ears, eyes and digestive tract, a pattern similar but distinct to that found in mammals. (c) 2005 Elsevier B.V. All rights reserved.

Growth hormone binding protein correlates strongly with leptin and percentage body fat in GH-deficient adults, is increased by GH replacement but does not predict IGF-I response

GH-binding protein (GHBP) corresponds to the extracellular domain of the GH receptor (GHR) and has been shown to be closely related to body fat. This study aimed to examine the inter-relationship between GHBP, leptin and body fat, and to test the hypothesis that GHBP is modified by GH replacement in GH-deficient adults and predicts IGF-I response. Twenty adults, mean age 47 years (range 20-69) with proven GH deficiency were randomly allocated to either GH (up to 0.25 U/kg/week in daily doses) or placebo for 3 months before cross-over to the opposite treatment. Plasma GHBP and leptin were measured at baseline and 2, 4, 8 and 12 weeks after each treatment. Whole body composition was measured at baseline by dual-energy X-ray absorptiometry (DEXA). There was a strong correlation between baseline leptin and GHBP (r = 0.88, P < 0.0001) and between baseline GHBP and percentage body fat, (r = 0.83, P < 0.0001). Mean GHBP levels were higher on GH compared with placebo, 1.53 +/- 0.28 vs 1.41 +/- 0.25 nM, P = 0.049. There was no correlation between baseline IGF-I and GHBP (r = -0.049, P = 0.84), and GHBP did not predict IGF-I response to GH replacement. The close inter-relationship between GHBP, leptin and body fat suggests a possible role for GHBP in the regulation of body composition. GHBP is increased by GH replacement in GH-deficient adults, but does not predict biochemical response to GH replacement. (C) 1999 Churchill Livingstone.

Nutrient partitioning during treatment of tuberculosis: gain in body fat mass but not in protein mass

Background: Tuberculosis is an important cause of wasting. The functional consequences of wasting and recovery may depend on the distribution of lost and gained nutrient stores between protein and fat masses. Objective: The goal was to study nutrient partitioning, ie, the proportion of weight change attributable to changes in fat mass (FM) versus protein mass (PM), during anti mycobacterial treatment. Design: Body-composition measures were made of 21 men and 9 women with pulmonary tuberculosis at baseline and after 1 and 6 mo of treatment. All subjects underwent dual-energy X-ray absorptiometry and deuterium bromide dilution tests, and a four-compartment model of FM, total body water (TBW), bone minerals (BM), and PM was derived. The ratio of PM to FM at any time was expressed as the energy content (p-ratio). Changes in the p-ratio were related to disease severity as measured by radiologic criteria. Results: Patients gained 10% in body weight (P < 0.001) from baseline to month 6. This was mainly due to a 44% gain in FM (P < 0.001); PM, BM, and TBW did not change significantly. Results were similar in men and women. The p-ratio decreased from baseline to month 1 and then fell further by month 6. Radiologic disease severity was not correlated with changes in the p-ratio. Conclusions: Microbiological cure of tuberculosis does not restore PM within 6 mo, despite a strong anabolic response. Change in the p-ratio is a suitable parameter for use in studying the effect of disease on body composition because it allows transformation of such effects into a normal distribution across a wide range of baseline proportion between fat and protein mass.

A role for protein kinase B beta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKB beta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKB alpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKB beta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKB beta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKB beta in insulin-stimulated glucose transport in adipocytes.

Placental growth hormone (GH), GH-binding protein, and insulin-like growth factor axis in normal, growth-retarded, and diabetic pregnancies: Correlations with fetal growth

We previously described significant changes in GH-binding protein (GHBP) in pathological human pregnancy. There was a substantial elevation of GHBP in cases of noninsulin-dependent diabetes mellitus and a reduction in insulin-dependent diabetes mellitus. GHBP has the potential to modulate the proportion of free placental GH (PGH) and hence the impact on the maternal GH/insulin-like growth factor I (IGF-I) axis, fetal growth, and maternal glycemic status. The present study was undertaken to investigate the relationship among glycemia, GHBP, and PGH during pregnancy and to assess the impact of GHBP on the concentration of free PGH. We have extended the analysis of specimens to include measurements of GHBP, PGH, IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFSP-2, and IGFBP-3 and have related these to maternal characteristics, fetal growth, and glycemia. The simultaneous measurement of GHBP and PGH has for the first time allowed calculation of the free component of PGH and correlation of the free component to indexes of fetal growth and other endocrine markers. PGH, free PGH, IGF-I, and IGF-II were substantially decreased in IUGR at 28-30 weeks gestation (K28) and 36-38 weeks gestation (K36). The mean concentration (+/-SEM) of total PGH increased significantly from K28 to K36 (30.0 +/- 2.2 to 50.7 +/- 6.2 ng/mL; n = 40), as did the concentration of free PGH (23.4 +/- 2.3 to 43.7 +/- 6.0 ng/mL; n = 38). The mean percentage of free PGH was significantly less in IUGR than in normal subjects (67% vs. 79%; P < 0.01). Macrosomia was associated with an increase in these parameters that did not reach statistical significance. Multiple regression analysis revealed that PGH/IGF-I and IGFBP-5 account for 40% of the variance in birth weight. IGFBP-3 showed a significant correlation with IGF-I, IGF-II, and free and total PGK at K28 and K36. Noninsulin-dependent diabetes mellitus patients had a lower mean percentage of free PGH (65%; P < 0.01), and insulin-dependent diabetics had a higher mean percentage of free PGH (87%; P < 0.01) than normal subjects. Mean postprandial glucose at K28 correlated positively with PGH and free PGH (consistent with the hyperglycemic action of GH). GHBP correlated negatively with both postprandial and fasting glucose. Although GHBP correlated negatively with PGH (r = -0.52; P

Characterization of peroxisome proliferator-activated receptor alpha in normal rat mammary gland and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary gland tumors from rats fed high and low fat diets

Normal Sprague-Dau ley rat mammary gland epithelial cells and mammary gland carcinomas induced by 2-amino-1 -methyl-6-phenylimidazo[4,5-b]pyridine, a carcinogen found in the diet, were examined for the expression of peroxisome proliferator-activated receptor alpha (PPAR alpha). PPAR alpha mRNA and protein was detected in normal and tumor tissue by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. By quantitative RT-PCR, carcinomas had a 12-fold higher expression than control mammary glands, a statistically significant difference. PPAR alpha expression was examined in carcinomas and normal tissues from rats on high fat (23.5/% corn oil) and low fat (5% corn oil) diets. Although neither carcinomas, nor control tissues showed statistically significant differences between the two diet groups, PPAR alpha expression was the highest in carcinomas from rats on the high fat diet. The expression of PPAR alpha in normal mammary gland and its significant elevation in mammary gland carcinomas raises the possibility of its involvement in mammary gland physiology and pathophysiology. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.

Characterization of a novel protein with homology to C-type lectins expressed by the Cotesia rubecula bracovirus in larvae of the lepidopteran host, Pieris rapae

Polydnaviruses are essential for the survival of many Ichneumonoid endoparasitoids, providing active immune suppression of the host in which parasitoid larvae develop. The Cotesia rubecula bracovirus is unique among polydnaviruses in that only four major genes are detected in parasitized host ( Pieris rapae) tissues, and gene expression is transient. Here we describe a novel C. rubecula bracovirus gene (CrV3) encoding a lectin monomer composed of 159 amino acids, which has conserved residues consistent with invertebrate and mammalian C-type lectins. Bacterially expressed CrV3 agglutinated sheep red blood cells in a divalent ion-dependent but Ca2+-independent manner. Agglutination was inhibited by EDTA but not by biological concentrations of any saccharides tested. Two monomers of similar to14 and similar to17 kDa in size were identified on SDS-PAGE in parasitized P. rapae larvae. The 17-kDa monomer was found to be an N-glyscosylated form of the 14-kDa monomer. CrV3 is produced in infected hemocytes and fat body cells and subsequently secreted into hemolymph. We propose that CrV3 is a novel lectin, the first characterized from an invertebrate virus. CrV3 shows over 60% homology with hypothetical proteins isolated from polydnaviruses in two other Cotesia wasps, indicating that these proteins may also be C-type lectins and that a novel polydnavirus lectin family exists in Cotesia-associated bracoviruses. CrV3 is probably interacting with components in host hemolymph, resulting in suppression of the Pieris immune response. The high similarity of CrV3 with invertebrate lectins, as opposed to those from viruses, may indicate that some bracovirus functions were acquired from their hosts.

Increase of fat oxidation and weight loss in obese mice caused by chronic treatment with human growth hormone or a modified C-terminal fragment

OBJECTIVE: To observe the chronic effects of human growth hormone (hGH) and AOD9604 (a C-terminal fragment of hGH) on body weight, energy balance, and substrate oxidation rates in obese (ob/ob) and lean C57BL/6Jmice. In vitro assays were used to confirm whether the effects of AOD9604 are mediated through the hGH receptor, and if this peptide is capable of cell proliferation via the hGH receptor. METHOD: Obese and lean mice were treated with hGH, AOD or saline for 14 days using mini-osmotic pumps. Body weight, caloric intake, resting energy expenditure, fat oxidation, glucose oxidation, and plasma glucose, insulin and glycerol were measured before and after treatment. BaF-BO3 cells transfected with the hGH receptor were used to measure in Vitro I-125-hGH receptor binding and cell proliferation. RESULTS: Both hGH and AOD significantly reduced body weight gain in obese mice. This was associated with increased in vivo fat oxidation and increased plasma glycerol levels (an index of lipolysis). Unlike hGH, however, AOD9604 did not induce hyperglycaemia or reduce insulin secretion. AOD9604 does not compete for the hGH receptor and nor does it induce cell proliferation, unlike hGH. CONCLUSIONS: Both hGH and its C-terminal fragment reduce body weight gain, increase fat oxidation, and stimulate lipolysis in obese mice, yet AOD9604 does not interact with the hGH receptor. Thus, the concept of hGH behaving as a pro-hormone is further confirmed. This data shows that fragments of hGH can act in a manner novel to traditional hGH-stimulated pathways.

Increased expression of the SNARE accessory protein Munc18c in lipid-mediated insulin resistance

Fatty acids inhibit insulin-mediated glucose metabolism in skeletal muscle, an effect largely attributed to defects in insulin-mediated glucose transport. Insulin-resistant mice transgenic for the overexpression of lipoprotein lipase (LPL) in skeletal muscle were used to examine the molecular mechanism(s) in more detail. Using DNA gene chip array technology, and confirmation by RT-PCR and Western analysis, increases in the yeast Sec1p homolog Munc18c mRNA and protein were found in the gastrocnemius muscle of transgenic mice, but not other tissues. Munc18c has been previously demonstrated to impair insulin-mediated glucose transport in mammalian cells in vitro. Of interest, stably transfected C2C12 cells overexpressing LPL not only demonstrated increases in Munc18c mRNA and protein but also in transcription rates of the Munc18c gene. jlr To confirm the relevance of fatty acid metabolism and insulin resistance to the expression of Munc18c in vivo, a 2-fold increase in Munc18c protein was demonstrated in mice fed a high-fat diet for 4 weeks. Together, these data are the first to implicate in vivo increases in Munc18c as a potential contributing mechanism to fatty acid-induced insulin resistance.

Protein utilisation and turnover in lines of chickens selected for different aspects of body composition

1. Protein utilisation and turnover were measured in male chickens sampled from a line selected for high breast yield and a randombred control line (lines QL and CL, experiment 1) and in male chickens sampled from lines selected for either high or low abdominal fatness (lines FL and LL, experiment 2). In each experiment, 18 birds per line were given iso-energetic (12.9 MJ ME/kg) diets containing either 120 or 220 g CP/kg from 21 to 29 d (experiment 1) and 33 to 43 d (experiment 2). 2. Measurements were made of growth rate, food intake, body composition, excreta production and N-tau-methylhistidine excretion as a measure of myofibrillar protein breakdown, and fractional rates (%/d) of protein deposition, breakdown and synthesis were calculated. 3. In experiment 1, there were no significant differences between the line means for the fractional measures of protein turnover, but there was marked differential response in the two lines in the fractional rates of protein deposition, breakdown and synthesis, to increase in protein intake. The positive slope of the regressions of fractional (%/d) protein deposition and synthesis rates on protein intake (g/d/kg BW) were approximately 1.4- and 2.0-fold higher respectively in the QL than the CL line birds, and the negative slope of the regression of fractional breakdown rate on protein intake was approximately threefold greater in the CL than the QL line birds. 4. In experiment 2, fractional deposition rate was 6.2% lower, but fractional breakdown rate 9.4% higher in the LL than the FL birds, whilst there was essentially no difference in response of the FL and LL birds in the components of protein turnover to increase in protein intake. Line differences in deposition and breakdown rates were thus a reflection of the considerably higher (20%) food and hence protein intake in the FL than the LL birds. 5. The differential line responses in protein turnover in the two experiments suggest that selection for increased breast muscle yield and for reduced body fatness manipulate different physiological pathways in relation to protein turnover, but neither selection strategy results in an improvement in net protein utilisation at typical levels of protein intake by birds on commercial broiler diets, through a reduction in protein breakdown rate.

Clinically active Crohn's disease in the presence of a low C-reactive protein

Objective. Clinical interest in C-reactive protein (CRP) - a component of the innate immune system - has focused mainly on its worth as an indicator of disease activity. There has been a resurgence of interest in CRP in the Crohn's disease ( CD) literature because several trials of new treatments for active CD have been characterized by both a large proportion of patients with low CRP ( < 10 mg/l) at entry to the trials and by a negative therapeutic outcome. It is therefore of interest to study the clinical characteristics of patients who are thought to have at the same time both active CD and a low CRP. Material and methods. Patients were prospectively recruited as part of the Brisbane IBD clinical and research programme. Subjects were included in the low CRP group only if there were complete datasets for CRP on all occasions of active CD, and CRP was < 10 mg/l. Active disease was defined as CD activity index (CDAI) > 200. The low CRP group was compared with patients in the raised CRP group for a range of clinical variables as well as the major NOD2 variants. Results. There were data sets for 223 CD patients, with a mean disease duration of 12 years. Of these, 22 patients fulfilled the criteria for low CRP. The low CRP group ( group 1) showed significant differences for disease site (p < 0.01) and for BMI (p = 0.006) compared to the raised CRP group ( group 2). Specifically, group 1 had a predominance of pure ileal disease (95% versus 53%) and lack of pure colonic disease (0% versus 24%) compared to group 2, and their BMI was significantly lower (20.3 kg/m(2) versus 25.0 kg/m(2)). Groups 1 and 2 did not differ with respect to Vienna behaviour at diagnosis, smoking, appendicectomy, extra-intestinal manifestations of CD, or NOD2 SNP variants. There was a trend for low CRP patients with previous ileal resection to evolve to a stricturing phenotype. Fat wrapping was noted in 11/13 (85%) of low CRP patients undergoing ileal resections. Conclusions. Patients with CD and a persistently low CRP in the face of active disease were characterized by an almost exclusive ileal disease distribution and a low BMI, compared to those with a raised CRP. These patients had a similar frequency and distribution of NOD2/CARD15 variants. Stricturing ( v inflammatory or penetrating) behaviour may explain some low CRP. Despite the abnormally low BMI, fat wrapping was noted in the majority of low CRP patients undergoing ileal resection.

Cross-sectional and prospective relationships of interleukin-6 and C-reactive protein with physical performance in elderly persons: MacArthur Studies of Successful aging

Although IL-6 has been shown to predict onset of disability in older persons and both IL-6 and CRP are associated with motality risk, these markers of inflammation have only limited associations with physical performance, except for walking measures and grip strength at baseline, and do not predict change in performance 7 years later in a high-functioning subset of older adults.