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David R Powell,1 Deon D Doree,1 Christopher M DaCosta,1 Kenneth A Platt,2 Robert Brommage,1 Lindsey Buhring,1 Jean-Pierre Revelli,1 Melanie K Shadoan1 1Department of Pharmaceutical Biology, Lexicon Pharmaceuticals, Inc, The Woodlands, TX, USA; 2Department of Molecular Biology, Lexicon Pharmaceuticals, Inc., The Woodlands, TX, USACorrespondence: David R PowellDepartment of Pharmaceutical Biology, Lexicon Pharmaceuticals, Inc., 2445 Technology Forest Boulevard, The Woodlands, TX, 77381, USATel +1 713 249 3972Fax +1 281 863 8115Email dpowell@lexpharma.comPurpose: Humans with haploinsufficiency of GPR75, an orphan GPCR, are thin. Gpr75 knockout (KO) mice are also thin with improved glucose homeostasis. We wanted to confirm these findings in Gpr75 KO mice and determine whether decreased energy intake and/or increased energy expenditure contributed to the thin phenotype.Methods: Gpr75 KO mice were generated by homologous recombination. All studies compared female and male Gpr75 KO mice to their wild type (WT) littermates. Body composition was measured by DXA and QMR technologies. Glucose homeostasis was evaluated by measuring glucose and insulin levels during oral glucose tolerance tests (OGTTs). Food intake was measured in group-housed mice. In singly housed mice, energy expenditure was measured in Oxymax indirect calorimetry chambers, and locomotor activity was measured in Oxymax and Photobeam Activity System chambers.Results: In all 12 cohorts of adult female or male mice, Gpr75 KO mice had less body fat; pooled data showed that, compared to WT littermates (n = 103), Gpr75 KO mice (n = 118) had 49% less body fat and 4% less LBM (P < 0.001 for each). KO mice also had 8% less body fat at weaning (P < 0.05), and during the month after weaning as the thin phenotype became more exaggerated, Gpr75 KO mice ate significantly less than, but had energy expenditure and activity levels comparable to, their WT littermates. During OGTTs, Gpr75 KO mice showed improved glucose tolerance (glucose AUC 23% lower in females, P < 0.05, and 26% lower in males, P < 0.001), accompanied by significantly decreased insulin levels and significantly increased insulin sensitivity indices.Conclusion: Gpr75 KO mice are thin at weaning, are hypophagic as the thin phenotype becomes more exaggerated, and exhibit improved glucose tolerance and insulin sensitivity as healthy-appearing adults. These results suggest that inhibiting GPR75 in obese humans may safely decrease energy intake and body fat while improving glucose tolerance and insulin sensitivity.Keywords: obesity, diabetes, druggable, insulin sensitivity, healthy thinness

David R Powell,1 Jean-Pierre Revelli,1 Deon D Doree,1 Christopher M DaCosta,1 Urvi Desai,1 Melanie K Shadoan,1 Lawrence Rodriguez,2 Michael Mullens,2 Qi M Yang,1 Zhi-Ming Ding,1 Laura L Kirkpatrick,3 Peter Vogel,1 Brian Zambrowicz,1– 3 Arthur T Sands,1– 3 Kenneth A Platt,3 Gwenn M Hansen,3 Robert Brommage1 1Department of Pharmaceutical Biology, Lexicon Pharmaceuticals, Inc, The Woodlands, TX, USA; 2Department of Information Technology, Lexicon Pharmaceuticals, Inc, The Woodlands, Tx, USA; 3Department of Molecular Biology, Lexicon Pharmaceuticals, Inc, The Woodlands, Tx, USACorrespondence: David R PowellDepartment of Pharmaceutical Biology, Lexicon Pharmaceuticals, Inc, 2445 Technology Forest Boulevard, The Woodlands, Tx, 77381, USATel +1 713 249 3972Fax +1 281 863 8115Email dpowell@lexpharma.comPurpose: Obesity is a major public health problem. Understanding which genes contribute to obesity may better predict individual risk and allow development of new therapies. Because obesity of a mouse gene knockout (KO) line predicts an association of the orthologous human gene with obesity, we reviewed data from the Lexicon Genome5000TM high throughput phenotypic screen (HTS) of mouse gene KOs to identify KO lines with high body fat.Materials and Methods: KO lines were generated using homologous recombination or gene trapping technologies. HTS body composition analyses were performed on adult wild-type and homozygous KO littermate mice from 3758 druggable mouse genes having a human ortholog. Body composition was measured by either DXA or QMR on chow-fed cohorts from all 3758 KO lines and was measured by QMR on independent high fat diet-fed cohorts from 2488 of these KO lines. Where possible, comparisons were made to HTS data from the International Mouse Phenotyping Consortium (IMPC).Results: Body fat data are presented for 75 KO lines. Of 46 KO lines where independent external published and/or IMPC KO lines are reported as obese, 43 had increased body fat. For the remaining 29 novel high body fat KO lines, Ksr2 and G2e3 are supported by data from additional independent KO cohorts, 6 (Asnsd1, Srpk2, Dpp8, Cxxc4, Tenm3 and Kiss1) are supported by data from additional internal cohorts, and the remaining 21 including Tle4, Ak5, Ntm, Tusc3, Ankk1, Mfap3l, Prok2 and Prokr2 were studied with HTS cohorts only.Conclusion: These data support the finding of high body fat in 43 independent external published and/or IMPC KO lines. A novel obese phenotype was identified in 29 additional KO lines, with 27 still lacking the external confirmation now provided for Ksr2 and G2e3 KO mice. Undoubtedly, many mammalian obesity genes remain to be identified and characterized.Keywords: obesity, druggable, homologous recombination, gene trapping

David R Powell, Deon D Doree, Christopher M DaCosta, Kenneth A Platt, Gwenn M Hansen, Isaac van Sligtenhorst, Zhi-Ming Ding, Jean-Pierre Revelli, Robert Brommage Lexicon Pharmaceuticals Inc, The Woodlands, TX, 77381, USACorrespondence: David R PowellLexicon Pharmaceuticals Inc., 8800 Technology Forest Place, The Woodlands, TX 77381, USATel +1 281 863 3060Fax +1 281 863 8115Email dpowell@lexpharma.comPurpose: In humans, single nucleotide polymorphisms (SNPs) near the adjacent protein kinase D1 (PRKD1) and G2/M-phase-specific E3 ubiquitin protein ligase (G2E3) genes on chromosome 14 are associated with obesity. To date, no published evidence links inactivation of either gene to changes in body fat. These two genes are also adjacent on mouse chromosome 12. Because obesity genes are highly conserved between humans and mice, we analyzed body fat in adult G2e3 and Prkd1 knockout (KO) mice to determine whether inactivating either gene leads to obesity in mice and, by inference, probably in humans.Methods: The G2e3 and Prkd1 KO lines were generated by gene trapping and by homologous recombination methodologies, respectively. Body fat was measured by DEXA in adult mice fed chow from weaning and by QMR in a separate cohort of mice fed high-fat diet (HFD) from weaning. Glucose homeostasis was evaluated with oral glucose tolerance tests (OGTTs) performed on adult mice fed HFD from weaning.Results: Body fat was increased in multiple cohorts of G2e3 KO mice relative to their wild-type (WT) littermates. When data from all G2e3 KO (n=32) and WT (n=31) mice were compared, KO mice showed increases of 11% in body weight (P< 0.01), 65% in body fat (P< 0.001), 48% in % body fat (P< 0.001), and an insignificant 3% decrease in lean body mass. G2e3 KO mice were also glucose intolerant during an OGTT (P< 0.05). In contrast, Prkd1 KO and WT mice had comparable body fat levels and glucose tolerance.Conclusion: Significant obesity and glucose intolerance were observed in G2e3, but not Prkd1, KO mice. The conservation of obesity genes between mice and humans strongly suggests that the obesity-associated SNPs located near the human G2E3 and PRKD1 genes are linked to variants that decrease the amount of functional human G2E3.Keywords: glucose tolerance, gene trap, homologous recombination, Prkd1, PRKD1, SNP

Introduction: Contrast computer tomography (CT) scanning is the investigation of choice for the further assessment of suspected cystic congenital lung lesions (CCLL). Its use to identify the presence of anomalous feeding vessels supplying the lesion is well documented, but data regarding its accuracy is limited. This study compares CT results to operative and pathological findings to determine the accuracy of CT in identifying these anomalous vessels. Methods: 51 consecutive cases of cystic congenital lung lesions managed in one hospital by a single consultant were reviewed. All cases had contrast CT scans performed preoperatively, as standard practice in this institution. We compared the results of these CT scans to the macroscopic appearance at surgery and histological findings postoperatively. We also compared the results of 2 CT protocols used in our institution between 1999-2007 and 2007-2009, respectively. Results: Anomalous vessels were reported on CT in 9 cases. All but 1 had concordant operative and pathological findings. In the remaining 42 cases, no anomalous vessels were seen on CT. Of these, 9 cases were found to have an anomalous blood supply at surgery, 6 of which were hybrid lesions and 3 isolated sequestrations. The specificity of CT in identifying feeding vessels in the study was 97% (95% CI: 0.83-0.99) and the sensitivity was 47% (95% CI: 0.23-0.71). The positive predictive value was 89% (95% CI: 0.50-0.99) and negative predictive value 79% (95% CI: 0.62-0.89). The most recent protocol yielded an improved sensitivity of 75% (95% CI: 0.22-0.98) and a specificity of 100% (95% CI: 0.46-1.0) with a 100% (95% CI: 0.31-1.0) positive and 83% (95% CI: 0.36-0.99) negative predictive value. Conclusion: CT is a specific investigation for identifying anomalous vessels in CCLL but lacks sensitivity, leading to a relatively low negative predictive value. This emphasises the need in every case to look for anomalous vessels at surgery to avoid morbidity and potential mortality. An improved protocol for CT scans leads to improved specificity and sensitivity and predictive values. [ABSTRACT FROM AUTHOR]

GREMLIN 2 (GREM2)—formerly, protein related to Dan and cerberus (PRDC)—is a potent antagonist of the bone morphogenetic proteins 2 and 4, but little else in known about its functions. We found that Grem2-/- mice developed small deformed mandibular and maxillary incisors, indicating that GREMLIN2 is required for normal tooth morphogenesis. Although DEXA scans suggested that bone mineral density might be increased in Grem2-/- mice, histology did not reveal any evident bone phenotype. Grem2-/- mice did not display any other notable phenotypes evaluated in a high-throughput screening process that encompassed a range of immunologic, metabolic, ophthalmic, and behavioral parameters. Our findings indicate that Grem2 can be added to the growing list of genes that affect tooth development in mice. [ABSTRACT FROM PUBLISHER]

Maternal high-fat diet consumption and obesity have been shown to program long-term obesity and lead to impaired glucose tolerance in offspring. Many rodent studies, however, use non-purified, cereal-based diets as the control for purified high-fat diets. In this study, primiparous ICR mice were fed purified control diet (10–11 kcal% from fat of lard or butter origin) and lard (45 or 60 kcal% fat) or butter (32 or 60 kcal% fat)-based high-fat diets for 4 weeks before mating, throughout pregnancy, and for 2 weeks of nursing. Before mating, female mice fed the 32 and 60% butter-based high-fat diets exhibited impaired glucose tolerance but those females fed the lard-based diets showed normal glucose disposal following a glucose challenge. High-fat diet consumption by female mice of all groups decreased lean to fat mass ratios during the 4th week of diet treatment compared with those mice consuming the 10–11% fat diets. All females were bred to male mice and pregnancy and offspring outcomes were monitored. The body weight of pups born to 45% lard-fed dams was significantly increased before weaning, but only female offspring born to 32% butter-fed dams exhibited long-term body weight increases. Offspring glucose tolerance and body composition were measured for at least 1 year. Minimal, if any, differences were observed in the offspring parameters. These results suggest that many variables should be considered when designing future high-fat diet feeding and maternal obesity studies in mice. [ABSTRACT FROM PUBLISHER]

The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepalocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethylsulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and before cryopreservation, and (5) removal of unvital hepatocytes by Percoll centrifugation after thawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryopreserved by our standard procedure have a viability ≥ 80%. Metabolic capacity of cryopreserved hepatocytes determined by testosterone hydroxylation, 7-ethoxyresorufin-O-de-ethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transferase, sulfotransferase, and epoxide hydrolase activities is ≥60% of freshly isolated cells. Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the complex pattern of benzo[a]pyrene metabolites including phase II metabolites formed by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryopreserved hepatocyte cultures are required. We present a technique with cryopreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme activities of induced hepatocytes and solvent controls were smaller in the cryopreserved cells. In conclusion,... [ABSTRACT FROM AUTHOR]

David R Powell, Jason P Gay, Melinda Smith, Nathaniel Wilganowski, Angela Harris, Autumn Holland, Maricela Reyes, Laura Kirkham, Laura L Kirkpatrick, Brian Zambrowicz, Gwenn Hansen, Kenneth A Platt, Isaac van Sligtenhorst, Zhi-Ming Ding, Urvi Desai Metabolism Research, Lexicon Pharmaceuticals, Inc., The Woodlands, TX, USA Abstract: Delta-5 desaturase (D5D) and delta-6 desaturase (D6D), encoded by fatty acid desaturase 1 (FADS1) and FADS2 genes, respectively, are enzymes in the synthetic pathways for w3, w6, and w9 polyunsaturated fatty acids (PUFAs). Although PUFAs appear to be involved in mammalian metabolic pathways, the physiologic effect of isolated D5D deficiency on these pathways is unclear. After generating >4,650 knockouts (KOs) of independent mouse genes and analyzing them in our high-throughput phenotypic screen, we found that Fads1 KO mice were among the leanest of 3,651 chow-fed KO lines analyzed for body composition and were among the most glucose tolerant of 2,489 high-fat-diet-fed KO lines analyzed by oral glucose tolerance test. In confirmatory studies, chow- or high-fat-diet-fed Fads1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 38% and 31% lower in Fads1 male and female KO mice, respectively. Fads1 KO mice also had lower glucose and insulin excursions during oral glucose tolerance tests along with lower fasting glucose, insulin, triglyceride, and total cholesterol levels. In additional studies using a vascular injury model, Fads1 KO mice had significantly decreased femoral artery intima/media ratios consistent with a decreased inflammatory response in their arterial wall. Based on this result, we bred Fads1 KO and WT mice onto an ApoE KO background and fed them a Western diet for 14 weeks; in this atherogenic environment, aortic trees of Fads1 KO mice had 40% less atheromatous plaque compared to WT littermates. Importantly, PUFA levels measured in brain and liver phospholipid fractions of Fads1 KO mice were consistent with decreased D5D activity and normal D6D activity. The beneficial metabolic phenotype demonstrated in Fads1 KO mice suggests that selective D5D inhibitors may be useful in the treatment of human obesity, diabetes, and atherosclerotic cardiovascular disease. Keywords: delta-5 desaturase, delta-6 desaturase, obesity, diabetes, atherosclerosis

The development of oil bodies and oil droplets in fruits of olive was examined at the ultra structural level. Both oil bodies that form in young fruits and oil droplets that develop with fruit maturation are cytoplasmic bodies. The formation of the small oil bodies occurs in localized regions of the cytoplasm. These bodies are closely associated and fuse together, forming a small oil droplet that protruded against and indented the vacuolar membrane. As the fruit matures, new oil bodies appear to form and fuse with the oil droplet, resulting in the formation of a single large oil droplet of about 30 μm in diameter in most mature mesocarp cells. The cytoplasmic region where the oil bodies formed had a granulate, ultrastructural appearance, and cytoplasmic components such as membranes and ribosomes were noticeably absent in these regions. The granulate material coated the oil bodies and oil droplets, and appeared as a thin, compressed band between the round inner surface of the droplets and the indented tonoplast. We suggest that this granulate material is involved in the synthesis of the oil and, with enlargement of the oil bodies, this coat becomes thinner in regions where they are closely associated, resulting in zones where confluence of the oil occurs. [ABSTRACT FROM AUTHOR]

Leaves of desiccated ‘resurrection plants’,Selaginella lepidophylla, were hydrated either through the roots of intact plants or as isolated organs. Air-dry tissue and samples at 1, 4, 8 and 24 h (both detached and intact) of hydration were prepared for electron microscopy using aldehyde fixatives of different osmotic strengths. Both dry and hydrated tissues were also prepared using freeze substitution. Significant differences in the ultrastructural preservation of these different samples were noted. There was a direct correlation between the osmolality of both the fixative and the tissue with the quality of ultrastructural preservation. When the osmolality of the fixative was slightly (or even considerably) higher than that of the tissue, optimal preservation was achieved. Freeze substitution, however, gave the most faithful preservation of all subcellular compartments, despite the frequent presence of small ice crystals. Additionally, hydration of detached leaves for more than 4 h resulted in swelling damage of the organelles and cytoplasm, regardless of the fixation protocol.Broadly interpreted, the results of this study indicate that an optimal preservation of plant cell and organelle ultrastructure can be achieved by the use of high osmolality fixatives or, preferably, freeze substitution. These results are also important in determining the method of hydration of poikilohydric samples for physiological studies and for interpretation of functional changes as related to the structural condition of the organelles.Copyright 1997 Annals of Botany Company [ABSTRACT FROM AUTHOR]

Dry (7-10% water content) leaves of the spikemoss Selaginella lepidophylla ('resurrection plant') and of the desiccationtolerant moss, Tortula ruralis were examined by freeze fracture electron microscopy. As has been described for dry seeds, the cells of these dehydrated leaves were shrunken, with highly convoluted walls and membranes. The membranes of all samples had a lipid bilayer organization with dispersed intramembranous particles (IMPs). Lipid droplets were very closely associated with the plasmamembrane. Chloroplasts were surrounded by a double membrane envelope and contained well-organized grana. Mitochondria were irregular in outline, and endoplasmic reticulum and cytoplasmic vesicles were present. [ABSTRACT FROM AUTHOR]

. The dissemination of dengue (DEN) 3 virus in parenterally infected female Aedes aegypti mosquitoes was studied imrnunocytochemically. Antigen was first detected in fat body cells near the thoracic site of virus inoculation. The intussuscepted foregut, salivary glands and nervous tissue were the first major tissues infected. Nervous tissue appeared to be the primary site of amplification. Muscles, tracheae, Malphigian tubules and the posterior midgut did not become infected. The only part of the reproductive system to be infected was the calyx (71% of specimens 16-22 days post-infection) consistent with low rates of vertical transmission. After 7 days post-inoculation the salivary glands of 100% of the specimens examined were infected. Virus dissemination was slow and the most common sequence of infection following intrathoracic inoculation was as follows: thoracic fat body, intussuscepted foregut, salivary glands, cardial epithelium, thoracic ganglion, brain, compound eye, anterior midgut, intermediate midgut/anterior abdominal ganglia, and calyx/hindgut/posterior abdominal ganglia. Fat body and intussuscepted foregut tissues lost infections after 16 days post-inoculation. [ABSTRACT FROM AUTHOR]