Mice Lacking Mrs2 Magnesium Transporter are Hypophagic and Thin When Maintained on a High-Fat Diet.

Genes regulating body fat are shared with high fidelity by mice and humans, indicating that mouse knockout (KO) phenotyping might identify valuable antiobesity drug targets. Male Mrs2 magnesium transporter (Mrs2) KO mice were recently reported as thin when fed a high-fat diet (HFD). They also exhibited increased energy expenditure (EE)/body weight and had beiged adipocytes that, along with isolated hepatocytes, demonstrated increased oxygen consumption, suggesting that increased EE drove the thin phenotype. Here we provide our data on these and additional assays in Mrs2 KO mice. We generated Mrs2 KO mice by homologous recombination. HFD-fed male and female Mrs2 KO mice had significantly less body fat, measured by quantitative magnetic resonance, than wild-type (WT) littermates. HFD-fed Mrs2 KO mice did not demonstrate increased EE by indirect calorimetry and could not maintain body temperature at 4 °C, consistent with their decreased brown adipose tissue stores but despite increased beige white adipose tissue. Instead, when provided a choice between HFD and low-fat diet (LFD), Mrs2 KO mice showed a significant 15% decrease in total energy intake resulting from significantly lower HFD intake that offset numerically increased LFD intake. Food restriction studies performed using WT mice suggested that this decrease in energy intake could explain the loss of body fat. Oral glucose tolerance test studies revealed significantly improved insulin sensitivity in Mrs2 KO mice. We conclude that HFD-fed Mrs2 KO mice are thin with improved insulin sensitivity, and that this favorable metabolic phenotype is driven by hypophagia. Further evaluation is warranted to determine the suitability of MRS2 as a drug target for antiobesity therapeutics.

Mice Lacking Gpr75 are Hypophagic and Thin.

PURPOSE: 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.

High-Throughput Screening of Mouse Gene Knockouts Identifies Established and Novel High Body Fat Phenotypes.

PURPOSE: 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.

Obesity of G2e3 Knockout Mice Suggests That Obesity-Associated Variants Near Human G2E3 Decrease G2E3 Activity.

PURPOSE: 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.

Physiological Predictors of Performance on the CrossFit "Murph" Challenge.

We examined physiological predictors of performance on the CrossFit Murph challenge (1-mile run, 100 pullups, 200 pushups, 300 air squats, 1-mile run). Male CrossFit athletes (n = 11, 27 ± 3 years) performed a battery of physical assessments including: (1) body composition, (2) upper and lower body strength, (3) upper body endurance, (4) anaerobic power, and (5) maximal oxygen consumption. No less than 72 h later, participants completed the Murph challenge, heart rate was monitored throughout, and blood lactate was obtained pre-post. Correlations between physiological parameters and total Murph time, and Murph subcomponents, were assessed using Pearson's correlations. Murph completion time was 43.43 ± 4.63 min, and maximum and average heart rate values were 185.63 ± 7.64 bpm and 168.81 ± 6.41 bpm, respectively, and post-Murph blood lactate was 10.01 ± 3.04 mmol/L. Body fat percentage was the only physiological parameter significantly related to total Murph time (r = 0.718; p = 0.013). Total lift time (25.49 ± 3.65 min) was more strongly related (r = 0.88) to Murph time than total run time (17.60 ± 1.97 min; r = 0.65). Greater relative anaerobic power (r = -0.634) and less anaerobic fatigue (r = 0.649) were related to total run time (p < 0.05). Individuals wanting to enhance overall Murph performance are advised to focus on minimizing body fat percentage and improving lift performance. Meanwhile, performance on the run subcomponent may be optimized through improvements in anaerobic power.

NOTUM inhibition increases endocortical bone formation and bone strength.

The disability, mortality and costs caused by non-vertebral osteoporotic fractures are enormous. Existing osteoporosis therapies are highly effective at reducing vertebral but not non-vertebral fractures. Cortical bone is a major determinant of non-vertebral bone strength. To identify novel osteoporosis drug targets, we phenotyped cortical bone of 3 366 viable mouse strains with global knockouts of druggable genes. Cortical bone thickness was substantially elevated in Notum -/- mice. NOTUM is a secreted WNT lipase and we observed high NOTUM expression in cortical bone and osteoblasts but not osteoclasts. Three orally active small molecules and a neutralizing antibody inhibiting NOTUM lipase activity were developed. They increased cortical bone thickness and strength at multiple skeletal sites in both gonadal intact and ovariectomized rodents by stimulating endocortical bone formation. Thus, inhibition of NOTUM activity is a potential novel anabolic therapy for strengthening cortical bone and preventing non-vertebral fractures.

LX2761, a Sodium/Glucose Cotransporter 1 Inhibitor Restricted to the Intestine, Improves Glycemic Control in Mice.

LX2761 is a potent sodium/glucose cotransporter 1 inhibitor restricted to the intestinal lumen after oral administration. Studies presented here evaluated the effect of orally administered LX2761 on glycemic control in preclinical models. In healthy mice and rats treated with LX2761, blood glucose excursions were lower and plasma total glucagon-like peptide-1 (GLP-1) levels higher after an oral glucose challenge; these decreased glucose excursions persisted even when the glucose challenge occurred 15 hours after LX2761 dosing in ad lib-fed mice. Further, treating mice with LX2761 and the dipeptidyl-peptidase 4 inhibitor sitagliptin synergistically increased active GLP-1 levels, suggesting increased LX2761-mediated release of GLP-1 into the portal circulation. LX2761 also lowered postprandial glucose, fasting glucose, and hemoglobin A1C, and increased plasma total GLP-1, during long-term treatment of mice with either early- or late-onset streptozotocin-diabetes; in the late-onset cohort, LX2761 treatment improved survival. Mice and rats treated with LX2761 occasionally had diarrhea; this dose-dependent side effect decreased in severity and frequency over time, and LX2761 doses were identified that decreased postprandial glucose excursions without causing diarrhea. Further, the frequency of LX2761-associated diarrhea was greatly decreased in mice either by gradual dose escalation or by pretreatment with resistant starch 4, which is slowly digested to glucose in the colon, a process that primes the colon for glucose metabolism by selecting for glucose-fermenting bacterial species. These data suggest that clinical trials are warranted to determine if LX2761 doses and dosing strategies exist that provide improved glycemic control combined with adequate gastrointestinal tolerability in people living with diabetes.

LP-925219 maximizes urinary glucose excretion in mice by inhibiting both renal SGLT1 and SGLT2.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of oral anti-diabetic agents that improve glycemic control by inhibiting SGLT2-mediated renal glucose reabsorption. Currently available agents increase urinary glucose excretion (UGE) to <50% of maximal values because they do not inhibit SGLT1, which reabsorbs >50% of filtered glucose when SGLT2 is completely inhibited. This led us to test whether LP-925219, a small molecule dual SGLT1/SGLT2 inhibitor, increases UGE to maximal values in wild-type (WT) mice. We first tested LP-925219 inhibition of glucose transport by HEK293 cells expressing SGLT1 or SGLT2, and then characterized LP-925219 pharmacokinetics. We found that LP-925219 was a potent inhibitor of mouse SGLT1 (IC50 = 22.6 nmol/L) and SGLT2 (IC50 = 0.5 nmol/L), and that a 10 mg/kg oral dose was bioavailable (87%) with a long half-life (7 h). We next delivered LP-925219 by oral gavage to WT, SGLT1 knockout (KO), SGLT2 KO, and SGLT1/SGLT2 double KO (DKO) mice and measured their 24-h UGE. We found that, in vehicle-treated mice, DKO UGE was maximal and SGLT2 KO, SGLT1 KO, and WT UGEs were 30%, 2%, and 0.2% of maximal, respectively; we also found that LP-925219 dosed at 60 mg/kg twice daily increased UGE of SGLT1 KO, SGLT2 KO, and WT mice to DKO UGE levels. These findings show that orally available dual SGLT1/SGLT2 inhibitors can maximize 24-h UGE in mammals, and suggest that such agents merit further evaluation for their potential, in diabetic patients, to achieve better glycemic control than is achieved using selective SGLT2 inhibitors.

New DArT markers for oat provide enhanced map coverage and global germplasm characterization.

BACKGROUND: Genomic discovery in oat and its application to oat improvement have been hindered by a lack of genetic markers common to different genetic maps, and by the difficulty of conducting whole-genome analysis using high-throughput markers. This study was intended to develop, characterize, and apply a large set of oat genetic markers based on Diversity Array Technology (DArT). RESULTS: Approximately 19,000 genomic clones were isolated from complexity-reduced genomic representations of pooled DNA samples from 60 oat varieties of global origin. These were screened on three discovery arrays, with more than 2000 polymorphic markers being identified for use in this study, and approximately 2700 potentially polymorphic markers being identified for use in future studies. DNA sequence was obtained for 2573 clones and assembled into a non-redundant set of 1770 contigs and singletons. Of these, 705 showed highly significant (Expectation < 10E-10) BLAST similarity to gene sequences in public databases. Based on marker scores in 80 recombinant inbred lines, 1010 new DArT markers were used to saturate and improve the 'Kanota' x 'Ogle' genetic map. DArT markers provided map coverage approximately equivalent to existing markers. After binning markers from similar clones, as well as those with 99% scoring similarity, a set of 1295 non-redundant markers was used to analyze genetic diversity in 182 accessions of cultivated oat of worldwide origin. Results of this analysis confirmed that major clusters of oat diversity are related to spring vs. winter type, and to the presence of major breeding programs within geographical regions. Secondary clusters revealed groups that were often related to known pedigree structure. CONCLUSION: These markers will provide a solid basis for future efforts in genomic discovery, comparative mapping, and the generation of an oat consensus map. They will also provide new opportunities for directed breeding of superior oat varieties, and guidance in the maintenance of oat genetic diversity.

High-throughput screening of mouse knockout lines identifies true lean and obese phenotypes.

We developed a high-throughput approach to knockout (KO) and phenotype mouse orthologs of the 5,000 potential drug targets in the human genome. As part of the phenotypic screen, dual-energy X-ray absorptiometry (DXA) technology estimates body-fat stores in eight KO and four wild-type (WT) littermate chow-fed mice from each line. Normalized % body fat (nBF) (mean KO % body fat/mean WT littermate % body fat) values from the first 2322 lines with viable KO mice at 14 weeks of age showed a normal distribution. We chose to determine how well this screen identifies body-fat phenotypes by selecting 13 of these 2322 KO lines to serve as benchmarks based on their published lean or obese phenotype on a chow diet. The nBF values for the eight benchmark KO lines with a lean phenotype were > or =1 s.d. below the mean for seven (perilipin, SCD1, CB1, MCH1R, PTP1B, GPAT1, PIP5K2B) but close to the mean for NPY Y4R. The nBF values for the five benchmark KO lines with an obese phenotype were >2 s.d. above the mean for four (MC4R, MC3R, BRS3, translin) but close to the mean for 5HT2cR. This screen also identifies novel body-fat phenotypes as exemplified by the obese kinase suppressor of ras 2 (KSR2) KO mice. These body-fat phenotypes were confirmed upon studying additional cohorts of mice for KSR2 and all 13 benchmark KO lines. This simple and cost-effective screen appears capable of identifying genes with a role in regulating mammalian body fat.

mdmx is a negative regulator of p53 activity in vivo.

Regulation of p53 protein activity is required for normal embryogenesis, tumor suppression, and cellular response to DNA damage. Here we report that loss of mdmx, a p53-binding protein, results in midgestational embryo lethality, a phenotype that is completely rescued by the absence of p53. Mice homozygous for both mdmx and p53 null mutations are viable and appear developmentally normal. Fibroblasts derived from embryos with reduced mdmx expression demonstrate a decreased growth rate and increased UV-induced apoptosis compared with wild-type cells and contain elevated levels of p53 and several p53 target proteins including the proapoptotic bax protein. These observations demonstrate that mdmx functions as a critical negative regulator of p53 in vivo.

Cross-talk between IL-1 and IL-6 signaling pathways in rheumatoid arthritis synovial fibroblasts.

The balance between pro- and anti-inflammatory cytokines plays an important role in determining the severity of inflammation in rheumatoid arthritis (RA). Antagonism between opposing cytokines at the level of signal transduction plays an important role in many other systems. We have begun to explore the possible contribution of signal transduction cross-talk to cytokine balance in RA by examining the effects of IL-1, a proinflammatory cytokine, on the signaling and action of IL-6, a pleiotropic cytokine that has both pro- and anti-inflammatory actions, in RA synovial fibroblasts. Pretreatment with IL-1 suppressed Janus kinase-STAT signaling by IL-6, modified patterns of gene activation, and blocked IL-6 induction of tissue inhibitor of metalloproteases 1 expression. These results suggest that proinflammatory cytokines may contribute to pathogenesis by modulating or blocking signal transduction by pleiotropic or anti-inflammatory cytokines. The mechanism of inhibition did not require de novo gene activation and did not depend upon tyrosine phosphatase activity, but, instead, was dependent on the p38 stress kinase. These results identify a molecular basis for IL-1 and IL-6 cross-talk in RA synoviocytes and suggest that, in addition to levels of cytokine expression, modulation of signal transduction also plays a role in regulating cytokine balance in RA.

Total synthesis of (+)-arteanniun M using the tandem oxy-Cope/ene reaction.

[see reaction]. The first total synthesis of (+)-arteannuin M was accomplished using the tandem oxy-Cope/transannular ene reaction as the key step to construct the bicyclic core of the natural product. The tandem reaction proceeded with high diastereo- and enantiomeric excess.