EPHB4 kinase-inactivating mutations cause autosomal dominant lymphatic-related hydrops fetalis.

Hydrops fetalis describes fluid accumulation in at least 2 fetal compartments, including abdominal cavities, pleura, and pericardium, or in body tissue. The majority of hydrops fetalis cases are nonimmune conditions that present with generalized edema of the fetus, and approximately 15% of these nonimmune cases result from a lymphatic abnormality. Here, we have identified an autosomal dominant, inherited form of lymphatic-related (nonimmune) hydrops fetalis (LRHF). Independent exome sequencing projects on 2 families with a history of in utero and neonatal deaths associated with nonimmune hydrops fetalis uncovered 2 heterozygous missense variants in the gene encoding Eph receptor B4 (EPHB4). Biochemical analysis determined that the mutant EPHB4 proteins are devoid of tyrosine kinase activity, indicating that loss of EPHB4 signaling contributes to LRHF pathogenesis. Further, inactivation of Ephb4 in lymphatic endothelial cells of developing mouse embryos led to defective lymphovenous valve formation and consequent subcutaneous edema. Together, these findings identify EPHB4 as a critical regulator of early lymphatic vascular development and demonstrate that mutations in the gene can cause an autosomal dominant form of LRHF that is associated with a high mortality rate

Lipid derivatives activate GPR119 and trigger GLP-1 secretion in primary murine L-cells.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from proglucagon, which is released from intestinal L-cells and increases insulin secretion in a glucose dependent manner. GPR119 is a lipid derivative receptor present in L-cells, believed to play a role in the detection of dietary fat. This study aimed to characterize the responses of primary murine L-cells to GPR119 agonism and assess the importance of GPR119 for the detection of ingested lipid. METHODS: GLP-1 secretion was measured from murine primary cell cultures stimulated with a panel of GPR119 ligands. Plasma GLP-1 levels were measured in mice lacking GPR119 in proglucagon-expressing cells and controls after lipid gavage. Intracellular cAMP responses to GPR119 agonists were measured in single primary L-cells using transgenic mice expressing a cAMP FRET sensor driven by the proglucagon promoter. RESULTS: L-cell specific knockout of GPR119 dramatically decreased plasma GLP-1 levels after a lipid gavage. GPR119 ligands triggered GLP-1 secretion in a GPR119 dependent manner in primary epithelial cultures from the colon, but were less effective in the upper small intestine. GPR119 agonists elevated cAMP in ∼70% of colonic L-cells and 50% of small intestinal L-cells. CONCLUSIONS/INTERPRETATION: GPR119 ligands strongly enhanced GLP-1 release from colonic cultures, reflecting the high proportion of colonic L-cells that exhibited cAMP responses to GPR119 agonists. Less GPR119-dependence could be demonstrated in the upper small intestine. In vivo, GPR119 in L-cells plays a key role in oral lipid-triggered GLP-1 secretion.This work was funded by grants from the Wellcome Trust (WT088357/Z/09/Z and WT084210/Z/07/Z), the MRC Metabolic Diseases Unit (MRC_MC_UU_12012/3), Full4Health (FP7/2011-2015, grant agreement n° 266408) and a BBSRC/AstraZeneca CASE studentship to CEM.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.peptides.2015.06.01

Plasma analyses.

<p>Values are presented as group mean ± SEM. WT mice n = 8, <i>Fxr</i> KO mice n = 8. Statistical analysis performed by Student's t-test.</p>*<p>p<0.05;</p>**<p>p<0.01;</p>***<p>p<0.001 <i>Fxr</i> KO vs. WT mice.</p><p>NEFA; Non-Esterified Fatty Acids, ALT; alanine aminotransferase, BA; bile acids.</p

Tissue weight and triglyceride content.

<p>(A) Weight of liver, liver triglyceride (TG) content and weight of the gall bladder in WT (<i>n</i> = 8, black bars) and <i>Fxr</i> KO mice (<i>n</i> = 8, grey bars). Statistical analysis was performed using Student's T-test. ** p<0.01 <i>Fxr</i> KO vs. WT mice. (B) Representative slides of livers from WT (<i>n</i> = 8) and <i>Fxr</i> KO mice (<i>n</i> = 8) as indicated. The arrows indicate perisinusoidal/sinusoidal foam cells (FC), lobular inflammation (LI) and ballooning degeneration (BD).</p

Sequences of primers & probes.

<p>Ppara; peroxisome proliferator activated receptor alpha;</p><p>Acox1; acyl-Coenzyme A oxidase 1, palmitoyl;</p><p>Acadm; acyl-Coenzyme A dehydrogenase, medium chain;</p><p>Cyp7a1; cytochrome P450, family 7, subfamily a, polypeptide 1;</p><p>Cyp8b1; cytochrome P450, family 8, subfamily b, polypeptide 1;</p><p>Abcb11; ATP-binding cassette, sub-family B (MDR/TAP), member 11;</p><p>Srepb1c; sterol regulatory element binding protein 1c;</p><p>Fasn; fatty acid synthase;</p><p>Scd1; stearoyl CoA desaturase 1;</p><p>Hmox1; heme oxygenase 1;</p><p>Tnfa; tumor necrosis factor alpha;</p><p>Itgax; cd11c or Itgax integrin alpha x;</p><p>Ccl5; rantes;</p><p>Tlr4; toll-like receptor 4.</p

Body weight development.

<p>Body weight development over 39 weeks in WT (<i>n</i> = 8, black solid line) and <i>Fxr</i> KO mice (<i>n</i> = 8, grey dashed line). Statistical analysis was done by a repeated Student' T-test. * p<0.05 <i>Fxr</i> KO vs. WT mice.</p

Absolute and relative (Rel.) tissue weights.

<p>Values are presented as group mean ± SEM. WT mice n = 8, <i>Fxr</i> KO mice n = 8. Statistical analysis performed by Student's t-test.</p>*<p>p<0.05;</p>**<p>p<0.01;</p>***<p>p<0.001 <i>Fxr</i> KO vs. WT mice.</p><p>WAT; white adipose tissue, Epi; Epididymal, Retro; retroperitoneal, BAT; brown adipose tissue, bw; body weight, Rel.; relative.</p

Body composition at 30 weeks of age.

<p>Body composition assessed by DEXA. Values are presented as group mean ± SEM. Body weight at assessment for WT mice (n = 8): 36.1±1.5 g, <i>Fxr</i> KO mice (n = 8): 28.2±1.4 g. Statistical analysis performed by Student's t-test.</p>*<p>p<0.05;</p>***<p>p<0.001 <i>Fxr</i> KO vs. WT mice.</p><p>bw; body weight, bl; body length.</p

Adipose tissue cell size, inflammation and density assessment.

<p>(A) Representative slides of basic fuchsin stained WAT from WT (<i>n</i> = 8) and <i>Fxr</i> KO mice (<i>n</i> = 8) as indicated. (B) Representative slides of WAT stained for Mac2 (Macrophage 2 antigen, Galectin-3) from WT (<i>n</i> = 8) and <i>Fxr</i> KO mice (<i>n</i> = 8) as indicated. (C) Representative slides of hematoxylin- eosin stained BAT from WT (<i>n</i> = 8) and <i>Fxr</i> KO mice (<i>n</i> = 8) as indicated. Statistical analysis was performed using Student's T-test. ** p<0.01 <i>Fxr</i> KO vs. WT mice.</p

Indirect calorimetry assessment.

<p>(A) Energy expenditure relative to body weight assessed in kilocalories per hour and kilogram body weight (kcal/Hr/kg) and (B) energy expenditure relative to lean body mass (lbm) in WT (<i>n</i> = 8, black solid line) and <i>Fxr</i> KO mice (<i>n</i> = 8, grey dashed line). Black bars at the X-axis represent light off. Statistical analysis was performed using a 2 way ANOVA mixed model.</p