The Vasopressin Receptor 2 Mutant R137L Linked to the Nephrogenic Syndrome of Inappropriate Antidiuresis (NSIAD) Signals through an Alternative Pathway that Increases AQP2 Membrane Targeting Independently of S256 Phosphorylation

NSIAD is a rare X-linked condition, caused by activating mutations in the AVPR2 gene coding for the vasopressin V2 receptor (V2R) associated with hyponatremia, despite undetectable plasma vasopressin levels. We have recently provided in vitro evidence that, compared to V2R-wt, expression of activating V2R mutations R137L, R137C and F229V cause a constitutive redistribution of the AQP2 water channel to the plasma membrane, higher basal water permeability and significantly higher basal levels of p256-AQP2 in the F229V mutant but not in R137L or R137C. In this study, V2R mutations were expressed in collecting duct principal cells and the associated signalling was dissected. V2R-R137L and R137C mutants had significantly higher basal pT269-AQP2 levels -independently of S256 and PKA-which were reduced to control by treatment with Rho kinase (ROCK) inhibitor. Interestingly, ROCK activity was found significantly higher in V2R-R137L along with activation of the Gα12/13-Rho-ROCK pathway. Of note, inhibition of ROCK reduced the basal elevated osmotic water permeability to control. To conclude, our data demonstrate for the first time that the gain-of-function mutation of the V2R, R137L causing NSIAD, signals through an alternative PKA-independent pathway that increases AQP2 membrane targeting through ROCK-induced phosphorylation at S/T269 independently of S256 of AQP2

Olive Leaf Extract (OLE) impaired vasopressin-induced aquaporin-2 trafficking through the activation of the calcium-sensing receptor

Vasopressin (AVP) increases water permeability in the renal collecting duct through the regulation of aquaporin-2 (AQP2) trafficking. Several disorders, including hypertension and inappropriate antidiuretic hormone secretion (SIADH), are associated with abnormalities in water homeostasis. It has been shown that certain phytocompounds are beneficial to human health. Here, the effects of the Olive Leaf Extract (OLE) have been evaluated using in vitro and in vivo models. Confocal studies showed that OLE prevents the vasopressin induced AQP2 translocation to the plasma membrane in MCD4 cells and rat kidneys. Incubation with OLE decreases the AVP-dependent increase of the osmotic water permeability coefficient (Pf). To elucidate the possible effectors of OLE, intracellular calcium was evaluated. OLE increases the intracellular calcium through the activation of the Calcium Sensing Receptor (CaSR). NPS2143, a selective CaSR inhibitor, abolished the inhibitory effect of OLE on AVP-dependent water permeability. In vivo experiments revealed that treatment with OLE increases the expression of the CaSR mRNA and decreases AQP2 mRNA paralleled by an increase of the AQP2-targeting miRNA-137. Together, these findings suggest that OLE antagonizes vasopressin action through stimulation of the CaSR indicating that this extract may be beneficial to attenuate disorders characterized by abnormal CaSR signaling and affecting renal water reabsorption

Hypoxia and adipose tissue function and dysfunction in obesity

The rise in the incidence of obesity has led to a major interest in the biology of white adipose tissue. The tissue is a major endocrine and signalling organ, with adipocytes, the characteristic cell type, secreting a multiplicity of protein factors – the adipokines. Increases in the secretion of a number of adipokines occurs in obesity, underpinning inflammation in white adipose tissue and the development of obesity-associated diseases. There is substantial evidence, particularly from animal studies, that hypoxia develops in adipose tissue as the tissue mass expands, and the reduction in pO2 is considered to underlie the inflammatory response. Exposure of white adipocytes to hypoxic conditions in culture induces changes in the expression of >1,000 genes. The secretion of inflammation-related adipokines is up-regulated by hypoxia, and there is a switch from oxidative metabolism to anaerobic glycolysis. Glucose utilisation is increased in hypoxic adipocytes with corresponding increases in lactate production. Importantly, hypoxia induces insulin resistance in fat cells and leads to the development of adipose tissue fibrosis. Many of the responses of adipocytes to hypoxia are initiated at pO2 levels above the normal physiological range for adipose tissue. The other cell types within the tissue also respond to hypoxia, with the differentiation of preadipocytes to adipocytes being inhibited and preadipocytes being transformed into leptin-secreting cells. Overall, hypoxia has pervasive effects on the function of adipocytes and appears to be a key factor in adipose tissue dysfunction in obesity

In vitro and in vivo nutraceutical characterization of two chickpea accessions: Differential effects on hepatic lipid over-accumulation

Dietary habits are crucially important to prevent the development of lifestyle-associated diseases. Diets supplemented with chickpeas have numerous benefits and are known to improve body fat composition. The present study was undertaken to characterize two genetically and phenotypically distinct accessions, MG_13 and PI358934, selected from a global chickpea collection. Rat hepatoma FaO cells treated with a mixture of free fatty acids (FFAs) (O/P) were used as an in vitro model of hepatic steatosis. In parallel, a high-fat diet (HFD) animal model was also established. In vitro and in vivo studies revealed that both chickpea accessions showed a significant antioxidant ability. However, only MG_13 reduced the lipid over-accumulation in steatotic FaO cells and in the liver of HFD fed mice. Moreover, mice fed with HFD + MG_13 displayed a lower level of glycemia and aspartate aminotransferase (AST) than HFD mice. Interestingly, exposure to MG_13 prevented the phosphorylation of the inflammatory nuclear factor kappa beta (NF-kB) which is upregulated during HFD and known to be linked to obesity. To conclude, the comparison of the two distinct chickpea accessions revealed a beneficial effect only for the MG_13. These findings highlight the importance of studies addressing the functional characterization of chickpea biodiversity and nutraceutical properties

Curative Effect of 18β-Glycyrrhetinic Acid in Experimental Visceral Leishmaniasis Depends on Phosphatase-Dependent Modulation of Cellular MAP Kinases

We earlier showed that 18β-glycyrrhetinic acid (GRA), a pentacyclic triterpenoid from licorice root, could completely cure visceral leishmaniasis in BALB/c mouse model. This was associated with induction of nitric oxide and proinflammatory cytokine production through the up regulation of NF-κB. In the present study we tried to decipher the underlying cellular mechanisms of the curative effect of GRA. Analysis of MAP kinase pathways revealed that GRA caused strong activation of p38 and to a lesser extent, ERK in bone marrow-derived macrophages (BMDM). Almost complete abrogation of GRA-induced cytokine production in presence of specific inhibitors of p38 and ERK1/2 confirmed the involvement of these MAP kinases in GRA-mediated responses. GRA induced mitogen- and stress-activated protein kinase (MSK1) activity in a time-dependent manner suggested that GRA-mediated NF-κB transactivation is mediated by p38, ERK and MSK1 pathway. As kinase/phosphatase balance plays an important role in modulating infection, the effect of GRA on MAPK directed phosphatases (MKP) was studied. GRA markedly reduced the expression and activities of three phosphatases, MKP1, MKP3 and protein phosphatase 2A (PP2A) along with a substantial reduction of p38 and ERK dephosphorylation in infected BMDM. Similarly in the in vivo situation, GRA treatment of L. donovani-infected BALB/c mice caused marked reduction of spleen parasite burden associated with concomitant decrease of individual phosphatase levels. However, activation of kinases also played an important role as the protective effect of GRA was significantly abrogated by pharmacological inhibition of p38 and ERK pathway. Curative effect of GRA may, therefore, be associated with restoration of proper cellular kinase/phosphatase balance, rather than modulation of either kinases or phosphatases

Regulation of N-WASP and the Arp2/3 Complex by Abp1 Controls Neuronal Morphology

Polymerization and organization of actin filaments into complex superstructures is indispensable for structure and function of neuronal networks. We here report that knock down of the F-actin-binding protein Abp1, which is important for endocytosis and synaptic organization, results in changes in axon development virtually identical to Arp2/3 complex inhibition, i.e., a selective increase of axon length. Our in vitro and in vivo experiments demonstrate that Abp1 interacts directly with N-WASP, an activator of the Arp2/3 complex, and releases the autoinhibition of N-WASP in cooperation with Cdc42 and thereby promotes N-WASP-triggered Arp2/3 complex-mediated actin polymerization. In line with our mechanistical studies and the colocalization of Abp1, N-WASP and Arp2/3 at sites of actin polymerization in neurons, we reveal an essential role of Abp1 and its cooperativity with Cdc42 in N-WASP-induced rearrangements of the neuronal cytoskeleton. We furthermore show that introduction of N-WASP mutants lacking the ability to bind Abp1 or Cdc42, Arp2/3 complex inhibition, Abp1 knock down, N-WASP knock down and Arp3 knock down, all cause identical neuromorphological phenotypes. Our data thus strongly suggest that these proteins and their complex formation are important for cytoskeletal processes underlying neuronal network formation

A Feedback Loop between Dynamin and Actin Recruitment during Clathrin-Mediated Endocytosis

A live-cell imaging study reveals that a positive feedback loop between dynamin and actin contributes to efficient endocytic membrane scission

A High-Speed Congenic Strategy Using First-Wave Male Germ Cells

BACKGROUND: In laboratory mice and rats, congenic breeding is essential for analyzing the genes of interest on specific genetic backgrounds and for analyzing quantitative trait loci. However, in theory it takes about 3-4 years to achieve a strain carrying about 99% of the recipient genome at the tenth backcrossing (N10). Even with marker-assisted selection, the so-called 'speed congenic strategy', it takes more than a year at N4 or N5. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a new high-speed congenic system using round spermatids retrieved from immature males (22-25 days of age). We applied the technique to three genetically modified strains of mice: transgenic (TG), knockin (KI) and N-ethyl-N-nitrosourea (ENU)-induced mutants. The donor mice had mixed genetic backgrounds of C57BL/6 (B6):DBA/2 or B6:129 strains. At each generation, males used for backcrossing were selected based on polymorphic marker analysis and their round spermatids were injected into B6 strain oocytes. Backcrossing was repeated until N4 or N5. For the TG and ENU-mutant strains, the N5 generation was achieved on days 188 and 190 and the proportion of B6-homozygous loci was 100% (74 markers) and 97.7% (172/176 markers), respectively. For the KI strain, N4 was achieved on day 151, all the 86 markers being B6-homozygous as early as on day 106 at N3. The carrier males at the final generation were all fertile and propagated the modified genes. Thus, three congenic strains were established through rapid generation turnover between 41 and 44 days. CONCLUSIONS/SIGNIFICANCE: This new high-speed breeding strategy enables us to produce congenic strains within about half a year. It should provide the fastest protocol for precise definition of the phenotypic effects of genes of interest on desired genetic backgrounds