Increased Gut Permeability and Microbiota Change Associate with Mesenteric Fat Inflammation and Metabolic Dysfunction in Diet-Induced Obese Mice

We investigated the relationship between gut health, visceral fat dysfunction and metabolic disorders in diet-induced obesity. C57BL/6J mice were fed control or high saturated fat diet (HFD). Circulating glucose, insulin and inflammatory markers were measured. Proximal colon barrier function was assessed by measuring transepithelial resistance and mRNA expression of tight-junction proteins. Gut microbiota profile was determined by 16S rDNA pyrosequencing. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNA levels were measured in proximal colon, adipose tissue and liver using RT-qPCR. Adipose macrophage infiltration (F4/80+) was assessed using immunohistochemical staining. HFD mice had a higher insulin/glucose ratio (P = 0.020) and serum levels of serum amyloid A3 (131%; P = 0.008) but reduced circulating adiponectin (64%; P = 0.011). In proximal colon of HFD mice compared to mice fed the control diet, transepithelial resistance and mRNA expression of zona occludens 1 were reduced by 38% (P<0.001) and 40% (P = 0.025) respectively and TNF-α mRNA level was 6.6-fold higher (P = 0.037). HFD reduced Lactobacillus (75%; P<0.001) but increased Oscillibacter (279%; P = 0.004) in fecal microbiota. Correlations were found between abundances of Lactobacillus (r = 0.52; P = 0.013) and Oscillibacter (r = −0.55; P = 0.007) with transepithelial resistance of the proximal colon. HFD increased macrophage infiltration (58%; P = 0.020), TNF-α (2.5-fold, P<0.001) and IL-6 mRNA levels (2.5-fold; P = 0.008) in mesenteric fat. Increased macrophage infiltration in epididymal fat was also observed with HFD feeding (71%; P = 0.006) but neither TNF-α nor IL-6 was altered. Perirenal and subcutaneous adipose tissue showed no signs of inflammation in HFD mice. The current results implicate gut dysfunction, and attendant inflammation of contiguous adipose, as salient features of the metabolic dysregulation of diet-induced obesity

H-Ras mediates the inhibitory effect of epidermal growth factor on the epithelial Na+ channel.

The present study investigates the role of small G-proteins of the Ras family in the epidermal growth factor (EGF)-activated cellular signalling pathway that downregulates activity of the epithelial Na+ channel (ENaC). We found that H-Ras is a key component of this EGF-activated cellular signalling mechanism in M1 mouse collecting duct cells. Expression of a constitutively active H-Ras mutant inhibited the amiloride-sensitive current. The H-Ras-mediated signalling pathway that inhibits activity of ENaC involves c-Raf, and that the inhibitory effect of H-Ras on ENaC is abolished by the MEK1/2 inhibitor, PD98059. The inhibitory effect of H-Ras is not mediated by Nedd4-2, a ubiquitin protein ligase that regulates the abundance of ENaC at the cell surface membrane, or by a negative effect of H-Ras on proteolytic activation of the channel. The inhibitory effects of EGF and H-Ras on ENaC, however, were not observed in cells in which expression of caveolin-1 (Cav-1) had been knocked down by siRNA. These findings suggest that the inhibitory effect of EGF on ENaC-dependent Na+ absorption is mediated via the H-Ras/c-Raf, MEK/ERK signalling pathway, and that Cav-1 is an essential component of this EGF-activated signalling mechanism. Taken together with reports that mice expressing a constitutive mutant of H-Ras develop renal cysts, our findings suggest that H-Ras may play a key role in the regulation of renal ion transport and renal development

Inhibition of ENaC by H-Ras is not mediated by Nedd4-2.

<p><i>A</i>, I_{ami (normalized)} in FRT cells transfected with wild-type α-, β-, and γ-ENaC (αβγ) subunits or with ENaC comprising one truncation mutant (α-ENaC mutant that is truncated at amino acid Pro⁶⁴⁶ (α_T); β-ENaC mutant that is truncated at amino acid Cys⁵⁹⁴ (β_T); or γ-ENaC mutant that is truncated at amino acid Phe⁶¹⁰ (γ_T)). The <i>solid bar</i> indicates that the monolayer were co-transfected with H-Ras^G12V. <i>B</i>, I_{ami (normalized)} in FRT cells co-transfected with wild-type ENaC subunits, and with pcDNA3.1 or pSM2c empty vector (control for shRNA-treated group) or with H-Ras^G12V together with or without an shRNA directed against Nedd4-2 (shNd4-2) in pSM2c as indicated. <i>C</i>, <i>upper panel</i>, Immunoblot analysis of membrane α-ENaC in HEK293 cells transfected with HA (N-terminus) and V5 (C-terminus) tagged α-ENaC (_HAα_V5) and β- and γ-ENaC with FLAG at the C-terminal. Cells were co-transfected with the pcDNA3.1 empty vector, wt-H-Ras, H-Ras^G12V or H-Ras^S17N. Biotinylated membrane ENaC was isolated and immunoblotted with an antibody directed against HA, allowing detection of the 95 kDa un-cleaved and 30 kDa cleaved α-ENaC. <i>C</i>, <i>lower panel</i>, Immunoblot analysis of α-ENaC in total cell lysate. <i>D</i>, <i>upper panel</i>, Immunoblot analysis of membrane γ-ENaC in HEK293 cells transfected with HA (N-terminus) and V5 (C-terminus) tagged γ-ENaC (_HAγ_V5) and α- and β-ENaC with FLAG tagged at the C-terminal. <i>D</i>, <i>lower panel</i>, Immunoblot analysis of γ-ENaC in total cell lysate.</p

Caveolin-1 is involved in the regulation of ENaC by EGF and H-Ras.

<p><i>A</i>. Immuno blot analysis of phosphorylated ERK1/2 (phospho-ERK1/2) and total ERK1/2 in FRT cells transfected with a scrambled siRNA or the siRNA directed against Cav-1 treated with (+) or without (-) EGF. <i>B</i>. Immunoblot analysis of phosphorylated ERK1/2 and total ERK1/2 in FRT cells transfected with wt-Cav-1. C. I_{ami (normalized)} in FRT cells transfected with wild-type α-, β-, and γ-ENaC subunits. Cells were co-transfected with the wtCav-1, scrambled siRNA, siRNA directed against Cav-1, H-Ras^G12V or H-Ras^S17N as indicated.</p

Regulation of the Epithelial Na+ Channel by the RH Domain of G Protein-coupled Receptor Kinase, GRK2, and Gαq/11*

The G protein-coupled receptor kinase (GRK2) belongs to a family of protein kinases that phosphorylates agonist-activated G protein-coupled receptors, leading to G protein-receptor uncoupling and termination of G protein signaling. GRK2 also contains a regulator of G protein signaling homology (RH) domain, which selectively interacts with α-subunits of the Gq/11 family that are released during G protein-coupled receptor activation. We have previously reported that kinase activity of GRK2 up-regulates activity of the epithelial sodium channel (ENaC) in a Na+ absorptive epithelium by blocking Nedd4-2-dependent inhibition of ENaC. In the present study, we report that GRK2 also regulates ENaC by a mechanism that does not depend on its kinase activity. We show that a wild-type GRK2 (wtGRK2) and a kinase-dead GRK2 mutant (K220RGRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (ΔRH-GRK2) or a GRK2 mutant that cannot interact with Gαq/11/14 (D110AGRK2), increase activity of ENaC. GRK2 up-regulates the basal activity of the channel as a consequence of its RH domain binding the α-subunits of Gq/11. We further found that expression of constitutively active Gαq/11 mutants significantly inhibits activity of ENaC. Conversely, co-expression of siRNA against Gαq/11 increases ENaC activity. The effect of Gαq on ENaC activity is not due to change in ENaC membrane expression and is independent of Nedd4-2. These findings reveal a novel mechanism by which GRK2 and Gq/11 α-subunits regulate the activity ENaC

Effect of H-Ras on exogenous ENaC expressed in FRT cells.

<p><i>A</i>, Immunoblot analysis detecting expression of H-Ras, <i>upper panel</i>, and corresponding I_{ami (normalized)}, <i>lower panel</i>, in FRT cells co-transfected with all three wild-type α-, β- and γ-ENaC subunits (ENaC) together with pcDNA3.1, wild-type H-Ras (wt-H-Ras), H-Ras^G12V or a constitutively inactive mutant of H-Ras (H-Ras^S17N). <i>B</i>, I_{ami (normalized)} in FRT cells transfected with wild-type α-, β- and γ-ENaC together with empty pcDNA3.1 empty vector, K-Ras^G12V or H-Ras^G12V, and with or without the dominant negative mutant of PI3K, PI3KΔp85. In some experiments, cells were pre-treated with insulin (100 nM) for 2 hr. *, ** and *** denote <i>P</i> < 0.05, <i>P</i> < 0.01 and <i>P</i> < 0.001, respectively.</p

Roles of the C termini of alpha-, beta-, and gamma-subunits of epithelial Na(+) channels (ENaC) in regulating ENaC and mediating its inhibition by cytosolic Na(+)

The amiloride-sensitive epithelial Na+ channels (ENaC) in the intralobular duct cells of mouse mandibular glands are inhibited by the ubiquitin-protein ligase, Nedd4, which is activated by increased intracellular Na+. In this study we have used whole-cell patch clamp methods in mouse mandibular duct cells to investigate the role of the C termini of the α-, β-, and γ-subunits of ENaC in mediating this inhibition. We found that peptides corresponding to the C termini of the β- and γ-subunits, but not the α-subunit, inhibited the activity of the Na+ channels. This mechanism did not involve Nedd4 and probably resulted from the exogenous C termini interfering competitively with the protein-protein interactions that keep the channels active. In the case of the C terminus of mouse β-ENaC, the interacting motif included βSer631, βAsp632, and βSer633. In the C terminus of mouse γ-ENaC, it included γSer640. Once these motifs were deleted, we were able to use the C termini of β- and γ-ENaC to prevent Nedd4-mediated down-regulation of Na+ channel activity. The C terminus of the α-subunit, on the contrary, did not prevent Nedd4-mediated inhibition of the Na+ channels. We conclude that mouse Nedd4 interacts with the β- and γ-subunits of ENaC.Anuwat Dinudom,Kieran F. Harvey,Permsak Komwatana,Corina N. Jolliffe,John A. Young,Sharad Kumar,and David I. Coo

Pathogenesis of chytridiomycosis, a cause of catastrophic amphibian declines

The pathogen Batrachochytrium dendrobatidis (Bd), which causes the skin disease chytridiomycosis, is one of the few highly virulent fungi in vertebrates and has been implicated in worldwide amphibian declines. However, the mechanism by which Bd causes death has not been determined. We show that Bd infection is associated with pathophysiological changes that lead to mortality in green tree frogs (Litoria caerulea). In diseased individuals, electrolyte transport across the epidermis was inhibited by >50%, plasma sodium and potassium concentrations were respectively reduced by ~20% and ~50%, and asystolic cardiac arrest resulted in death. Because the skin is critical in maintaining amphibian homeostasis, disruption to cutaneous function may be the mechanism by which Bd produces morbidity and mortality across a wide range of phylogenetically distant amphibian taxa