Protein expression profile of Gasterophilus intestinalis larvae causing horse gastric myiasis and characterization of horse immune reaction

Background Little information is available on the immunological aspect of parasitic Gasterophilus intestinalis (Diptera, Oestridae) larvae causing horse gastric myiasis. The objectives of this research were to analyze the protein content of larval crude extracts of the migrating second and third larvae (L2 and L3) of G. intestinalis in order to characterize the immune response of horses. Results The proteomic profile of L2 and L3, investigated by using one and two dimensional approaches, revealed a migration pattern specific to each larval stage. Furthermore, Western blots were performed with horse sera and with sera of Balb/c mice immunised with the larval crude extracts of L2 or L3, revealing a different immune reaction in naturally infected horses vs. artificially induced immune reaction in mice. The comparisons of the immunoblot profiles demonstrate that the stage L2 is more immunogenic than the stage L3 most likely as an effect of the highest enzymatic production of L2 while migrating through the host tissues. Fifteen proteins were identified by mass spectrometry. Conclusion This work provides further information into the understanding of the interaction between G. intestinalis and their host and by contributing a novel scheme of the proteomic profile of the main larval stages

The KCNQ1 potassium channel is down-regulated by ubiquitylating enzymes of the Nedd4/Nedd4-like family

Objective: The voltage-gated KCNQ1 potassium channel regulates key physiological functions in a number of tissues. In the heart, KCNQ1 α-subunits assemble with KCNE1 β-subunits forming a channel complex constituting the delayed rectifier current IKs. In epithelia, KCNQ1 channels participate in controlling body electrolyte homeostasis. Several regulatory mechanisms of the KCNQ1 channel complexes have been reported, including protein kinase A (PKA)-phosphorylation and β-subunit interactions. However, the mechanisms controlling the membrane density of KCNQ1 channels have attracted less attention. Methods and results: Here we demonstrate that KCNQ1 proteins expressed in HEK293 cells are down-regulated by Nedd4/Nedd4-like ubiquitin-protein ligases. KCNQ1 and KCNQ1/KCNE1 currents were reduced upon co-expression of Nedd4-2, the isoform among the nine members of the Nedd4/Nedd4-like family displaying the highest expression level in human heart. In vivo expression of a catalytically inactive form of Nedd4-2, able to antagonize endogenous Nedd4-2 in guinea-pig cardiomyocytes, increased IKs significantly, but did not modify IK1. Concomitant with the reduction in current induced by Nedd4-2, an increased ubiquitylation as well as a decreased total level of KCNQ1 proteins were observed in HEK293 cells. Pull-down and co-immunoprecipitation experiments showed that Nedd4-2 interacts with the C-terminal part of KCNQ1. The Nedd4/Nedd4-like-mediated regulation of the KCNQ1 channel complexes is strictly dependent on a PY motif located in the distal part of the C-terminal domain. When this motif was mutated, the current and ubiquitylation levels were unaffected by Nedd4-2, and Nedd4-2 proteins were neither pulled-down nor co-immunoprecipitated. Conclusions: These results suggest that KCNQ1 internalization and stability is physiologically regulated by its Nedd4/Nedd4-like-dependent ubiquitylation. This mechanism may thereby be important in regulating the surface density of the KCNQ1 channels in cardiomyocytes and other cell type

PAR2 absence completely rescues inflammation and ichthyosis caused by altered CAP1/Prss8 expression in mouse skin

Altered serine protease activity is associated with skin disorders in humans and in mice. The serine protease channel-activating protease-1 (CAP1; also termed protease serine S1 family member 8 (Prss8)) is important for epidermal homeostasis and is thus indispensable for postnatal survival in mice, but its roles and effectors in skin pathology are poorly defined. In this paper, we report that transgenic expression in mouse skin of either CAP1/Prss8 (K14-CAP1/Prss8) or protease-activated receptor-2 (PAR2; Grhl3PAR2/+), one candidate downstream target, causes epidermal hyperplasia, ichthyosis and itching. K14-CAP1/Prss8 ectopic expression impairs epidermal barrier function and causes skin inflammation characterized by an increase in thymic stromal lymphopoietin levels and immune cell infiltrations. Strikingly, both gross and functional K14-CAP1/Prss8-induced phenotypes are completely negated when superimposed on a PAR2-null background, establishing PAR2 as a pivotal mediator of pathogenesis. Our data provide genetic evidence for PAR2 as a downstream effector of CAP1/Prss8 in a signalling cascade that may provide novel therapeutic targets for ichthyoses, pruritus and inflammatory skin diseases

Trigonelline is a novel NAD+ precursor that improves muscle function during ageing and is reduced in human sarcopenia

Mitochondrial dysfunction and low nicotinamide adenine dinucleotide (NAD+) levels are hallmarks of skeletal muscle aging and sarcopenia1-3, but it remains unclear whether these defects result from local changes or can be mediated by systemic or dietary cues. Here we reporta functional link between circulating levels of the natural alkaloid trigonelline, which is structurally related to nicotinic acid4, NAD+ levels and muscle health in multiple species. In humans, serum trigonelline levels are reduced with sarcopenia, and correlate positively with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle. Using naturally-occuring and isotopically-labelled trigonelline, we demonstrate that trigonelline incorporates into the NAD+ pool, and increases NAD+ levels in C. elegans, mice and primary myotubes from healthy and sarcopenic humans. Mechanistically, trigonelline does not activate GPR109A but is metabolized via the NAPRT/Preiss-Handler pathway5,6 across models. In C. elegans, trigonelline improves mitochondrial respiration and biogenesis, reduces age-related muscle wasting and increases lifespan and mobility through an NAD+-dependent mechanism requiring sirtuin. Dietary trigonelline supplementation in male mice enhances muscle strength and prevents fatigue during aging. Collectively, we identify nutritional supplementation of trigonelline as a novel NAD+-boosting strategy with therapeutic potential for age-associated muscle decline

Gut microbiota modulation with norfloxacin and ampicillin enhances glucose tolerance in mice

Recent data suggest that the gut microbiota plays a significant role in fat accumulation. However, it is not clear whether gut microbiota is involved in the pathophysiology of type 2 diabetes. To assess this issue, we modulated gut microbiota via antibiotics administration in two different mouse models with insulin resistance. Results from dose-determination studies showed that a combination of norfloxacin and ampicillin, at a dose of 1 g/L, maximally suppressed the numbers of cecal aerobic and anaerobic bacteria in ob/ob mice. After a 2-wk intervention with the antibiotic combination, both ob/ob and diet-induced obese and insulin-resistant mice showed a significant improvement in fasting glycemia and oral glucose tolerance. The improved glycemic control was independent of food intake or adiposity because pair-fed ob/ob mice were as glucose intolerant as the control ob/ob mice. Reduced liver triglycerides and increased liver glycogen correlated with improved glucose tolerance in the treated mice. Concomitant reduction of plasma lipopolysaccharides and increase of adiponectin further supported the antidiabetic effects of the antibiotic treatment in ob/ob mice. In summary, modulation of gut microbiota ameliorated glucose tolerance of mice by altering the expression of hepatic and intestinal genes involved in inflammation and metabolism, and by changing the hormonal, inflammatory, and metabolic status of the host

Resolving microbial membership using Abundance and Variability In Taxonomy (‘AVIT )

Development of NGS has revolutionized the analysis in microbial ecology contributing to our deeper understanding of microbiota in health and disease

High fat diet drives obesity regardless the composition of gut microbiota in mice

The gut microbiota is involved in many aspects of host physiology but its role in body weight and glucose metabolism remains unclear. Here we studied the compositional changes of gut microbiota in diet-induced obesity mice that were conventionally raised or received microbiota transplantation. In conventional mice, the diversity of the faecal microbiota was weakly associated with 1 week weight gain but transferring the microbiota of mice with contrasting weight gain to germfree mice did not change obesity development or feed efficiency of recipients regardless whether the microbiota was taken before or after 10 weeks high fat (HF) feeding. Interestingly, HF-induced glucose intolerance was influenced by microbiota inoculation and improved glucose tolerance was associated with a low Firmicutes to Bacteroidetes ratio. Transplantation of Bacteroidetes rich microbiota compared to a control microbiota ameliorated glucose intolerance caused by HF feeding. Altogether, our results demonstrate that gut microbiota is involved in the regulation of glucose metabolism and the abundance of Bacteroidetes significantly modulates HF-induced glucose intolerance but has limited impact on obesity in mice. Our results suggest that gut microbiota is a part of complex aetiology of insulin resistance syndrome, individual microbiota composition may cause phenotypic variation associated with HF feeding in mice

Trigonelline is an NAD+ precursor that improves muscle function during ageing and is reduced in human sarcopenia.

Mitochondrial dysfunction and low nicotinamide adenine dinucleotide (NAD+) levels are hallmarks of skeletal muscle ageing and sarcopenia1-3, but it is unclear whether these defects result from local changes or can be mediated by systemic or dietary cues. Here we report a functional link between circulating levels of the natural alkaloid trigonelline, which is structurally related to nicotinic acid4, NAD+ levels and muscle health in multiple species. In humans, serum trigonelline levels are reduced with sarcopenia and correlate positively with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle. Using naturally occurring and isotopically labelled trigonelline, we demonstrate that trigonelline incorporates into the NAD+ pool and increases NAD+ levels in Caenorhabditis elegans, mice and primary myotubes from healthy individuals and individuals with sarcopenia. Mechanistically, trigonelline does not activate GPR109A but is metabolized via the nicotinate phosphoribosyltransferase/Preiss-Handler pathway5,6 across models. In C. elegans, trigonelline improves mitochondrial respiration and biogenesis, reduces age-related muscle wasting and increases lifespan and mobility through an NAD+-dependent mechanism requiring sirtuin. Dietary trigonelline supplementation in male mice enhances muscle strength and prevents fatigue during ageing. Collectively, we identify nutritional supplementation of trigonelline as an NAD+-boosting strategy with therapeutic potential for age-associated muscle decline

Compound- and fiber type-selective requirement of AMPKγ3 for insulin-independent glucose uptake in skeletal muscle

Objective The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and potentially in BAT. Herein, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed whether γ3 plays a role in adipose thermogenesis and browning. Methods Global AMPKγ3 knockout (KO) mice were generated. A systematic whole-body, tissue, and molecular phenotyping linked to glucose homeostasis was performed in γ3 KO and wild-type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo as well as blood glucose clearance in response to small molecule AMPK activators that target the nucleotide-binding domain of the γ subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the α and β subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a β3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics, and function. Results Genetic ablation of γ3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high-fat diet. γ3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in γ3 KO mice showed a partial loss of AMPKα2 activity, which was associated with reduced levels of AMPKα2 and β2 subunit isoforms. Notably, γ3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected γ3 in BAT and found that it preferentially interacts with α2 and β2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and γ3 KO mice. Conclusions These results demonstrate that γ3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that γ3 is dispensable for β3-adrenergic receptor agonist-induced thermogenesis and browning of iWAT