Characterization of two rat models of cystic fibrosis—KO and F508del CFTR—Generated by Crispr-Cas9

Background: Genetically engineered animals are essential for gaining a proper understanding of the disease mechanisms of cystic fibrosis(CF). The rat is a relevant laboratory model for CF because of its zootechnical capacity, size, and airway characteristics, including the presence of submucosal glands.Methods: We describe the generation of a CF rat model(F508 del) homozygous for the p.Phe508 del mutation in the transmembrane conductance regulator(Cftr) gene. This model was compared to new Cftr-/-rats(CFTR KO). Target organs in CF were examined by histological staining of tissue sections and tooth enamel was quantified by micro-computed tomography. The activity of CFTR was evaluated by nasal potential difference(NPD) and short-circuit current measurements. The effect of VX-809 and VX-770 was analyzed on nasal epithelial primary cell cultures from F508 del rats.Results: Both newborn F508 del and Knock out(KO) animals developed intestinal obstruction that could be partly compensated by special diet combined with an osmotic laxative. The two rat models exhibited CF phenotypic anomalies such as vas deferens agenesis and tooth enamel defects. Histology of the intestine, pancreas, liver, and lungs was normal. Absence of CFTR function in KO rats was confirmed ex vivo by short-circuit current measurements on colon mucosae and in vivo by NPD, whereas residual CFTR activity was observed in F508 del rats. Exposure of F508 del CFTR nasal primary cultures to a combination of VX-809 and VX-770 improved CFTR-mediated Cl-transport.Conclusions: The F508 del rats reproduce the phenotypes observed in CFTR KO animals and represent a novel resource to advance the development of CF therapeutics.

High doses of catecholamines activate glucose transport in human adipocytes independently from adrenoceptor stimulation or vanadium addition

BACKGROUND When combined with vanadium salts,catecholamines strongly activate glucose uptake in rat and mouse adipocytes.AIM To test whether catecholamines activate glucose transport in human adipocytes.METHODS The uptake of 2-deoxyglucose(2-DG)was measured in adipocytes isolated from pieces of abdominal subcutaneous tissue removed from women undergoing reconstructive surgery.Pharmacological approaches with amine oxidase inhibitors,adrenoreceptor agonists and antioxidants were performed to unravel the mechanisms of action of noradrenaline or adrenaline(also named epinephrine).RESULTS In human adipocytes,45-min incubation with 100μmol/L adrenaline or noradrenaline activated 2-DG uptake up to more than one-third of the maximal response to insulin.This stimulation was not reproduced with millimolar doses of dopamine or serotonin and was not enhanced by addition of vanadate to the incubation medium.Among various natural amines and adrenergic agonists tested,no other molecule was more efficient than adrenaline and noradrenaline in stimulating 2-DG uptake.The effect of the catecholamines was not impaired by pargyline and semicarbazide,contrarily to that of benzylamine or methylamine,which are recognized substrates of semicarbazide-sensitive amine oxidase.Hydrogen peroxide at 1 mmol/L activated hexose uptake but not pyrocatechol or benzoquinone,and only the former was potentiated by vanadate.Catalase and the phosphoinositide 3-kinase inhibitor wortmannin inhibited adrenaline-induced activation of 2-DG uptake.CONCLUSION High doses of catecholamines exert insulin-like actions on glucose transport in human adipocytes.At submillimolar doses,vanadium did not enhance this catecholamine activation of glucose transport.Consequently,this dismantles our previous suggestion to combine the metal ion with catecholamines to improve the benefit/risk ratio of vanadium-based antidiabetic approaches.

Increased monoamine oxidase activity and imidazoline binding sites in insulin-resistant adipocytes from obese Zucker rats

BACKGROUND Despite overt insulin resistance,adipocytes of genetically obese Zucker rats accumulate the excess of calorie intake in the form of lipids.AIM To investigate whether factors can replace or reinforce insulin lipogenic action by exploring glucose uptake activation by hydrogen peroxide,since it is produced by monoamine oxidase(MAO)and semicarbazide-sensitive amine oxidase(SSAO)in adipocytes.METHODS 3H-2-deoxyglucose uptake(2-DG)was determined in adipocytes from obese and lean rats in response to insulin or MAO and SSAO substrates such as tyramine and benzylamine.14C-tyramine oxidation and binding of imidazolinic radioligands[3H-Idazoxan,3H-(2-benzofuranyl)-2-imidazoline]were studied in adipocytes,the liver,and muscle.The influence of in vivo administration of tyramine+vanadium on glucose handling was assessed in lean and obese rats.RESULTS 2-DG uptake and lipogenesis stimulation by insulin were dampened in adipocytes from obese rats,when compared to their lean littermates.Tyramine and benzylamine activation of hexose uptake was vanadate-dependent and was also limited,while MAO was increased and SSAO decreased.These changes were adipocyte-specific and accompanied by a greater number of imidazoline I2 binding sites in the obese rat,when compared to the lean.In vitro,tyramine precluded the binding to I2 sites,while in vivo,its administration together with vanadium lowered fasting plasma levels of glucose and triacylglycerols in obese CONCLUSION The adipocytes from obese Zucker rats exhibit increased MAO activity and imidazoline binding site number.However,probably as a consequence of SSAO down-regulation,the glucose transport stimulation by tyramine is decreased as much as that of insulin in these insulin-resistant adipocytes.The adipocyte amine oxidases deserve more studies with respect to their putative contribution to the management of glucose and lipid handling.