Background Choline, an essential nutrient, is required for cell membranes, lipoprotein secretion, and methyl-group metabolism. Recently, it has been proposed that excess dietary choline consumption is metabolized to trimethylamine (TMA) by the gut microbiota; TMA is then oxidized to trimethylamine N-oxide (TMAO) in the liver. Epidemiological studies have clearly shown a positive correlation between plasma TMAO concentrations and cardiovascular events. Furthermore, some studies have shown an association between excess dietary choline, plasma TMAO concentrations, and atherosclerotic lesion size in apoE knockout (Apoe−/−) mice. Objective The aim of this study was to further investigate the relation between dietary choline and atherosclerosis in 2 atherogenic mouse models, the LDL receptor knockout (Ldlr−/−) and Apoe−/− mice. Methods Six feeding trials were performed in Ldlr−/− (40% high-fat diet) and Apoe−/− (unpurified diet) male mice, aged 8–10 wk. Mice randomly received control diet (0.1% choline), or choline- (1% choline), betaine- (0.1% choline and 0.9% betaine), or TMAO- (0.1% choline and 0.12% or 0.2% TMAO) supplemented diet for ≤28 wk. After the dietary intervention, the animals were killed and tissues and blood collected. Aortic atherosclerotic plaque area, plasma lipids, and choline metabolites were quantified. Results In Ldlr−/− mice, dietary supplementation for 8 wk with choline or TMAO increased plasma TMAO concentrations by 1.6- and 4-fold, respectively. After 16 wk, there was a 2-fold increase in plasma TMAO after dietary TMAO supplementation. In Apoe−/− mice, dietary supplementation with choline, betaine, or TMAO for 12 wk did not increase plasma TMAO concentrations. However, choline and TMAO supplementation for 28 wk significantly increased plasma TMAO concentrations by 1.8- and 1.5-fold, respectively. Contrary to predictions, atherosclerotic lesion size was not altered by any of the dietary interventions, irrespective of mouse model. Conclusions In our study, high intakes of dietary choline or TMAO supplementation did not influence atherosclerosis development in Ldlr−/− or Apoe−/− male mice.

Background Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42, and they regulate cytoskeletal dynamics, cell polarity, and transcription. We previously demonstrated that Pak1 and Pak2 function redundantly to promote skeletal myoblast differentiation during postnatal development and regeneration in mice. However, the roles of Pak1 and Pak2 in adult muscle homeostasis are unknown. Choline kinase β (Chk β) is important for adult muscle homeostasis, as autosomal recessive mutations in CHKβ are associated with two human muscle diseases, megaconial congenital muscular dystrophy and proximal myopathy with focal depletion of mitochondria. Methods We analyzed mice conditionally lacking Pak1 and Pak2 in the skeletal muscle lineage (double knockout (dKO) mice) over 1 year of age. Muscle integrity in dKO mice was assessed with histological stains, immunofluorescence, electron microscopy, and western blotting. Assays for mitochondrial respiratory complex function were performed, as was mass spectrometric quantification of products of choline kinase. Mice and cultured myoblasts deficient for choline kinase β (Chk β) were analyzed for Pak1/2 phosphorylation. Results dKO mice developed an age-related myopathy. By 10 months of age, dKO mouse muscles displayed centrally-nucleated myofibers, fibrosis, and signs of degeneration. Disease severity occurred in a rostrocaudal gradient, hindlimbs more strongly affected than forelimbs. A distinctive feature of this myopathy was elongated and branched intermyofibrillar (megaconial) mitochondria, accompanied by focal mitochondrial depletion in the central region of the fiber. dKO muscles showed reduced mitochondrial respiratory complex I and II activity. These phenotypes resemble those of rmd mice, which lack Chkβ and are a model for human diseases associated with CHKβ deficiency. Pak1/2 and Chkβ activities were not interdependent in mouse skeletal muscle, suggesting a more complex relationship in regulation of mitochondria and muscle homeostasis. Conclusions Conditional loss of Pak1 and Pak2 in mice resulted in an age-dependent myopathy with similarity to mice and humans with CHKβ deficiency. Protein kinases are major regulators of most biological processes but few have been implicated in muscle maintenance or disease. Pak1/Pak2 dKO mice offer new insights into these processes. Electronic supplementary material The online version of this article (10.1186/s13395-019-0191-4) contains supplementary material, which is available to authorized users.

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