Non-lethal Inhibition of Gut Microbial Trimethylamine Production for the Treatment of Atherosclerosis.

Trimethylamine (TMA) N-oxide (TMAO), a gut-microbiota-dependent metabolite, both enhances atherosclerosis in animal models and is associated with cardiovascular risks in clinical studies. Here, we investigate the impact of targeted inhibition of the first step in TMAO generation, commensal microbial TMA production, on diet-induced atherosclerosis. A structural analog of choline, 3,3-dimethyl-1-butanol (DMB), is shown to non-lethally inhibit TMA formation from cultured microbes, to inhibit distinct microbial TMA lyases, and to both inhibit TMA production from physiologic polymicrobial cultures (e.g., intestinal contents, human feces) and reduce TMAO levels in mice fed a high-choline or L-carnitine diet. DMB inhibited choline diet-enhanced endogenous macrophage foam cell formation and atherosclerotic lesion development in apolipoprotein e(-/-) mice without alterations in circulating cholesterol levels. The present studies suggest that targeting gut microbial production of TMA specifically and non-lethal microbial inhibitors in general may serve as a potential therapeutic approach for the treatment of cardiometabolic diseases.

The Nutritional Supplement L-Alpha Glycerylphosphorylcholine Promotes Atherosclerosis.

L-alpha glycerylphosphorylcholine (GPC), a nutritional supplement, has been demonstrated to improve neurological function. However, a new study suggests that GPC supplementation increases incident stroke risk thus its potential adverse effects warrant further investigation. Here we show that GPC promotes atherosclerosis in hyperlipidemic Apoe-/- mice. GPC can be metabolized to trimethylamine N-oxide, a pro-atherogenic agent, suggesting a potential molecular mechanism underlying the observed atherosclerosis progression. GPC supplementation shifted the gut microbial community structure, characterized by increased abundance of Parabacteroides, Ruminococcus, and Bacteroides and decreased abundance of Akkermansia, Lactobacillus, and Roseburia, as determined by 16S rRNA gene sequencing. These data are consistent with a reduction in fecal and cecal short chain fatty acids in GPC-fed mice. Additionally, we found that GPC supplementation led to an increased relative abundance of choline trimethylamine lyase (cutC)-encoding bacteria via qPCR. Interrogation of host inflammatory signaling showed that GPC supplementation increased expression of the proinflammatory effectors CXCL13 and TIMP-1 and activated NF-κB and MAPK signaling pathways in human coronary artery endothelial cells. Finally, targeted and untargeted metabolomic analysis of murine plasma revealed additional metabolites associated with GPC supplementation and atherosclerosis. In summary, our results show GPC promotes atherosclerosis through multiple mechanisms and that caution should be applied when using GPC as a nutritional supplement.

Impaired Ribosomal Biogenesis by Noncanonical Degradation of ß-Catenin during Hyperammonemia.

Increased ribosomal biogenesis occurs during tissue hypertrophy, but whether ribosomal biogenesis is impaired during atrophy is not known. We show that hyperammonemia, which occurs in diverse chronic disorders, impairs protein synthesis as a result of decreased ribosomal content and translational capacity. Transcriptome analyses, real-time PCR, and immunoblotting showed consistent reductions in the expression of the large and small ribosomal protein subunits (RPL and RPS, respectively) in hyperammonemic murine skeletal myotubes, HEK cells, and skeletal muscle from hyperammonemic rats and human cirrhotics. Decreased ribosomal content was accompanied by decreased expression of cMYC, a positive regulator of ribosomal biogenesis, as well as reduced expression and activity of ß-catenin, a transcriptional activator of cMYC. However, unlike the canonical regulation of ß-catenin via glycogen synthase kinase 3ß (GSK3ß)-dependent degradation, GSK3ß expression and phosphorylation were unaltered during hyperammonemia, and depletion of GSK3ß did not prevent ammonia-induced degradation of ß-catenin. Overexpression of GSK3ß-resistant variants, genetic depletion of IκB kinase ß (IKKß) (activated during hyperammonemia), protein interactions, and in vitro kinase assays showed that IKKß phosphorylated ß-catenin directly. Overexpressing ß-catenin restored hyperammonemia-induced perturbations in signaling responses that regulate ribosomal biogenesis. Our data show that decreased protein synthesis during hyperammonemia is mediated via a novel GSK3ß-independent, IKKß-dependent impairment of the ß-catenin-cMYC axis.