A Nutraceutical Mechanistic Model Receives a Gut Check

The recent study in Cellular and Molecular Gastroenterology and Hepatology by Sánchez et al¹ from the Bergheim laboratory at the University of Vienna explores the mechanisms underlying the potentially pathogenic role for the depletion of liver phosphatidylcholine under metabolic stress.

It has been known for some time that liver phosphatidylcholine levels are lower in human MASLD metabolic dysfunction-associated steatotic liver disease (MASLD) patients compared with nonsteatotic controls.² The effect of dietary supplementation of phosphatidylcholine and related phospholipids has been an area of active exploration in the nutraceutical space, with soy-derived polyenyl-phosphatidylcholine supplementation showing significant clinical benefit in a randomized clinical trial in a MASLD/metabolic dysfunction–associated steatohepatitis (MASH) cohort.³ Phosphatidylcholine supplementation enhances barrier function in cultured intestinal epithelial cells,⁴ and can rescue intestinal barrier defects and decrease inflammation in patients with ulcerative colitis.⁵ Despite this, the mechanisms underlying both the decrease in liver phosphatidylcholine and the protective effect of its restoration remain unclear, which are areas that Sánchez et al addressed in their recent study.

Sánchez et al used a fast-food–type diet (high fat, high fructose, and high cholesterol; FFC) in female C57BL/6J mice to induce MASLD/MASH. After 8 weeks on the diet (16 weeks of age), the mouse livers showed extensive macrosteatosis and the onset of MASH, with increased alanine aminotransferase levels and increased neutrophil granulocyte infiltration, and increased myeloperoxidase activity. The authors also noted effects on the nitric oxide signaling pathway, with increased nitrogen oxides and a reciprocal decrease in arginase activity (a counter-regulator of nitric oxide signaling). Relative to the standard diet group, the FFC-fed female mice showed a roughly 2-fold decrease in their liver phosphatidylcholine content. Strikingly, the markers of MASH onset were attenuated significantly by dietary co-administration of phosphatidylcholine with the FFC diet.

Sánchez et al next measured lipopolysaccharide from the portal blood of 4 groups of mice: control and fast-food type diet, with and without phosphatidylcholine supplementation. As expected, they found that lipopolysaccharide in portal blood was increased by a fast-food–type diet, however, interestingly, the increased lipopolysaccharide levels were not attenuated by phosphatidylcholine co-administration, suggesting that the effects on the liver markers did not reflect changes in gut permeability. With this in mind, Sánchez et al focused on the effect of phosphatidylcholine on the Toll-like receptor 4 (TLR4) pathway, which transduces the pathogenic effect of lipopolysaccharide to its output in inflammatory signaling via nitric oxide and inflammatory cytokines. In contrast to the absence of an effect on lipopolysaccharide levels in blood, the pathway downstream of TLR4 was affected by phosphatidylcholine treatment: levels of tumor necrosis factor α (TNF-α) protein of the ratio of phosphorylated-IκBα to total IκBα were both increased significantly by the FCC diet, and these effects were blunted by co-administration of phosphatidylcholine. In addition, Pparg2 Peroxisome Proliferator Activated Receptor Gamma 2 (Pparg2) messenger RNA and PPARG2 protein levels were increased by FFC, and this induction was suppressed in the fast-food–type plus phosphatidylcholine group, but there were no differences between groups with respect to the transcript levels of either Pparg1 or Nr5a2 (the latter encodes the Liver Receptor Homolog-1 [LRH-1]protein).

Lastly, Sánchez et al probed the effects of known modulators of the TLR4–TNF-α axis on the protective effect of phosphatidylcholine. Using lipopolysaccharide as a stressor in the murine macrophage-like cell line, J774A.1, and in primary human macrophages, they measured TNF-α protein and the concentration of nitrogen oxides as end points (relative to lipopolysaccharide-treated cells) and found that agonists of PPARG and LRH-1 both attenuated the protective effect of phosphatidylcholine administration (or potentiated the inflammatory effect of lipopolysaccharide).

Sánchez et al are appropriately cautious both in the inferences drawn from their data and in their acknowledgment of the limitations of the study. Nonetheless, their observation that the levels of lipopolysaccharide in portal blood are unaffected by co-administration of phosphatidylcholine with the fast-food–type diet raises an interesting challenge to the dogma that phosphatidylcholine supplementation promotes intestinal barrier function and thereby secondarily decreases endotoxemia. Sánchez et al show that phosphatidylcholine affects the pathway downstream of TLR4, suggesting an alternate hypothesis in which the phosphatidylcholine-mediated rescue of intestinal barrier function instead is secondary to a primary effect in decreasing inflammation, and that the impact of phosphatidylcholine supplementation in liver therefore is separable from, but analogous to that in, the gut.

Notably, the transcription factors implicated in this study are both nuclear receptors that are responsive to endogenous lipid species that are dysregulated in MASLD (PPARG to fatty acids,⁶ LRH-1 to phospholipids⁷), and both are central regulators in liver, intestine, and the immune system. Fortunately, a wealth of lineage-specific genetic tools and pharmacologic agents already have been developed, and these will be essential to teasing apart the implications of the current work both in healthy and disease conditions.