mInd the gAP: Intestinal Alkaline Phosphatase Puts the Breaks on Atherosclerosis

Type 2 diabetes, obesity, non-alcoholic fatty liver disease (NALFD), and atherosclerosis, share many common hallmark features and are increasing in prevalence at a rapid rate.^2–5 Western diets, high in fat and cholesterol, cause disturbances in gut microbial homeostasis (dysbiosis) and intestinal barrier function, leading to the increased translocation of the bacterial endotoxin lipopolysaccharide (LPS) into the systemic circulation.⁶ Patients with obesity, type 2 diabetes, NAFLD, and CVD have increased circulating levels of LPS suggesting a relationship between metabolic disease and intestinal barrier dysfunction.^7–9

The gut-liver axis plays a key role in the movement of LPS from the subepithelial lamina propria to the liver via the portal vein. The portal system connects the gut and the liver, such that any substance that can cross the intestinal barrier can subsequently reach the liver and eventually peripheral cells and tissues. These include important and essential dietary components, but in the setting of a damaged intestinal barrier, can also include toxins such as LPS. The healthy intestinal barrier consists of four layers; (i) luminal intestinal alkaline phosphatase (IAP) and the mucosal layer (extracellular layer), (ii) a single layer of epithelial cells that facilitates selective uptake of nutrients, water, and electrolytes (cellular layer), (iii) antimicrobial proteins and immune cells (immunological layer), and (iv) vascular endothelial and glial cells (vascular layer). IAP is secreted by enterocytes onto the apical surface of the epithelium where it dephosphorylates toxins such as LPS, and the pattern-associated molecular patterns (PAMPs) flagellin and CpG DNA. IAP removes one of the two phosphate groups from the toxic lipid A moiety of LPS. The resulting monophosphoryl-LPS cannot activate Toll-like Receptor 4 (TLR4) signaling, thereby reducing local intestinal inflammation.^10,11 However, loss of this protective effect results in translocation of LPS across the intestinal barrier and into the systemic circulation, leading to increased local and systemic inflammation which incites intestinal barrier dysfunction and facilitates more LPS entering the circulation, establishing a vicious cycle that continually promotes inflammation and metabolic endotoxemia.^6,8,12

Ghosh et al.¹ investigated whether increased levels of IAP could protect from the development of atherosclerosis. The authors used intestine-specific IAP-overexpressing mice on an Ldlr^–/– hyperlipidemic, atherosclerosis-prone background (Ldlr^–/–IAP^Tg). In wildtype mice, IAP expression is highest in the duodenum, declining progressively along the intestinal tract. Interestingly, the lowest levels of IAP are found in the colon, the region of the intestine with the highest concentration of bacteria. The authors have previously reported that IAP^Tg mice have increased IAP expression along the whole intestinal tract, including the colon.¹³ The colonic mucous layer provides a physical barrier between bacteria/bacterial toxins and intestinal epithelial cells. Western diet feeding, which is low in fiber, forces the bacteria to utilize the nutrient-rich mucous for energy, ultimately destroying the mucosal layer. Therefore, the authors first assess the mucosal layer in Western diet-fed IAP^Tg mice. IAP^Tg mice fed a Western diet maintained the integrity of the colonic mucosal layer, compared to control animals, suggesting increased levels of IAP in the colon modulate the stability of the mucosal layer. Second, they measure levels of LPS in the circulation as a function of IAP activity, and demonstrate reduced plasma LPS in IAP^Tg mice, likely due to improved maintenance of the intestinal barrier and increased detoxification of LPS. However, this decrease in circulating LPS did not result in reduced systemic inflammation, as indicated by the lack of change in plasma IL-6 levels.

Previous studies by the authors have shown that transgenic overexpression of IAP in Western diet-fed C57BL6/J had no effect on plasma lipid levels. However, in the present study, the authors demonstrate that hyperlipidemic Ldlr^–/–IAP^Tg mice had lower plasma cholesterol and triglyceride levels compared to Ldlr^–/– control mice, which was accompanied by reduced hepatic lipid accumulation. The authors also observed reduced expression of macrophage marker gene expression and the inflammatory cytokine MIP-1α in the liver, indicating decreased MIP-1α production by resident macrophages either due to reduced activation by LPS or reduced numbers of macrophages. The authors then go on to demonstrate alterations in fatty acid and cholesterol transporter gene expression in the intestines of Ldlr^–/–IAP^Tg mice, which coupled with the changes in plasma lipids suggested a possible reduction in the absorption of fatty acids and cholesterol. To determine whether intestinal lipid absorption is affected by overexpression of IAP, IAP^Tg mice were given a bolus of triglyceride in the form of corn oil in combination with the lipoprotein lipase inhibitor Tyloxapol. The addition of the lipase inhibitor allows for an excursion of plasma triglycerides that cannot be cleared due to the Tyloxapol-mediated inhibition of lipoprotein lipase. Transgenic overexpression of IAP resulted in reduced plasma triglycerides over time, suggesting reduced intestinal lipid absorption in these mice. Assessment of the impact of IAP expression on the development of atherosclerosis, showed that in addition to reduced plasma cholesterol levels and improved intestinal barrier function, Ldlr^–/–IAP^Tg mice developed significantly reduced atherosclerotic lesions.

Several intestinal diseases have been associated with increased risk of CVD, although individuals often do not have all the “classic” risk factors. However, there is mounting evidence linking dietary lipid intake, gut homeostasis, and atherosclerosis.^7,14,15 Intestinal lipid absorption is affected by multiple parameters, including bile acid composition (that act as detergents to facilitate lipid absorption), gut microbiota, and intestinal barrier function. Notably, the work by Ghosh et al.¹ suggests that the regulation of atherosclerosis progression by intestine-specific IAP expression is likely the combined effect of reduced intestinal lipid absorption, improved intestinal barrier function and reduced circulating LPS, and decreased plasma lipid levels. It is well appreciated that the liver is a central hub for handling lipids and is a major target for therapeutics for metabolic disease. This study lends further support to the notion that intestinal function, in particular a healthy intestinal barrier and optimal lipid absorption, is key to metabolic health. Furthermore, this study also supports the idea that targeting intestinal homeostasis also harbors significant therapeutic potential in the fight against cardiometabolic disease.

Figure 1. Overexpression of intestinal alkaline phosphatase improves intestinal barrier function and attenuates atherosclerosis.

(Left hand side) Increased expression of intestinal alkaline phosphatase (IAP) at the apical surface results in increased detoxification of LPS, maintenance of the colonic mucosal layer, reduced lipid absorption, and decreased atherosclerotic lesions. (Right hand side) During metabolic endotoxemia, increased dietary cholesterol and fat cause dysbiosis and dysregulation of the intestinal barrier, increased systemic inflammation and circulating levels of LPS, resulting in increased progression of atherosclerosis.