Abeba Teshager

Intestinal alkaline phosphatase prevents metabolic syndrome in mice

Metabolic syndrome comprises a cluster of related disorders that includes obesity, glucose intolerance, insulin resistance, dyslipidemia, and fatty liver. Recently, gut-derived chronic endotoxemia has been identified as a primary mediator for triggering the low-grade inflammation responsible for the development of metabolic syndrome. In the present study we examined the role of the small intestinal brush-border enzyme, intestinal alkaline phosphatase (IAP), in preventing a high-fat-diet–induced metabolic syndrome in mice. We found that both endogenous and orally supplemented IAP inhibits absorption of endotoxin (lipopolysaccharides) that occurs with dietary fat, and oral IAP supplementation prevents as well as reverses metabolic syndrome. Furthermore, IAP supplementation improves the lipid profile in mice fed a standard, low-fat chow diet. These results point to a potentially unique therapy against metabolic syndrome in at-risk humans.

Intestinal alkaline phosphatase promotes gut bacterial growth by reducing the concentration of luminal nucleotide triphosphates

The intestinal microbiota plays a pivotal role in maintaining human health and well-being. Previously, we have shown that mice deficient in the brush-border enzyme intestinal alkaline phosphatase (IAP) suffer from dysbiosis and that oral IAP supplementation normalizes the gut flora. Here we aimed to decipher the molecular mechanism by which IAP promotes bacterial growth. We used an isolated mouse intestinal loop model to directly examine the effect of exogenous IAP on the growth of specific intestinal bacterial species. We studied the effects of various IAP targets on the growth of stool aerobic and anaerobic bacteria as well as on a few specific gut organisms. We determined the effects of ATP and other nucleotides on bacterial growth. Furthermore, we examined the effects of IAP on reversing the inhibitory effects of nucleotides on bacterial growth. We have confirmed that local IAP bioactivity creates a luminal environment that promotes the growth of a wide range of commensal organisms. IAP promotes the growth of stool aerobic and anaerobic bacteria and appears to exert its growth promoting effects by inactivating (dephosphorylating) luminal ATP and other luminal nucleotide triphosphates. We observed that compared with wild-type mice, IAP-knockout mice have more ATP in their luminal contents, and exogenous IAP can reverse the ATP-mediated inhibition of bacterial growth in the isolated intestinal loop. In conclusion, IAP appears to promote the growth of intestinal commensal bacteria by inhibiting the concentration of luminal nucleotide triphosphates.

Intestinal alkaline phosphatase inhibits the proinflammatory nucleotide uridine diphosphate

Uridine diphosphate (UDP) is a proinflammatory nucleotide implicated in inflammatory bowel disease. Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor capable of inhibiting intestinal inflammation. We used the malachite green assay to show that IAP dephosphorylates UDP. To study the anti-inflammatory effect of IAP, UDP or other proinflammatory ligands (LPS, flagellin, Pam3Cys, or TNF-α) in the presence or absence of IAP were applied to cell cultures, and IL-8 was measured. UDP caused dose-dependent increase in IL-8 release by immune cells and two gut epithelial cell lines, and IAP treatment abrogated IL-8 release. Costimulation with UDP and other inflammatory ligands resulted in a synergistic increase in IL-8 release, which was prevented by IAP treatment. In vivo, UDP in the presence or absence of IAP was instilled into a small intestinal loop model in wild-type and IAP-knockout mice. Luminal contents were applied to cell culture, and cytokine levels were measured in culture supernatant and intestinal tissue. UDP-treated luminal contents induced more inflammation on target cells, with a greater inflammatory response to contents from IAP-KO mice treated with UDP than from WT mice. Additionally, UDP treatment increased TNF-α levels in intestinal tissue of IAP-KO mice, and cotreatment with IAP reduced inflammation to control levels. Taken together, these studies show that IAP prevents inflammation caused by UDP alone and in combination with other ligands, and the anti-inflammatory effect of IAP against UDP persists in mouse small intestine. The benefits of IAP in intestinal disease may be partly due to inhibition of the proinflammatory activity of UDP.

Prevention of antibiotic-associated metabolic syndrome in mice by intestinal alkaline phosphatase.

To examine whether co‐administration of intestinal alkaline phosphatase (IAP) with antibiotics early in life may have a preventive role against metabolic syndrome (MetS) in mice.

Tu1620 Intestinal Alkaline Phosphatase Is an Endogenous Anti-Inflammatory Factor

Introduction: Intestinal alkaline phosphatase (IAP) is an intestinal brush border enzyme known to have the ability to detoxify in-vitro many pro-inflammatory bacterial components, including lipopolysaccharides (LPS), lipoteichoic acid (LTA), flagellin, CpG-DNA and uridine diphosphate (UDP). Gastrointestinal tract inflammation and endotoxemia due to elevated bacterial toxic components in the gut and disruption of intestinal permeability play a crucial role in the development and progression of a wide spectrum of diseases. In this study we sought to determine whether the endogenous IAP enzyme functions as an anti-inflammatory factor. Methods: We established a novel intestinal loop model to study the impact of endogenous IAP on the inflammatory activity of different bacterial components within a physiologic in vivo environment. The model was set up in wild type (WT) vs. IAP knockout (KO) mice of approximately 25 grams (n=5 for all groups). In another setting, we applied a fast (48 hours) vs. fed mouse model. Under general anesthesia, a 5 cm segment of proximal jejunum was carefully tied off at the proximal and distal ends, to isolate the loop. Different concentrations of LPS (100 ng/ml), LTA (5 μg/ml), flagellin (100 ng/ml), CpG-DNA (100 μg/ml) or UDP (1 mM) were injected into the loop and the luminal content was collected 2 hours later. Then, the supernatants were applied to RAW264.7 murine macrophage cells in triplicate and incubated overnight. LPS, LTA, Flagellin, CpG-DNA or UDP were directly applied to the cells as positive controls and endotoxin-free water was applied as a negative control. Tumor necrosis factor-alpha (TNF-α) levels were subsequently measured by sandwich ELISA. Results: All studied bacterial components induced a marked increase in TNFα levels from the RAW264.7 cells, whereas little TNF-α was seen in the case of endotoxinfree water alone. The luminal contents from the WT mice resulted in significantly lower TNF-α levels compared to the luminal contents from the KO mice for all studied bacterial toxins: LPS (600.9±75.47 vs. 946.2±55.99 pg/ml, p= 0.006), LTA (223.1±62.85 vs. 536.2±54.64 pg/ml, p= 0.005), flagellin (679.9±60.05 vs. 1008.8±61.15 pg/ml, p= 0.005), CpG-DNA (638.7±61.81 vs. 949.6±57.36 pg/ml , p= 0.006) and UDP (212.5±15.77 vs. 312.6±26.60 pg/ml, p= 0.012). Luminal contents from fed mice resulted in lower TNF-a levels compared to fasted mice for LPS (585.4±76.35 vs. 900.2±63.62 pg/ml, p=0.013). Conclusions: IAP detoxifies and prevents the inflammatory effects of LPS, LTA, flagellin, CpG-DNA and UDP in the gut. The loss of IAP expression that occurs with fasting could be responsible for the systemic sepsis syndrome seen in critically ill patients.