Sulaiman R. Hamarneh

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.

Intestinal Alkaline Phosphatase Prevents Antibiotic-Induced Susceptibility to Enteric Pathogens

Objective:To determine the efficacy of oral supplementation of the gut enzyme intestinal alkaline phosphatase (IAP) in preventing antibiotic-associated infections from Salmonella enterica serovar Typhimurium (S. Typhimurium) and Clostridium difficile. Background:The intestinal microbiota plays a pivotal role in human health and well-being. Antibiotics inherently cause dysbiosis, an imbalance in the number and composition of intestinal commensal bacteria, which leads to susceptibility to opportunistic bacterial infections. Previously, we have shown that IAP preserves the normal homeostasis of intestinal microbiota and that oral supplementation with calf IAP (cIAP) rapidly restores the normal gut flora. We hypothesized that oral IAP supplementation would protect against antibiotic-associated bacterial infections. Methods:C57BL/6 mice were treated with antibiotic(s) ± cIAP in the drinking water, followed by oral gavage of S. Typhimurium or C. difficile. Mice were observed for clinical conditions and mortality. After a defined period of time, mice were killed and investigated for hematological, inflammatory, and histological changes. Results:We observed that oral supplementation with cIAP during antibiotic treatment protects mice from infections with S. Typhimurium as well as with C. difficile. Animals given IAP maintained their weight, had reduced clinical severity and gut inflammation, and showed improved survival. Conclusions:Oral IAP supplementation protected mice from antibiotic-associated bacterial infections. We postulate that oral IAP supplementation could represent a novel therapy to protect against antibiotic-associated diarrhea (AAD), C. difficile-associated disease (CDAD), and other enteric infections in humans.

A Novel Approach to Maintain Gut Mucosal Integrity Using an Oral Enzyme Supplement

Objective:To determine the role of intestinal alkaline phosphatase (IAP) in enteral starvation-induced gut barrier dysfunction and to study its therapeutic effect as a supplement to prevent gut-derived sepsis. Background:Critically ill patients are at increased risk for systemic sepsis and, in some cases, multiorgan failure leading to death. Years ago, the gut was identified as a major source for this systemic sepsis syndrome. Previously, we have shown that IAP detoxifies bacterial toxins, prevents endotoxemia, and preserves intestinal microbiotal homeostasis. Methods:WT and IAP-KO mice were used to examine gut barrier function and tight junction protein levels during 48-hour starvation and fed states. Human ileal fluid samples were collected from 20 patients postileostomy and IAP levels were compared between fasted and fed states. To study the effect of IAP supplementation on starvation-induced gut barrier dysfunction, WT mice were fasted for 48 hours +/− IAP supplementation in the drinking water. Results:The loss of IAP expression is associated with decreased expression of intestinal junctional proteins and impaired barrier function. For the first time, we demonstrate that IAP expression is also decreased in humans who are deprived of enteral feeding. Finally, our data demonstrate that IAP supplementation reverses the gut barrier dysfunction and tight junction protein losses due to a lack of enteral feeding. Conclusions:IAP is a major regulator of gut mucosal permeability and is able to ameliorate starvation-induced gut barrier dysfunction. Enteral IAP supplementation may represent a novel approach to maintain bowel integrity in critically ill patients.

Inhibition of the gut enzyme intestinal alkaline phosphatase may explain how aspartame promotes glucose intolerance and obesity in mice

Diet soda consumption has not been associated with tangible weight loss. Aspartame (ASP) commonly substitutes sugar and one of its breakdown products is phenylalanine (PHE), a known inhibitor of intestinal alkaline phosphatase (IAP), a gut enzyme shown to prevent metabolic syndrome in mice. We hypothesized that ASP consumption might contribute to the development of metabolic syndrome based on PHEs inhibition of endogenous IAP. The design of the study was such that for the in vitro model, IAP was added to diet and regular soda, and IAP activity was measured. For the acute model, a closed bowel loop was created in mice. ASP or water was instilled into it and IAP activity was measured. For the chronic model, mice were fed chow or high-fat diet (HFD) with/without ASP in the drinking water for 18 weeks. The results were that for the in vitro study, IAP activity was lower (p < 0.05) in solutions containing ASP compared with controls. For the acute model, endogenous IAP activity was reduced by 50% in the ASP group compared with controls (0.2 ± 0.03 vs 0.4 ± 0.24) (p = 0.02). For the chronic model, mice in the HFD + ASP group gained more weight compared with the HFD + water group (48.1 ± 1.6 vs 42.4 ± 3.1, p = 0.0001). Significant difference in glucose intolerance between the HFD ± ASP groups (53 913 ± 4000.58 (mg·min)/dL vs 42 003.75 ± 5331.61 (mg·min)/dL, respectively, p = 0.02). Fasting glucose and serum tumor necrosis factor-alpha levels were significantly higher in the HFD + ASP group (1.23- and 0.87-fold increases, respectively, p = 0.006 and p = 0.01). In conclusion, endogenous IAPs protective effects in regard to the metabolic syndrome may be inhibited by PHE, a metabolite of ASP, perhaps explaining the lack of expected weight loss and metabolic improvements associated with diet drinks.

Selective and reversible suppression of intestinal stem cell differentiation by pharmacological inhibition of BET bromodomains.

Absorptive and secretory cells of the small intestine are derived from a single population of Lgr5-expressing stem cells. While key genetic pathways required for differentiation into specific lineages have been defined, epigenetic programs contributing to this process remain poorly characterized. Members of the BET family of chromatin adaptors contain tandem bromodomains that mediate binding to acetylated lysines on target proteins to regulate gene expression. In this study, we demonstrate that mice treated with a small molecule inhibitor of BET bromodomains, CPI203, exhibit greater than 90% decrease in tuft and enteroendocrine cells in both crypts and villi of the small intestine, with no changes observed in goblet or Paneth cells. BET bromodomain inhibition did not alter the abundance of Lgr5-expressing stem cells in crypts, but rather exerted its effects on intermediate progenitors, in part through regulation of Ngn3 expression. When BET bromodomain inhibition was combined with the chemotherapeutic gemcitabine, pervasive apoptosis was observed in intestinal crypts, revealing an important role for BET bromodomain activity in intestinal homeostasis. Pharmacological targeting of BET bromodomains defines a novel pathway required for tuft and enteroendocrine differentiation and provides an important tool to further dissect the progression from stem cell to terminally differentiated secretory cell.

Intestinal alkaline phosphatase at the crossroad of intestinal health and disease – a putative role in type 1 diabetes

Patients with type 1 diabetes have shown an increase in circulating cytokines, altered lipoprotein metabolism and signs of vascular dysfunction in response to high‐fat meals. Intestinal alkaline phosphatase (IAP) regulates lipid transport and inflammatory responses in the gastrointestinal tract. We therefore hypothesized that changes in IAP activity could have profound effects on gut metabolic homeostasis in patients with type 1 diabetes.

Relationship between serum IGF-1 and skeletal muscle IGF-1 mRNA expression to phosphocreatine recovery after exercise in obese men with reduced GH.

CONTEXT GH and IGF-1 are believed to be physiological regulators of skeletal muscle mitochondria. OBJECTIVE The objective of this study was to examine the relationship between GH/IGF-1 and skeletal muscle mitochondria in obese subjects with reduced GH secretion in more detail. DESIGN Fifteen abdominally obese men with reduced GH secretion were treated for 12 weeks with recombinant human GH. Subjects underwent (31)P-magnetic resonance spectroscopy to assess phosphocreatine (PCr) recovery as an in vivo measure of skeletal muscle mitochondrial function and percutaneous muscle biopsies to assess mRNA expression of IGF-1 and mitochondrial-related genes at baseline and 12 weeks. RESULTS At baseline, skeletal muscle IGF-1 mRNA expression was significantly associated with PCr recovery (r = 0.79; P = .01) and nuclear respiratory factor-1 (r = 0.87; P = .001), mitochondrial transcription factor A (r = 0.86; P = .001), peroxisome proliferator-activated receptor (PPAR)γ (r = 0.72; P = .02), and PPARα (r = 0.75; P = .01) mRNA expression, and trended to an association with PPARγ coactivator 1-α (r = 0.59; P = .07) mRNA expression. However, serum IGF-1 concentration was not associated with PCr recovery or any mitochondrial gene expression (all P > .10). Administration of recombinant human GH increased both serum IGF-1 (change, 218 ± 29 μg/L; P < .0001) and IGF-1 mRNA in muscle (fold change, 2.1 ± 0.3; P = .002). Increases in serum IGF-1 were associated with improvements in total body fat (r = -0.53; P = .04), trunk fat (r = -0.55; P = .03), and lean mass (r = 0.58; P = .02), but not with PCr recovery (P > .10). Conversely, increase in muscle IGF-1 mRNA was associated with improvements in PCr recovery (r = 0.74; P = .02), but not with body composition parameters (P > .10). CONCLUSION These data demonstrate a novel association of skeletal muscle mitochondria with muscle IGF-1 mRNA expression, but independent of serum IGF-1 concentrations.

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.