Intestinal alkaline phosphatase targets the gut barrier to prevent aging.

Gut barrier dysfunction and gut-derived chronic inflammation play crucial roles in human aging. The gut brush border enzyme intestinal alkaline phosphatase (IAP) functions to inhibit inflammatory mediators and also appears to be an important positive regulator of gut barrier function and microbial homeostasis. We hypothesized that this enzyme could play a critical role in regulating the aging process. We tested the role of several IAP functions for prevention of age-dependent alterations in intestinal homeostasis by employing different loss-of-function and supplementation approaches. In mice, there is an age-related increase in gut permeability that is accompanied by increases in gut-derived portal venous and systemic inflammation. All these phenotypes were significantly more pronounced in IAP-deficient animals. Oral IAP supplementation significantly decreased age-related gut permeability and gut-derived systemic inflammation, resulted in less frailty, and extended lifespan. Furthermore, IAP supplementation was associated with preserving the homeostasis of gut microbiota during aging. These effects of IAP were also evident in a second model system, Drosophilae melanogaster. IAP appears to preserve intestinal homeostasis in aging by targeting crucial intestinal alterations, including gut barrier dysfunction, dysbiosis, and endotoxemia. Oral IAP supplementation may represent a novel therapy to counteract the chronic inflammatory state leading to frailty and age-related diseases in humans.

Loss of Intestinal Alkaline Phosphatase Leads to Distinct Chronic Changes in Bone Phenotype.

BACKGROUND: The gut is becoming increasingly recognized as the source of various systemic diseases, and recently, it has been linked to bone metabolism via the so-called gut-bone axis. The microbiome and gut-derived mediators are thought to impact upon bone metabolism, and administration of probiotics has been shown to have beneficial effects in bone. The gut brush border enzyme intestinal alkaline phosphatase (IAP) plays an important role in controlling calcium absorption, inhibiting lipopolysaccharides, and other inflammatory mediators responsible for endotoxemia and appears to preserve the normal gut microbiota. Interestingly, IAP-deficient mice (AKP3-/-) also display a significant decrease in fecal Lactobacillus, the genus shown to be beneficial to bone. MATERIALS AND METHODS: IAP mRNA levels in mouse bone were measured using quantitative real-time polymerase chain reaction. Femurs of IAP-knockout (KO) and wild-type (WT) mice were analyzed by microcomputed tomography and histopathology. Serum levels of alkaline phosphatase, calcium, and phosphorus were measured. Target cell response upon exposure to serum from IAP-KO and WT mice was quantified using primary bone marrow macrophages. RESULTS: IAP was not significantly expressed in bones of WT or KO animals. IAP (alkaline phosphatase 3) expression in bone was vanishingly low compared to the duodenum (bone versus duodenum, 56.9 ± 17.7 versus 25,430.3 ± 10,884.5 relative expression, P = 0.01). Bone histology of younger IAP-KO and WT animals was indistinguishable, whereas older IAP-deficient mice showed a distinctly altered phenotype on histology and computed tomography scan. Younger KO mice did not display any abnormal levels in blood chemistry. Older IAP-KO animals showed an isolated increase in serum alkaline phosphatase levels reflecting an environment of active bone formation (IAP-WT versus IAP-KO, 80 ± 27.4 U/I versus 453 ± 107.5 U/I, P = 0.004). There was no significant difference in serum calcium or phosphorus levels between KO and WT mice. Serum from IAP-KO mice induced a significantly higher inflammatory target cell response. CONCLUSIONS: Through its multiple functions, IAP seems to play a crucial role in connecting the gut to the bone. IAP deficiency leads to chronic changes in bone formation, most likely through dysbiosis and systemic dissemination of proinflammatory mediators.

Intestinal Alkaline Phosphatase Attenuates Alcohol-Induced Hepatosteatosis in Mice.

BACKGROUND AND AIMS: Bacterially derived factors from the gut play a major role in the activation of inflammatory pathways in the liver and in the pathogenesis of alcoholic liver disease. The intestinal brush-border enzyme intestinal alkaline phosphatase (IAP) detoxifies a variety of bacterial pro-inflammatory factors and also functions to preserve gut barrier function. The aim of this study was to investigate whether oral IAP supplementation could protect against alcohol-induced liver disease. METHODS: Mice underwent acute binge or chronic ethanol exposure to induce alcoholic liver injury and steatosis ± IAP supplementation. Liver tissue was assessed for biochemical, inflammatory, and histopathological changes. An ex vivo co-culture system was used to examine the effects of alcohol and IAP treatment in regard to the activation of hepatic stellate cells and their role in the development of alcoholic liver disease. RESULTS: Pretreatment with IAP resulted in significantly lower serum alanine aminotransferase compared to the ethanol alone group in the acute binge model. IAP treatment attenuated the development of alcohol-induced fatty liver, lowered hepatic pro-inflammatory cytokine and serum LPS levels, and prevented alcohol-induced gut barrier dysfunction. Finally, IAP ameliorated the activation of hepatic stellate cells and prevented their lipogenic effect on hepatocytes. CONCLUSIONS: IAP treatment protected mice from alcohol-induced hepatotoxicity and steatosis. Oral IAP supplementation could represent a novel therapy to prevent alcoholic-related liver disease in humans.

Intestinal alkaline phosphatase decreases intraperitoneal adhesion formation.

BACKGROUND: Damage to the peritoneum initiates an inflammatory response leading to the formation of adhesions, which subsequently cause significant morbidity in some patients. Intestinal alkaline phosphatase (IAP) is a gut enzyme capable of detoxifying various inflammatory mediators such as lipopolysaccharide, lipoteichoic acid, CpG DNA, and adenosine triphosphate. In this study, we aimed to examine the anti-inflammatory effects of IAP on postoperative adhesions in mice. METHODS: C57BL/6 mice were subjected to a midline laparotomy and then six musculoperitoneal buttons (MPBs) were created by pinching and ligating the peritoneum and underlying muscle. The buttons were half-excised and E-cauterized, and then cecal abrasion was performed. Five hundred microliters of vehicle with IAP 5000 U or vehicle alone were applied over the peritoneal cavity. In some experiments, the mice were euthanized on the first and second postoperative day (POD), and cytokines analysis was done on the MPB, peritoneal tissue, and peritoneal fluid. In separate experiments, the mice were sacrificed on the 21st POD, and adhesion to each button was scored based on type and tenacity. RESULTS: IAP group mice had significantly lower adhesion scores compared with controls (21.5 ± 1.7 versus 13.2 ± 1.3; P = 0.0014, n = 15). MPB from IAP group mice had significantly lower interleukin-1ß and tumor necrosis factor-α protein level compared to control mice (105.66 ± 4.5 versus 69.8 ± 4.8 versus pg/mg, P = 0.0001; 45.25 ± 2.8 pg/mg versus 24.88 ± 4.1 pg/mg; P = 0.0007, n = 10). IAP treatment significantly decreased interleukin-1ß and tumor necrosis factor-α mRNA expression in MPB in the first POD (1.14 ± 0.25 versus 0.33 ± 0.07; P = 0.0068; 1.33 ± 0.31 versus 0.33 ± 0.08; P = 0.0064, n = 10). CONCLUSIONS: Application of IAP during laparotomy could represent a novel approach to prevent postoperative adhesions.

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 PHE's 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 IAP's 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.

Intestinal Alkaline Phosphatase Regulates Tight Junction Protein Levels.

BACKGROUND: Intestinal alkaline phosphatase (IAP) plays a pivotal role in maintaining gut health and well-being. Oral supplementation with IAP in mice improves gut barrier function and prevents luminal proinflammatory factors from gaining access to the circulation. In this study, we sought to explore the relationship between IAP and tight junction protein (TJP) expression and function. STUDY DESIGN: The effect of IAP deletion on TJP levels was studied in mouse embryonic fibroblasts (MEFs) generated from IAP-knockout and wild type mice. Regulation of TJPs by IAP was assayed in the human colon cancer Caco-2 and T84 cells by overexpressing the human IAP gene. Tight junction protein levels and localization were measured by using RT q-PCR and antibodies targeting the specific TJPs. Finally, the effect of IAP on inflammation-induced intestinal permeability was measured by in vitro trans-well epithelial electrical resistance (TEER). RESULTS: Intestinal alkaline phosphatase gene deletion in MEFs resulted in significantly lower levels of ZO-1, ZO-2, and Occludin compared with levels in wild-type control cells; IAP overexpression in Caco-2 and T84 cells resulted in approximate 2-fold increases in the mRNA levels of ZO-1 and ZO-2. The IAP treatment ameliorated lipopolysaccharide-induced increased permeability in the Caco-2 trans-well system. Furthermore, IAP treatment preserved the localization of the ZO-1 and Occludin proteins during inflammation and was also associated with improved epithelial barrier function. CONCLUSIONS: Intestinal alkaline phosphatase is a major regulator of gut mucosal permeability and appears to work at least partly through improving TJP levels and localization. These data provide a strong foundation to develop IAP as a novel therapy to maintain gut barrier function.

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.

FNDC5 relates to skeletal muscle IGF-I and mitochondrial function and gene expression in obese men with reduced growth hormone.

OBJECTIVE: To investigate the relationship of skeletal muscle FNDC5 mRNA expression and circulating irisin to the GH/IGF-I axis and to skeletal muscle mitochondrial function and mitochondria-related gene expression in obese men. DESIGN: Fifteen abdominally obese men with reduced growth hormone received 12weeks of recombinant human GH (rhGH). Before and after treatment, they underwent (31)P-magnetic resonance spectroscopy to evaluate phosphocreatine (PCr) recovery as a measure of mitochondrial function and skeletal muscle biopsy to assess expression of mitochondrial-related genes. Serum irisin and IGF-I and skeletal muscle FNDC5 and IGF-I mRNA were measured. RESULTS: At baseline, skeletal muscle FNDC5 mRNA was significantly and positively associated with IGF-I mRNA (ρ=0.81, P=0.005) and rate of PCr recovery (ρ=0.79, P=0.006). Similar relationships of circulating irisin to IGF-I mRNA (ρ=0.63, P=0.05) and rate of PCr recovery (ρ=0.48, P=0.08) were demonstrated, but were not as robust as those with muscle FNDC5 expression. Both serum irisin and skeletal muscle FNDC5 mRNA were significantly associated with PPARγ (ρ=0.73, P=0.02 and ρ=0.85, P=0.002), respectively. In addition, FNDC5 mRNA was correlated with skeletal muscle PGC-1α (ρ=0.68, P=0.03), NRF1 (ρ=0.66, P=0.04) and TFAM (ρ=0.79, P=0.007) mRNA. Neither serum irisin nor muscle mRNA expression of FNDC5 changed with rhGH treatment. CONCLUSION: These novel data in skeletal muscle demonstrate that local expression of FNDC5 is associated with mRNA expression of IGF-I and mitochondrial function and mitochondria-related gene expression in obese subjects with reduced growth hormone and suggest a potential role for FNDC5 acting locally in muscle in a low GH state. Further studies are needed to clarify the relationship between the GH/IGF-I axis and irisin.

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.

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.

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 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.

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 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.