Madhu S. Malo

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 preserves the normal homeostasis of gut microbiota

Background and aims The intestinal microbiota plays a critical role in maintaining human health; however, the mechanisms governing the normal homeostatic number and composition of these microbes are largely unknown. Previously it was shown that intestinal alkaline phosphatase (IAP), a small intestinal brush border enzyme, functions as a gut mucosal defence factor limiting the translocation of gut bacteria to mesenteric lymph nodes. In this study the role of IAP in the preservation of the normal homeostasis of the gut microbiota was investigated. Methods Bacterial culture was performed in aerobic and anaerobic conditions to quantify the number of bacteria in the stools of wild-type (WT) and IAP knockout (IAP-KO) C57BL/6 mice. Terminal restriction fragment length polymorphism, phylogenetic analyses and quantitative real-time PCR of subphylum-specific bacterial 16S rRNA genes were used to determine the compositional profiles of microbiotas. Oral supplementation of calf IAP (cIAP) was used to determine its effects on the recovery of commensal gut microbiota after antibiotic treatment and also on the colonisation of pathogenic bacteria. Results IAP-KO mice had dramatically fewer and also different types of aerobic and anaerobic microbes in their stools compared with WT mice. Oral supplementation of IAP favoured the growth of commensal bacteria, enhanced restoration of gut microbiota lost due to antibiotic treatment and inhibited the growth of a pathogenic bacterium (Salmonella typhimurium). Conclusions IAP is involved in the maintenance of normal gut microbial homeostasis and may have therapeutic potential against dysbiosis and pathogenic infections.

Intestinal Alkaline Phosphatase Has Beneficial Effects in Mouse Models of Chronic Colitis

Background: The brush border enzyme intestinal alkaline phosphatase (IAP) functions as a gut mucosal defense factor and is protective against dextran sulfate sodium (DSS)‐induced acute injury in rats. The present study evaluated the potential therapeutic role for orally administered calf IAP (cIAP) in two independent mouse models of chronic colitis: 1) DSS‐induced chronic colitis, and 2) chronic spontaneous colitis in Wiskott‐Aldrich Syndrome protein (WASP)‐deficient (knockout) mice that is accelerated by irradiation. Methods: The wildtype (WT) and IAP knockout (IAP‐KO) mice received four cycles of 2% DSS ad libitum for 7 days. Each cycle was followed by a 7‐day DSS‐free interval during which mice received either cIAP or vehicle in the drinking water. The WASP‐KO mice received either vehicle or cIAP for 6 weeks beginning on the day of irradiation. Results: Microscopic colitis scores of DSS‐treated IAP‐KO mice were higher than DSS‐treated WT mice (52 ± 3.8 versus 28.8 ± 6.6, respectively, P < 0.0001). cIAP treatment attenuated the disease in both groups (KO = 30.7 ± 6.01, WT = 18.7 ± 5.0, P < 0.05). In irradiated WASP‐KO mice cIAP also attenuated colitis compared to control groups (3.3 ± 0.52 versus 6.2 ± 0.34, respectively, P < 0.001). Tissue myeloperoxidase activity and proinflammatory cytokines were significantly decreased by cIAP treatment. Conclusions: Endogenous IAP appears to play a role in protecting the host against chronic colitis. Orally administered cIAP exerts a protective effect in two independent mouse models of chronic colitis and may represent a novel therapy for human IBD. (Inflamm Bowel Dis 2011)

Identification of specific targets for the gut mucosal defense factor intestinal alkaline phosphatase

Intestinal alkaline phosphatase (IAP) is a small intestinal brush border enzyme that has been shown to function as a gut mucosal defense factor, but its precise mechanism of action remains unclear. We investigated the effects of IAP on specific bacteria and bacterial components to determine its molecular targets. Purulent fluid from a cecal ligation and puncture model, specific live and heat-killed bacteria (Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes), and a variety of proinflammatory ligands (LPS, CpG DNA, Pam-3-Cys, flagellin, and TNF) were incubated with or without calf IAP (cIAP). Phosphate release was determined by using a malachite green assay. The various fluids were applied to target cells (THP-1, parent HT-29, and IAP-expressing HT-29 cells) and IL-8 secretion measured by ELISA. cIAP inhibited IL-8 induction by purulent fluid in THP-1 cells by >35% (P < 0.005). HT29-IAP cells had a reduced IL-8 response specifically to gram-negative bacteria; >90% reduction compared with parent cells (P < 0.005). cIAP had no effect on live bacteria but attenuated IL-8 induction by heat-killed bacteria by >40% (P < 0.005). cIAP exposure to LPS and CpG DNA caused phosphate release and reduced IL-8 in cell culture by >50% (P < 0.005). Flagellin exposure to cIAP also resulted in reduced IL-8 secretion by >40% (P < 0.005). In contrast, cIAP had no effect on TNF or Pam-3-Cys. The mechanism of IAP action appears to be through dephosphorylation of specific bacterial components, including LPS, CpG DNA, and flagellin, and not on live bacteria themselves. IAP likely targets these bacterially derived molecules in its role as a gut mucosal defense factor.

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.

Transcriptional activation of the enterocyte differentiation marker intestinal alkaline phosphatase is associated with changes in the acetylation state of histone H3 at a specific site within its promoter region in vitro.

Enterocyte differentiation is thought to occur through the transcriptional regulation of a small subset of specific genes. A recent growing body of evidence indicates that post-translational modifications of chromatin proteins (histones) play an important role in the control of gene transcription. Previous work has demonstrated that one such modification, histone acetylation, occurs in an in vitro model of enterocyte differentiation, butyrate-treated HT-29 cells. In the present work, we sought to determine if the epigenetic signal of histone acetylation occurs in an identifiable pattern in association with the transcriptional activation of the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). HT-29 cells were maintained under standard culture conditions and differentiated with sodium butyrate. The chromatin immunoprecipitation (ChIP) assay was used to compare the acetylation state of histones associated with specific regions of the IAP promoter in the two cell populations (undifferentiated vs. differentiated). Chromatin was extracted from cells and cleaved by sonication or enzymatic digestion to obtain fragments of approximately 200 to 600 base-pairs, as confirmed by polymerase chain reaction using primers designed to amplify the IAP segments of interest. The ChIP assay selects DNA sequences that are associated with acetylated histones by immunoprecipitation. Unbound segments represent DNA sequences whose histones are not acetylated. After immunoprecipitation, sequences were detected by radiolabeled polymerase chain reaction, and the relative intensity of the bands was quantified by densitometry. The relative acetylation state of histones at specific sites was determined by comparing the ratios of bound/unbound segments. We determined that in a segment of the IAP promoter between −378 and −303 base-pairs upstream from the transcriptional start site, the acetylation state of histone H3 increased twofold in the differentiated, IAP expressing cells, whereas that of histone H4 remained essentially constant. Additionally, at a distant site, between −1378 and −1303 base-pairs, the acetylation state of H3 and H4 did not change appreciably between the undifferentiated and differentiated cells. We conclude that butyrate-induced differentiation is associated with specific and localized changes in the histone acetylation state within the IAP promoter. These changes within the endogenous IAP gene may underlie its transcriptional activation in the context of the enterocyte differentiation program.

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.

A High Level of Intestinal Alkaline Phosphatase Is Protective Against Type 2 Diabetes Mellitus Irrespective of Obesity

Mice deficient in intestinal alkaline phosphatase (IAP) develop type 2 diabetes mellitus (T2DM). We hypothesized that a high level of IAP might be protective against T2DM in humans. We determined IAP levels in the stools of 202 diabetic patients and 445 healthy non-diabetic control people. We found that compared to controls, T2DM patients have approx. 50% less IAP (mean +/− SEM: 67.4 +/− 3.2 vs 35.3 +/− 2.5 U/g stool, respectively; p < 0.000001) indicating a protective role of IAP against T2DM. Multiple logistic regression analyses showed an independent association between the IAP level and diabetes status. With each 25 U/g decrease in stool IAP, there is a 35% increased risk of diabetes. The study revealed that obese people with high IAP (approx. 65 U/g stool) do not develop T2DM. Approx. 65% of the healthy population have < 65.0 U/g stool IAP, and predictably, these people might have ‘the incipient metabolic syndrome’, including ‘incipient diabetes’, and might develop T2DM and other metabolic disorders in the near future. In conclusion, high IAP levels appear to be protective against diabetes irrespective of obesity, and a ‘temporal IAP profile’ might be a valuable tool for predicting ‘the incipient metabolic syndrome’, including ‘incipient diabetes’.