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.etabolic syndrome is a complex syndrome composed of a cluster of disorders that includes obesity, glucose intolerance, insulin resistance, abnormal lipid profile (dyslipidemia), fatty liver, and hypertension (1, 2). Metabolic syndrome leads to type 2 diabetes, atherosclerosis, and nonalcoholic fatty liver disease (1, 2). Approximately 35-39% of the US population suffers from the syndrome (3). This epidemic of metabolic syndrome has devastating consequences in terms of mortality, morbidity, and total healthcare expenditures (4).Recently, "metabolic endotoxemia" has been proposed to be central to the pathogenesis of metabolic syndrome. The Gramnegative bacterial cell wall component lipopolysaccharide (LPS) is known as endotoxin, and metabolic endotoxemia is defined as a two-to threefold persistent increase in circulating endotoxin concentrations above the normal levels (5). Metabolic endotoxemia leads to low-grade systemic inflammation as evidenced by increased serum levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1, and IL-6 (5). It is well recognized that chronic inflammation causes damage to pancreatic beta cells (6), hepatocytes (7), and vascular endothelial cells (8), and dysfunction of these cells is thought to contribute to metabolic syndrome.A high-fat diet (HFD) has been shown to cause metabolic endotoxemia in animals and humans (5, 9), but the underlying molecular mechanisms remain incompletely understood. Ghoshal et al. (10) demonstrated that intestinal epithelial cells (enterocytes) internalize LPS from the apical surface, which is then transported to the Golgi apparatus where it complexes with chylomicrons, the lipoproteins that transport the absorbed longchain fatty acids in enterocytes. The chylomicron-LPS complex is then secreted into mesenteric lymph and makes its way into the systemic circulation. Excess chylomicron formation during highfat feeding leads to prolonged chylomicronemia (complexed with LPS) that ultimately induces systemic inflammation. Also, it has been shown that an HFD causes local intestinal inflammation (11). Systemic and local inf...

IAP is involved in the maintenance of normal gut microbial homeostasis and may have therapeutic potential against dysbiosis and pathogenic infections.

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’.

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 (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.

We have examined the role that the transcription factor gut-enriched Krüppel-like factor (KLF4 or GKLF) plays in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). A yeast one-hybrid screen was used to identify proteins interacting with a previously identified cis-element (IF-III) located within the human IAP gene promoter. DNA-protein interactions were determined by using EMSA. Northern blot analysis was used to study RNA expression in human colon cancer RKO cells engineered to overexpress KLF4. Transient transfections with IAP-luciferase reporter constructs were used to characterize the mechanisms by which KLF4 activates IAP transcription. The yeast one-hybrid screen and EMSA identified KLF4 as binding to IF-III. RKO cells induced to overexpress KLF4 demonstrated a corresponding dose-dependent increase in IAP expression, and EMSA with nuclear extract from these cells confirmed that KLF4 binds to the IF-III element. Transient transfections revealed that KLF4 transactivated the IAP gene largely via a critical segment in the IAP promoter that includes the IF-III cis-element. Mutant KLF4 constructs failed to fully activate IAP. We have identified the enterocyte differentiation marker IAP as a KLF4 target gene. IAP transactivation by KLF4 is likely mediated through a critical region located within the proximal IAP promoter region.

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 wild-type (WT) and IAP knockout (IAP-KO) mice received 4 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 vs. 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 vs. 6.2 ± 0.34, respectively, P < 0.001). Tissue myeloperoxidase activity and pro-inflammatory 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.

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