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Intestinal alkaline phosphatase: novel functions and
protective effects
Jean-Paul Lallès
Important protective roles of intestinal alkaline phosphatase (IAP) – including
regulation of intestinal surface pH, absorption of lipids, detoxification of free
nucleotides and bacterial lipopolysaccharide, attenuation of intestinal
inflammation, and possible modulation of the gut microbiota – have been reviewed
recently. IAP is modulated by numerous nutritional factors. The present review
highlights new findings on the properties of IAP and extends the list of its protective
functions. Critical assessment of data suggests that some IAP properties are a direct
result of dephosphorylation of proinflammatory moieties, while others (e.g., gut
barrier protection and microbiota shaping) may be secondary to IAP-mediated
downregulation of inflammation. IAP and tissue-nonspecific alkaline phosphatase
isoforms characterize the small intestine and the colon, respectively. Gastrointestinal
administration of exogenous IAP ameliorates gut inflammation and favors gut
tissue regeneration, whereas enteral and systemic IAP administration attenuates
systemic inflammation only. Finally, the IAP gene family has a strong evolutionary
link to food-driven changes in gastrointestinal tract anatomy and microbiota
composition. Therefore, stimulation of IAP activity by dietary intervention is a goal
for preserving gut homeostasis and health by minimizing low-grade inflammation.
© 2013 International Life Sciences Institute
INTRODUCTION
Obesity and metabolic disorders, including insulin resis-
tance, type-2 diabetes, hypertension, and cardiovascular
disease, are becoming more prevalent worldwide due to
lifestyle changes associated with overconsumption of
Western diets.1Bacterial lipopolysaccharide (LPS)
has been proposed as a causal link in diet-induced obesity
(DIO) because of its increased passage across the
intestinal barrier following high-fat diet intake, its
growth-promoting effects on adipose tissue, and its
proinflammatory properties.2More recently, a survey
pointed to the possible involvement of intestinal alkaline
phosphatase (IAP) in the development of obesity,3and
very recent experimental evidence demonstrates the
ability of IAP to prevent fat-induced metabolic syndrome
in mice.4IAP has been recently shown to display anti-
inflammatory properties that derive from its
dephosphorylating activity, which results in detoxifica-
tion of LPS and repression of the downstream Toll-like
receptor (TLR)-4-dependent and MyD88-dependent
inflammatory cascade.5,6 Given the large number of
important reports on IAP function in recent decades, the
first review on IAP and its nutritional modulation was
published 3 years ago.6Since then, many additional
reports have emerged, justifying an update on this topic.
Four major functions of IAP were described previously:
1) regulation of bicarbonate secretion and duodenal
surface pH; 2) modulation of intestinal LCFA absorption;
3) detoxification of LPS, resulting in amelioration of
intestinal and systemic inflammation; and 4) regulation
of gut microbial communities and their translocation
Affiliation: J-P Lallès is with the Institut National de la Recherche Agronomique, UR1341, Alimentation et Adaptations Digestives,
Nerveuses et Comportementales (ADNC), Saint-Gilles, France.
Correspondence: J-P Lallès, INRA UR1341, ADNC, Domaine de la Prise, F-35590 Saint-Gilles, France. E-mail: Jean-Paul.Lalles@rennes.inra.fr.
Phone: +33-2-23485359. Fax: +33-2-23485080.
Key words: alkaline phosphatase, disease, intestine, microbiota, nutrition
bs_bs_banner
Special Article
doi:10.1111/nure.12082
Nutrition Reviews® Vol. 72(2):82–9482
across the gut barrier.6Here,an update on these functions
is provided, in addition to novel functions ascribed to IAP
(Figure 1), and the entire literature is critically analyzed.
All these data, including a few translational studies per-
formed in humans, strongly suggest that IAP is a major
regulatory enzyme of gut homeostasis and health. As diet
is implicated as a causal determinant in metabolic syn-
drome and obesity,6knowledge of IAP modulation by
dietary factors is also updated.
NOVEL PROPERTIES OF IAP
IAP is dynamically secreted apically and basolaterally by
the enterocyte. IAP has been reported to modulate intes-
tinal absorption of dietary LCFAs by phosphorylating-
dephosphorylating the fatty acid (FA) transporter CD36.
It also limits fat-induced inflammation, metabolic syn-
drome, and obesity in rodent models.The involvement of
IAP in some of these properties, however, is probably
secondary to the anti-inflammatory action of IAP.
Presence of IAP in apical lipid-rich cellular
microdomains, and dynamic apical and basolateral
secretion of IAP
Lipid-rich microdomains, also called lipid rafts, are apical
membrane structures of intestinal epithelial cells (IECs)
that are rich in IAP and other functional proteins.7,8 IAP is
selectively endocytosed at the apical side of enterocytes
during intestinal fat absorption.9Recent investigations
suggest that lumenal FAs are inserted specifically into
these rafts,10 but the consequences of this insertion on raft
IAP dynamics remains unclear. Importantly, IAP-rich
lipid rafts have been recently linked to gut permeability
and inflammation.11 A kinetic study of different mouse
models of colitis revealed that lipid raft disruption pre-
cedes alteration in colonic permeability. Moreover,
IAP distribution into raft-enriched membrane fractions
tended to differ between ulcerative colitis patients in
remission and controls.11 Therefore, additional studies to
clarify the functional consequences of lipid-raft-
associated IAP in inflammation are needed.
It was previously reported that IAP is secreted from
the basolateral domain of IECs via so-called surfactant-
like particles, e.g.,during intestinal fat absorption.6At the
apical domain, however, IAP was thought to be released
into the intestinal lumen via a mechanism involving the
hydrolysis of its glycosyl-phosphatidyl-inositol linkage
and lysophosphatidylcholine.12 Recently described new
aspects of apical mechanisms of IAP release show that
enterocyte microvilli secrete 90-nm-diameter lumenal
vesicles highly enriched in functional proteins, including
IAP that preferentially locates in lipid rafts.13 These
vesicles were shown to dephosphorylate LPS from
various gram-negative bacteria; more importantly, they
prevented the adhesion of both pathogens and commen-
sal bacteria to IECs in vitro.14 Conversely, the presence of
pathogens (e.g., enteropathogenic Escherichia coli) stimu-
lated apical secretion of microvillar vesicles.
Collectively, these data provide new insight into a
unique defense mechanism: the secretion of IAP-rich
vesicles from both lumenal and basolateral domains of the
enterocytes (Figure 2). Lumenal vesicles influence gut
concentrations of pathogen-associated microbial patterns
(PAMPs), whereas basolateral-derived vesicles inactivate
PAMPs as they translocate into the interior milieu.Finally,
intracellular IAP blunts PAMP-stimulated nuclear factor
Figure 1 Summary of properties of intestinal alkaline phosphatase.
Abbreviations: IAP, intestinal alkaline phosphatase; LPS, lipopolysaccharide; NEC, necrotizing enterocolitis; TNAP,
tissue-nonspecific alkaline phosphatase.
Nutrition Reviews® Vol. 72(2):82–94 83
kappa B-mediated inflammatory responses.Whether IAP
has other functions within these lipid rafts is presently
unknown.
Ability of IAP to limit LCFA-induced inflammation,
metabolic syndrome, and obesity, and possible role of
IAP in the control of intestinal absorption of LCFAs
Modulation of intestinal absorption of LCFAs. A possible
role of IAP in intestinal FA absorption was proposed when
mice in which the Akp3 gene coding for the IAP isoform
was deleted were shown to absorb more fat and to gain
more weight than wild-type controls.15,16 Although a link
between IAP and FA transporter CD36 was suspected,6the
underlying mechanisms have, until recently, remained
obscure. Global IAP (gIAP),the product of the Akp6 gene
expressed throughout the small intestine, would be
responsible for the increased fat uptake in IAP Akp3
knockout mice.17,18 Both gIAP (but not duodenal IAP, or
dIAP) and CD36 were specifically increased in the
jejunum of mice, and gIAP (but not dIAP) was shown to
coprecipitate with CD36 when the fat content of the diet
was increased from 15% to 45%. It was thus suggested that
gIAP phosphorylates and dephosphorylates CD36, the
latter being responsible for an enhanced capacity for fat
absorption.17,18 However, this mechanism is likely to have
only a marginal effect on fat absorption, since deletion of
the CD36 gene in mice did not affect uptake of LCFAs.19An
alternative explanation has been proposed recently, sug-
gesting that increased FA absorption observed in Akp3
knockout mice could result from lower lumenal surface
pH and high free ATP (adenosine triphosphate) concen-
trations.20 Thus,the direct role of IAP isoform (from Akp3
gene) in the modulation of intestinal absorption of LCFAs
has been questioned.
Control of fat-induced inflammation, metabolic syndrome,
and obesity. Consumption of high-fat diets is known to
increase IAP, and this may be a mechanism of adaptation
to the parallel increase in intestinal entry of LPS.4Mice and
rats prone to DIO were reported to display lower intestinal
IAP activity after consuming a high-fat diet for a few
weeks, while IAP activity remained unaltered in DIO-
resistant animals.21,22 As this did not appear to be geneti-
cally driven, reduced IAP activity in DIO-prone rats was
suggested to be a consequence of fat-induced inflamma-
tion.22 In other words, DIO may develop in individuals
mounting higher inflammatory responses to fat, which in
turn would inhibit IAP activity and gene expression,
enhancing inflammation further.Inflammatory cytokines
such as interleukin (IL) 1βand tumor necrosis factor-α
(TNF-α) are known to downregulate IAP in IECs.23 Con-
versely, DIO-resistant rats are able to keep inflammation
under control through mechanisms that limit IAP repres-
sion. The molecular basis for interindividual differences in
the magnitude of DIO-induced innate immune responses
and inflammation remains poorly understood.
Intestinal resolution of inflammation. Tissue regeneration
following inflammation is regulated by a family of mol-
ecules called resolvins.24 Resolvin E1, a metabolite of the
omega-3 (n-3) FA eicosapentaenoic acid (EPA), interacts
with the leukotriene B4 receptor on immune cells to
downregulate inflammation.25 Resolvin E1 binds to the
ChemR23 receptor that is highly expressed on neutrophils
and oral epithelial cells.26 This receptor is also present in
IECs at the apical membrane above tight junctions and in
close vicinity to junctional actin.27 Importantly, resolvin
E1 stimulates IECs to express a mucosal protective factor
that was shown to be an IAP isoform.27 Resolvin E1 also
controlled chemically induced colitis in mice via the
upregulation of colonic IAP.27 Therefore, resolution of
inflammation at the gut level operates through two
complementary protective mechanisms: LPS detoxifica-
tion by IAP, and specific induction of cellular IAP expres-
sion by resolvin E1.
Possible indirect participation of IAP in the control of
intestinal barrier function
Intestinal barrier function facilitates vectorial transport
of nutrients, water, and ions while excluding potentially
toxic substances. Barrier defects are implicated in many
diseases of the gut and other organs.28 Indirect evidence
suggests a protective role of IAP on gut barrier function
by ameliorating inflammation.4,29,30 A direct role of IAP in
the control of intestinal permeability has also been
reported, e.g., in mice in which the Akp3 gene coding for
the IAP isoform in this animal species was deleted,15,16 but
this conclusion has been questioned because the Akp6
gene is upregulated in Akp3 knockout mice.17,18
Recently, a 15-fold increase in gut permeability
to macromolecules was observed in rat pups with
Figure 2 Production and circulation of intestinal
alkaline phosphatase in and around intestinal
epithelial cells.
Nutrition Reviews® Vol. 72(2):82–9484
necrotizing enterocolitis (NEC),31 confirming earlier find-
ings of a barrier defect in this disease.32 Administration of
a low dose of exogenous (bovine) IAP to pups with NEC
resulted in complete restoration of gut barrier function.31
Exogenous IAP had no significant effects on tissue-relative
concentrations of two major tight junction proteins
(claudin-1 and claudin-3) that were increased by NEC.31
Unfortunately, data on cellular localization of these pro-
teins were not reported, although protein localization is
essential for barrier functionality.33
A possible link between IAP and intestinal barrier
function is also suggested in cystic fibrosis (CF). This
disease afflicts Caucasians and involves a lack of func-
tional CF transmembrane conductance regulator anion
channel, with dramatic consequences on gut ion
exchange physiology and barrier function.34 Arecent
report indicates that mice with CF possess lower IAP
activity and lower Akp3 (but not Akp6) gene expression in
the small intestine.35 As CF is also associated with bacte-
rial overgrowth, antibiotic treatment of mice with CF was
shown to restore both Akp3 gene mRNA levels and IAP
activity.35 Oral administration of exogenous IAP restored
barrier function and damped bacterial overgrowth.35
However, experiments utilizing the IAP-specific inhibitor
L-phenylalanine failed to reduce gut permeability in wild-
type or CF mice, suggesting an indirect involvement of
IAP in the protective mechanism.35 Another possible
explanation for CF-associated intestinal barrier alteration
and reduced IAP could be the accumulation of free ATP
in the intestinal lumen.20 Indeed, CF transmembrane con-
ductance regulator anion channel is involved in the
control of bicarbonate secretion and surface pH.36 Free
ATP is a potent danger signal that induces strong inflam-
matory cytokine responses.37,38 These responses would in
turn inhibit IAP activity.23 Finally, two pig lines diver-
gently selected for residual feed intake (i.e., for higher
body-weight-gain to feed-intake efficiency ratio) were
shown to display different ileal IAP activities but similar
gut permeability to the marker probe fluorescein dextran
(molecular weight, 4,000 kDa) and to LPS.39
Collectively, these data do not support a direct link
between IAP activity and gut permeability. The beneficial
effects of IAP on intestinal barrier function may reflect
IAP-mediated inflammation downregulation.
Colonic alkaline phosphatase: association with TNAP
isoform, upregulation by oxidative stress, and
usefulness as a marker of inflammation
While many studies have focused on IAP in the small
intestine, conflicting results about IAP activity in the
colon have been reported, with both reduced27,40 and
increased41–43 IAP activity observed during colonic inflam-
mation. A consensual view has emerged, which indicates
that IAP and tissue-nonspecific alkaline phosphatase
(TNAP) isoforms are essentially expressed in the small
intestine and the colon, respectively.44 In response to
inflammation, colonic IAP is further depressed, while the
TNAP isoform is upregulated. An increase in TNAP
results from both colonic epithelial cells and neutrophils,
the latter being known to express the TNAP isoform and to
accumulate in the inflamed colon.
Importantly, the TNAP isoform is specifically up-
regulated by oxidative stressors, including monochlor-
amine, hydrogen peroxide, saponin, and deoxycholate, but
surprisingly, not by LPS in IECs.45 The major change
seems to be a chemical switch in N-glycosylation of alka-
line phosphatase (AP) isoforms, thus modulating AP
enzyme activity.45 The IAP isoform is found in both the cell
cytoplasm and the apical membrane,while TNAP is exclu-
sively cytosolic. Thus, oxidative stress may (in rats)43 or
may not (in mice)45 upregulate TNAP gene expression.
Collectively, these data indicate that the IAP isoform
is downregulated during colonic inflammation, while the
TNAP isoform is upregulated. Colonic upregulation of
TNAP by inflammation may be a protective adaptation
against oxidative stress and inflammation. Anti-
inflammatory treatments, e.g., oral IAP administration,
stimulate IAP expression and IAP-dependent resolvin-
E1-mediated amelioration of inflammation while reduc-
ing oxidative stress and TNAP expression and activity.
IAP AND GUT BACTERIA
IAP plays an important role in the crosstalk between the
gut microbiota and the host at the intestinal interface by
detoxifying proinflammatory bacterial and endogenous
components and by shaping the microbiota. This latter
effect, however, could be direct or indirect, since IAP is
anti-inflammatory and inflammation itself is known to
impact the composition of the gut microbiota46,47 and the
gut PAMP load.48
Detoxification of free nucleotides and various
bacterial PAMPs by IAP
Many nucleotides are proinflammatory, but adenosine is
anti-inflammatory.37,38 Previous work indicated that IAP
dephosphorylates ATP and other related adenosine
nucleotides.49 Additionally, IAP was recently shown to
dephosphorylate and, thus, detoxify, uridine diphosphate
nucleotide.50 The IAP isoform detoxifies LPS through
dephosphorylation of the lipid A moiety.6Recent investi-
gations showed that free IAP could also dephosphorylate
two other PAMPs, flagellin and CpG DNA motifs,but not
the Pam-3-Cys synthetic ligand in vitro (Table 1).51 These
PAMPs stimulate inflammation through TLR-4, TLR-5,
Nutrition Reviews® Vol. 72(2):82–94 85
TLR-9, and TLR-2 on IECs and various immune cells.52
These findings are important because both free
nucleotides and bacterial PAMPs have been implicated in
gut pathology in diseases such as CF20 and inflammatory
bowel disease (IBD).50
Role of IAP in shaping the gut microbiota
Earlier work in zebrafish and mice introduced the notion
that IAP participates in shaping the gut microbiota,6,53
and additional evidence on this role has now been pro-
vided. Free IAP dephosphorylated heat-killed bacteria of
both gram-negative and gram-positive origins, but it
did not seem to influence, by itself, the growth of
cultured Escherichia coli, Listeria monocytogenes,orSal-
monella typhimurium (Table 1).51 However, while IAP
expressed in IECs delayed the growth of E. coli,ithad
no effect on Clostridium difficile,S. typhimurium,or
Enterococcus faecalis.27 Importantly, when cellular pro-
duction of IAP was promoted, IECs were able to
downregulate IL-8 production induced by various gram-
negative bacteria like E. coli or S. typhimurium but not
that induced by gram-positive bacteria such as E. faecalis,
L. monocytogenes,orStaphylococcus aureus.51 Mice in
which the IAP Akp-3 gene product was deleted displayed
a fecal microbiota that is very different from that in wild-
type controls.54 IAP knockout mice had no detectable
E. coli and reduced numbers of total aerobic and anaero-
bic bacteria. Conversely,they showed relative increases in
the ratios of Clostridiales and increases in lactobacilli and
enterococci.54 Importantly, part of these effects may have
reflected changes in inflammatory tone in Akp3 knockout
mice. Intestinal inflammation could not be detected by
histology, but signs of chronic inflammation in the liver
were reported in Akp3 knockout mice.54 Among other
possible reasons for these changes, intestinal surface pH
might be proposed because bacterial growth is pH sensi-
tive, and IAP controls surface pH via an ATP-dependent
mechanism of bicarbonate secretion.36 Importantly, IAP
expression on intestinal cells has also been shown to spe-
cifically inhibit the intestinal pathogen S. typhimurium in
vivo.54 Finally, IAP-rich microvillar vesicles secreted into
the lumen by IECs may limit bacterial growth, but not
through a direct IAP-dependent mechanism.14
Prevention of bacterial translocation by IAP in the
gut of mice
IAP was shown earlier to limit intestinal bacterial trans-
location in mice, but little information was reported on
the possible mechanisms.5IAP-rich lumenal vesicles
secreted by enterocytes at the tips of microvilli were
recently reported to prevent adhesion of both pathogenic
and commensal bacteria to IECs in vitro, but this was
apparently not related to vesicle-bound IAP.14 Additional
data indicated that intrarectal administration of bovine
IAP could prevent bacterial translocation in different
models of colitis in mice.43 This may be indirectly linked
to IAP-dependent downregulation of inflammation.
Finally, increased bacterial translocation following
S. typhimurium challenge was observed in IAP knockout
mice and was not lethal, in contrast to results in wild-type
mice.55 These data were interpreted as evidence of toler-
ance to LPS in IAP Akp3-deficient mice.
Collectively, these data support a role for cellular-
induced IAP in regulating E. coli numbers and in inhib-
iting some gram-negative pathogens in mice.Conversely,
the role of free IAP in influencing gut microbiota
Table 1 Ability of bovine calf intestinal alkaline phosphatase to dephosphorylate bacterial pathogen-associated
microbial patterns (PAMPs) and live or heat-killed bacteria in vitro and to downregulate interleukin-8 (IL-8)
production by HT-29 cells.a
PAMP or bacteria Toll-like receptor Phosphate release in vitro Reduction of IL-8 production (HT-29)
Bacterial PAMPs
Pam-3-Cys TLR-2 No No
LPS TLR-4 Yes Yes
Flagellin TLR-5 Yes Yes
CpG DNA TLR-9 Yes Yes
Gram-negative bacteria Live Dead Live Dead
Escherichia coli Various No Yes No Yes
Salmonella typhimurium Various No Yes No Yes
Gram-positive bacteria Live Dead Live Dead
Listeria monocytogenes Various No Yes No Yes
Staphylococcus aureus Various NR NR No NR
Enterococcus faecalis Various NR NR No NR
Abbreviations: CpG DNA, cytosine nucleotide-phosphate-guanine nucleotide of bacterial DNA; LPS, lipopolysaccharide; NR, not
reported; Pam-3-Cys, synthetic substrate.
aAdapted from Chen et al. (2010).51
Nutrition Reviews® Vol. 72(2):82–9486
composition may be modest. The precise underlying
mechanisms are not yet well understood, but they may be
secondary to changes in gut inflammatory tone.46,47,54
IAP AND INFLAMMATORY DISEASES
As already mentioned, IAP plays a key role in ameliorat-
ing cellular inflammatory responses. Many reports show
that diverse inflammatory diseases are often associated
with abnormally low IAP expression or activity. More
precisely, a defect in the IAP isoform may be associated
with small intestinal inflammation, while overexpression
of the TNAP isoform, as seen in colonic diseases,44 merely
reflects inflammation-driven neutrophil infiltration of
tissue.
Possible involvement of an IAP defect in diseases of
the small intestine
NEC is a multifactorial pathological condition affecting
the ileum and colon of very-low-birthweight babies. This
disease involves genetic predisposition, abnormal micro-
bial colonization, ischemia, intestinal immaturity, and
feeding with milk formula, the last two being consistent
risk factors.56 Recently, Whitehouse et al.57 observed in a
rat model that pups with NEC displayed lower IAP
protein expression and activity levels than controls. They
suggested that IAP reduction precedes the onset of the
disease and may be involved causally. Although sup-
ported by data showing that exogenous (bovine) IAP
could prevent NEC,31 this has not been demonstrated
unequivocally. Celiac disease is a chronic inflammation of
the small intestine caused by gluten from wheat and most
other cereals.58 Depressed duodenal IAP protein expres-
sion and activity is especially marked in severe cases of
celiac disease in young patients.59,60 Importantly, tissue
co-localization between IAP and LPS-receptor TLR-4 was
reported.60 The IAP defect was reversed after a gluten-
free dietary regimen was intitiated.60 In farm animals, the
postweaning syndrome characterized by gut anatomical
and functional alterations is still a problem when rearing
pigs.61 A defect in IAP was reported recently and was
interpreted as a possible reason for intestinal inflamma-
tion and associated sensitivity to enteric infections.62
In summary, these data support the notion that IAP
could play a role in these diseases. However, it is still
unclear whether inhibition of IAP activity is a primary or
secondary factor, as already mentioned for DIO.22
Possible opposing effects of two AP isoforms in IBD
Chemicals such as dextran sulfate sodium (DSS) or
trinitrobenzene sulfonic acid are often used for inducing
colitis in mouse models of IBD. Importantly, some of
these compounds (DSS) upregulate the IAP isoform,
while others (trinitrobenzene sulfonic acid) induce the
TNAP isoform in colonocytes and in rodent colon.43 Mice
in which the IAP Akp3 gene product has been deleted
were more sensitive to chronic colitis in two IBD models
(one being DSS), thus suggesting a protective role for the
endogenous IAP isoform during colonic inflammation.63
Defects in IAP isoform expression were also reported in
other studies with DSS.27,40 Conversely, colonic expres-
sion of the TNAP isoform was reported to increase mark-
edly in chemical (DSS and trinitrobenzene sulfonic acid)
models of IBD.41–43 In humans, IAP protein levels were
lower in colonic biopsies of inflamed versus noninflamed
zones in young patients with Crohn’s disease or ulcerative
colitis.64 Such IAP defects are also found in adults with
Crohn’s disease or ulcerative colitis.40,65
Therefore, two intestinalAP isoforms operate in IBD.
IAP is reduced, probably as a direct consequence of
inflammation. In contrast, TNAP is upregulated. Impor-
tantly, the TNAP isoform has two origins: colonocytes and
neutrophils.While the former source is probably an adap-
tive response to counteract oxidative stress and inflamma-
tion, the latter source directly reflects the inflammatory
response, as indicated by neutrophil infiltration of colonic
tissues. Differences between studies suggest that genes,
proteins, and activities of both isoforms should be inves-
tigated more systematically in order to obtain clearer
answers.
POTENT ANTI-INFLAMMATORY ACTIVITY OF IAP IN THE
INTESTINE AND OTHER ORGANS
Emerging data suggest that exogenous IAP (most often
of bovine origin) is a potent anti-inflammatory agent for
treating inflammatory diseases of the gut and other
organs (Table 2).4,40,42,43,63,65–73 The activity of (human) IAP
is highly pH sensitive and is reduced in acidic environ-
ments like the stomach, a result of changes in enzyme site
structures and ion (e.g., zinc) movements.74 However, this
reduction in IAP activity, which depends on the duration
of exposure to extreme pH, is less than the reduction in
liver AP activity and,more importantly is partially revers-
ible.74 Then, IAP can be proteolyzed in the small intestine.
The recovery of polymerized inulin-protected bovine
IAP was evaluated to be around 30% in rat intestine.75
Increases in fecal concentrations of AP activity following
oral exogenous IAP administration may reflect partial
escape of the added IAP to bacterial fermentation in the
colon and (or) stimulation of endogenous IAP produc-
tion in the small intestine.5,6 In order to improve IAP
resistance to alterations in the gastrointestinal (GI) tract
and to avoid the use of heterologous (e.g., bovine) IAP, a
Nutrition Reviews® Vol. 72(2):82–94 87
Table 2 Summary of systemic effects of exogenous (bovine or placental) intestinal alkaline phosphatase (IAP) on survival and gut tissue, peritoneal fluid, or
plasma cytokine profiles in disease states.
Type of model or study Species, IAP form, and
route of administration
Effect on
survival
Cytokine Reference
IL-1βIL-4 IL-6 IL-8 IL-10 IFN-γTNF-α
Animal models
NEC Rat, bIAP, i.p. `` `Riggle et al. (2012)66
NEC Rat, bIAP, oral `` `Rentea et al. (2012)67
DSS colitis (acute)aRat, bIAP, oral `` `Tuin et al. (2009)40
DSS colitis (acute)bMouse, bIAP, oral ==`Bol-Schoenmakers et al. (2010)42
DSS colitis (chronic) Mouse, bIAP, oral ``Ramasamy et al. (2011)63
TNBS colitis Rat, bIAP, oral `Martínez-Moya et al. (2012)43
TNBS colitis Rat, bIAP, rectal `Martínez-Moya et al. (2012)43
WASP colitis Mouse, bIAP, oral `Ramasamy et al. (2011)63
PeritonitiscMouse, bIAP, i.p. ``Ebrahimi et al. (2011)68
Sepsis Mouse, placental, IAP Improved Verweij et al. (2004)69
Sepsis Mouse, bIAP, i.v. Improved `Beumer et al. (2003)70
Sepsis Pig, bIAP, i.v. Improved `Beumer et al. (2003)70
Sepsis Sheep, bIAP, i.p. Improved `Su et al. (2006)71
Metabolic syndromedMouse, bIAP, oral, i.p. ``Kaliannan et al. (2013)4
Human studies
IBDeHuman, bIAP, oral – === = Lukas et al. (2010)65
Coronary surgery Human, bIAP, i.v. – ``Kats et al. (2012)72
Renal failurefHuman, bIAP, i.v.+c.i. – `Pickkers et al. (2012)73
Abbreviations: bIAP, bovine intestinal alkaline phosphatase; c.i., continuous infusion; DSS, dextran sulfate sodium; IBD, inflammatory bowel disease; IFN, interferon; IL, interleukin; i.p.,
intraperitoneal; i.v., intravenous; NEC, necrotizing enterocolitis; TNF, tumor necrosis factor; TNBS, trinitrobenzene sulfonic acid; WASP, Wiskott-Aldrich syndrome protein; =, no change; `,
decreased.
aCytokine tissue gene expression. Gene expression of IL-10 receptor was decreased.
bCytokine tissue concentration.
cCytokines measured in peritoneal lavage fluid.
dIL-1βmeasured in plasma and tumor necrosis factor-αmeasured in both colonic digesta and plasma.
eCytokine levels measured after 7 days of treatment. Plasma C-reactive protein and fecal calprotectin decreased at day 21 and day 63 after treatment initiation.
fRenal failure associated with sepsis or septic shock. Bovine IAP administered as intravenous bolus plus intravenous continuous infusion for 48 h.
Nutrition Reviews® Vol. 72(2):82–9488
recombinant human chimeric AP that combines IAP
enzyme domain and placental AP stability domain is
being developed.76
Intestinal and colonic inflammation and peritonitis
To date, all of the published data report beneficial, anti-
inflammatory effects exerted by exogenous IAP, although
the magnitude of protection depends on the route of
administration. These effects include intestinal tissue
protection in rodent models of NEC31,57 and CF,35 and
colonic tissue protection in animal models of IBD.40,42,63
Exogenous IAP also reduces inflammation in patients
with ulcerative colitis65 and in peritonitis in a mouse
model.68
Systemic inflammation
Administration of exogenous IAP has been shown
to reduce systemic inflammation in various animal
models69–71,77 and in several diseases in humans78
(Table 2), as demonstrated in NEC,66,67 peritonitis,68
sepsis,69–71,78 and even brain disease.77
Sepsis is caused by infection and LPS-induced sys-
temic inflammation, which rapidly can lead to acute
kidney injury. Importantly,the TNAP (or kidney) isoform
is present in renal proximal tubules as a defense system
against LPS. Two double-blind, randomized, placebo-
controlled studies in septic patients reported improve-
ments following intravenous administration of IAP.73,78
The first study showed that IAP was able to downregulate
inducible nitric oxide synthase activity and to reduce
urinary excretion of nitric oxide metabolites.78 In the
second study, renal function was also improved and sys-
temic inflammation reduced.73 The beneficial effects of
IAP treatment were attributed to dephosphorylation-
dependent detoxification of both LPS and extracellular
ATP.76
Administration of exogenous IAP was also found to
be protective in patients undergoing coronary artery
bypass surgery,72 and exogenous IAP reduced neurologi-
cal alterations in a model of autoimmune encephalomy-
elitis in mice.77
As described above, while an increase in IAP in the
intestine is generally protective due to direct inhibition of
inflammation, high levels of TNAP expression and activ-
ity in the colon most probably reflect inflammation-
driven neutrophil infiltration of colonic tissues. Because
most serum AP is of bone and liver origin, the gut and
kidney contribute relatively little to this activity. Thus,
serum AP activity is considered a biomarker of inflam-
mation and cardiometabolic risk and has been positively
correlated with other systemic markers of inflammation
(e.g., C-reactive protein) in the general US population79 as
well as in patients with renal disease80 and Alzheimer’s
disease.81,82 Higher levels of plasma AP activity are also
indicative of elevated risk of mortality in the general US
population.83
Collectively, these results indicate that, although all
increases in AP activity may be an adaptive response, the
purpose of which is to control PAMPs (e.g., LPS),
increases in colonic (but not intestinal) and serum total
AP activities are clear signs of inflammation.
Influence of route of administration on effects of
exogenous IAP
Oral or enteral administration of exogenous IAP was able
to reduce the development of NEC, while systemic
administration of IAP strongly downregulated systemic
inflammation without reducing intestinal tissue injury
caused by NEC.66,67 Intrarectal administration of IAP was
more effective than oral administration in reducing colitis
and bacterial translocation, although both routes con-
ferred efficacy.43 A plausible reason for this difference is
that IAP given by the oral route may have been partially
digested in the intestine.5,75 Importantly, oral IAP admin-
istration was able to stimulate endogenous IAP in the
small intestine57 but had no effect on the colonic TNAP
isoform.43 Administration of exogenous IAP stimulated
the release of endogenous TNAP isoform into the sys-
temic circulation in patients undergoing cardiac vascular
surgery6,72 but not in the brain of mice.77 However, the
pathophysiological significance of TNAP isoform induc-
tion in these studies is unclear.
DIETARY MODULATION OF IAP: AN UPDATE
It was previously reported that dietary fat,protein, carbo-
hydrates, vitamins, and minerals are all important dietary
modulators of host IAP gene expression and enzyme
activity.6Since then, additional information has been
published.
Stimulation of IAP by free calcium, bound
phosphorus, and vitamins
Dietary calcium was recently shown to stimulate the
activity of IAP in vivo, which in turn limited intestinal
absorption of calcium as a percentage of total calcium
intake in rats.84 This is important because increased
calcium intake can be easily achieved, e.g.,by eating dairy
products. Beneficial effects of dietary calcium are well
documented in models of colonic inflammation.85,86
However, the role of IAP activity was not considered in
these studies, and it is unclear whether this activity may
have also contributed to the protective effects. Of note,
Nutrition Reviews® Vol. 72(2):82–94 89
calcium-induced protection against colonic inflamma-
tion requires a sufficiently high concentration of phos-
phorus in the colon.87 Earlier work concluded that free
dietary phosphorus had an inhibitory effect on IAP.88,89 By
contrast, bound phosphate (e.g.,esterified to glucose resi-
dues of some potato starch varieties) was able to stimulate
IAP activity dose-dependently throughout the small
intestine of rats.90
Vitamin K1(phylloquinone) and vitamin K2
(menaquinone-4) were already mentioned as IAP stimu-
lants in a previous review.6,91 Subsequent reports suggest
that these vitamins stimulate both the expression of Akp3
and Akp6 IAP genes and the activity of AP protein in the
mouse jejunum.92 Underlying mechanisms were sug-
gested to involve the nuclear steroid and xenobiotic
receptor SXR,92 but direct evidence is lacking.
Differential regulation of IAP by dietary FAs
As already mentioned, fat intake stimulates gut IAP in
rodents, and triglycerides with saturated LCFAs or
medium-chain FAs increase IAP expression and/or activ-
ity.6Conversely, polyunsaturated LCFAs were shown to
decrease IAP expression.6Recently, an important study
revealed that, while a diet enriched in (omega-6 [n-6])
polyunsaturated FAs promoted inflammation in mice, the
addition of (n-3) polyunsaturated FAs provided by fish oil
reduced inflammation but was also associated with higher
rates of sepsis-induced mortality.93 The authors con-
cluded that added fish oil depressed both gut IAP activity
and associated LPS dephosphorylation capacity, thus
favoring circulating LPS.93 In a rat model of chemically
induced colonic inflammation, however, the total activity
of AP (isoforms not specified) in the colon was shown to
decrease following partial replacement of linoleic acid
(C18:2 n-6) with α-linolenic acid (C18:3 n-3).94 There-
fore, these studies indicate that the role of dietary FAs is
critical and that clinical outcomes may depend on their
combination.
Stimulation of IAP by various plant
bioactive compounds
Different medicinal plants are able to modulate IAP activ-
ity, and various spices (e.g., black pepper, red pepper,
ginger, and bioactive compounds piperine and capsaicin)
have been shown to increase IAP activity in the small
intestine.95 Elevated IAP activity was associated with
elongation of intestinal microvilli, higher membrane flu-
idity, and a reduced cholesterol-to-phospholipid ratio.95
Caraway (Carum carvi L.) decreased the level of
colonic tissue AP in a rat model of chemically induced
colorectal cancer.96 An anti-inflammatory compound pre-
pared from fungi was shown to reduce colonic inflamma-
tion and alterations and to decrease colonic AP activity.97
More recently, narrow-leaved cattail (Typha angustifolia
L.) rhizome flour and green dwarf banana (Musa sp.AAA)
flour both were found to decrease colonic AP activity in a
rat model of colitis.98,99 Coumestrol, a phytoestrogen
known to regulate intestinal calcium absorption, was
found to inhibit IAP gene expression and enzyme activity,
although differential regulation was evident in the duode-
num and the jejunum in neonatal pups and lasted until 10
days postparturition.100,101 It was suggested that estrogen
receptor-alpha (ER-α) may be involved in this transient
inhibition.
Stimulation of IAP by some probiotic bacteria
In a chronic model of chemically induced colorectal car-
cinoma in rats, the probiotic mixture VSL#3 prevented
carcinoma development and limited colonic dysplasia.102
Probiotic-treated rats displayed a 50% reduction in fecal
AP activity,102 an observation interpreted as reflecting a
probiotic-mediated reduction in small intestine epithelial
cell death.103 An alternative explanation may be that this
treatment normalized colonic TNAP activity, which is
elevated during colonic inflammation.41,43 Lactobacillus
plantarum AS1 isolated from fermented food inhibited
chemically induced colorectal cancer in rats, and this was
associated with a reduction in colonic (TN)AP activity.104
Collectively, available data suggest that various plant
bioactive compounds and specific probiotics can contrib-
ute to gut protection, either directly, by influencing IAP-
mediated mechanisms, or indirectly, by ameliorating
inflammation, especially in the colon. However, effects of
FAs on IAP appear more complex and warrant further
investigation.
EARLY PROGRAMMING OF IAP
Nutrition and environment are increasingly suspected to
contribute to the development of various diseases of
adulthood, including obesity and associated metabolic
diseases.105 For example, intrauterine growth retardation
is a risk factor for metabolic syndrome and obesity.106
However, little is known about the early programming of
the GI tract in this context. Recently, it was hypothesized
that perinatal malnutrition programs key intestinal func-
tions, including IAP activity. In a rat model of intrauter-
ine growth retardation induced by maternal protein
deficiency during the perinatal period, it was shown that
adult offspring fed a high-fat diet failed to adapt to this
diet by increasing AP activity in jejunal tissue and cecal
content.107 Interestingly, mRNA levels of the transcrip-
tion factors Klf4 and Cdx1 were lower in jejunal epithelial
cells of intrauterine-growth-retarded rats fed the high-fat
Nutrition Reviews® Vol. 72(2):82–9490
diet.107 These are two major regulatory transcription
factors of IAP,108,109 and intestinal-specific deletion of the
Klf4 gene leads to constitutive reduction of IAP expres-
sion in mice.110 It was also hypothesized that early bacte-
rial colonization may influence gut programming, and a
swine model of antibiotic administration to mothers
around the time of parturition was developed. Results
showed that offspring born to antibiotic-treated sows dis-
played jejunal IAP activity at 6 months of age that was
lower than that in controls.111
Epigenetics has also been shown to regulate early
tissue programming. Epigenetic modifications include
DNA methylation of gene promoter regions, multiple
histone modifications, and noncoding microRNA expres-
sion, all leading to finely tuned modulation of gene
transcription.112 Epigenetics may be involved in IAP pro-
gramming.113 Indeed, it was recently reported that the
LPS receptor TLR4 gene is methylated in IECs and that
the frequency of methylation of this gene was higher in
differentiated cells expressing higher levels of IAP than
in undifferentiated cells.114 Interestingly, TLR4 gene
methylation is under the control of the transcription
factor Cdx2, required for intestinal development and IAP
expression.109 Epigenetic modifications of promoter
regions in the IAP gene after early malnutrition107 or after
early disturbances in bacterial colonization of the gut111
remain to be investigated.
With regard to early programming,balanced dietary
provision of methyl donors (betaine, choline, folate,
vitamin B12) during the perinatal period is important for
optimal methylation of genomic DNA and histones.113 It
was recently found that, following methyl donor defi-
ciency during gestation and lactation in rat dams, off-
spring displayed a dramatic reduction in IAP activity in
the distal (but not proximal) small intestine.115 This was
associated with many other anatomical and functional
alterations and a marked downregulation of the tran-
scription factor Cdx2.115,116 Thus, epigenetic modulation
appears to vary regionally along the gut, and, collectively,
these results suggest that IAP is sensitive to early pro-
gramming, probably through modifications of the intes-
tinal epigenome. Nutritional and environmental factors
susceptible to modulation by epigenetic factors are there-
fore likely to impact intestinal IAP function, with health
consequences potentially emerging during adulthood.
LINK BETWEEN EVOLUTIONARY HISTORY OF IAP AND
FOOD-DRIVEN CHANGES IN GUT MICROBIOTA
During evolution, AP genes have remained highly con-
served across animal species. Most likely, this reflects the
need to keep the gut microbiota and its PAMPs under
control for host well-being.117 As already reported,6
different AP isoforms are encoded by multiple genes that
display either ubiquitous or tissue-specific expression. In
a search to decipher the origins of two AP genes newly
discovered in zebrafish, Yang et al.117 published an out-
standing study on AP gene evolution across animal
species. They found that AP genes are organized in three
clades, except in mammals,which display only two clades
(AP-1 andAP-2).They also found that two major genome
duplications occurred in vertebrates during evolution,
generating these three clades. According to this phyloge-
netic analysis, AP genes of the intestines have been dupli-
cated and lost several times over different vertebrate
lineages. Yang et al.117 interpreted their findings as a
reflection of the dynamic evolutionary changes in the
dietary regimens that would have driven both adaptation
of GI tract anatomy and microbial symbiont composi-
tion. Although their analysis was based on fecal
microbiota data, which are not representative of bacterial
diversity in upstream compartments of the GI tract, Yang
et al.117 made some interesting observations. For example,
they found that domestic cats and dogs have only one
copy of the AP gene, matching a GI tract anatomy asso-
ciated with a monotonous dietary regimen. Mice and rats
appear more closely related to humans in terms of AP
gene content, belonging to clade 1.AP clade 2 displays the
most dynamic evolution. Indeed, artiodactyls with more
complex GI tract systems display a higher intestinal AP
gene diversity. Bovine ruminants with anatomically
complex forestomachs possess six AP gene copies. The
porcine species, an omnivorous monogastric animal, has
three copies, two of them being closely related. Interest-
ingly, one porcine AP gene closely resembles two bovine
AP genes, leading the researchers to suggest that swine
and bovine lineages had diverged after this tandem AP
gene duplication occurred.
Importantly, intestinal AP-dependent control of LPS
(and PAMP) bioactivity in these farm animal species is
still of major concern. Postweaning in livestock is a
period of high susceptibility to gut disorders and enteric
infections,61 and was recently shown to be associated with
the down regulation of IAP gene expression and activ-
ity.62 Furthermore, a pig line divergently selected for low
residual feed intake (or for higher body-weight-gain to
feed-intake ratio) showed higher ileal IAP and lower
intestinal (and systemic) inflammation associated with
lower plasma LPS than the line with high residual feed
intake.39 In dairy cattle, subclinical rumen acidosis is
characterized by ruminal and colonic blooms of gram-
negative bacteria and LPS, resulting in increased concen-
trations of circulating LPS; this leads to inflammation,
metabolic disorders, and a drop in milk fat production
that inversely correlates with plasma levels of LPS.118–120
However, little has been reported on AP gene expression
or activities in the GI tracts of ruminants.
Nutrition Reviews® Vol. 72(2):82–94 91
Collectively, these data support a dynamic phyloge-
netic evolution of AP gene family members involved in
the control of gut microbiota-host symbiosis and inflam-
mation, with evolution occurring across animal species.
CONCLUSION
Intestinal IAP is at the crossroads between diet, fat
absorption, the microbiota, LPS, and inflammation,
factors that have all been implicated as causal in obesity
and metabolic disorders. This review gathers important
new findings on IAP production, modulation, and circu-
lation in and around intestinal cells; on absorption of
calcium, fat, and minerals; on detoxification of novel
nucleotides and bacterial PAMPs; on amelioration of
intestinal inflammation; and, finally, on modulation of
the composition of gut microbiota (Figure 3). However,
the effects of IAP on some targets, such as gut permeabil-
ity and bacteria, might be indirect, through downregulat-
ion of inflammation and, possibly,through modulation of
intestinal epithelial surface pH. Members of the intestinal
AP gene family appear to reflect evolutionary changes in
dietary regimens across animal species and over time, as
well as changes in associated gut microbial symbionts.
This makes intestinal AP a crucial component of a detoxi-
fication system for the maintenance of both host and
microbiota homeostasis. Diet appears to be a major
modulator of the compositional and functional diversity
of the microbiota and may also be considered a regulator
of intestinal AP expression and activity in health and
disease. In that regard, many components of our diet,
including minerals, vitamins and micronutrients play a
beneficial role. Unbalanced diets, typified by the low fiber
intake of Western diets, promote gut dysbiosis, thereby
altering both intestinal barrier function and translocation
of bacterial PAMPs and thus contributing to inflamma-
tion, obesity, and metabolic disorders. Diets rich in
nondigestible but fermentable fiber components may
thus help prevent a number of clinical disorders. Intesti-
nal AP will continue to play a central role in this equilib-
rium and, perhaps, in apparently unrelated diseases.121
Finally, due to poor correlation between mouse models of
inflammation and human inflammatory diseases122 and
the limited data available from human studies, much
more evidence on the roles of IAP in humans is needed.
Acknowledgments
Dr. TC Savidge (Baylor College of Medicine, Houston,
Texas, USA) and Professor CR Stokes (Veterinary School,
University of Bristol, Bristol, UK) are acknowledged for
their valuable comments and suggestions on this review.
Funding. The author received no special funding for
writing this review.
Declaration of interest. The author has no relevant inter-
ests to declare.
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