Supplementation with Brush Border Enzyme Alkaline Phosphatase Slows Aging

Introduction

Gut integrity, specifically the structural integrity and function of the intestinal epithelial barrier, becomes impaired in older animals, ranging from nematodes and fruit flies to mice, monkeys, and humans. ^{1 –5} Normal gut functions include the adsorption of nutrients, ensuring that microbiota remain localized in the lumen of the intestine and protecting against microorganismal breaches by immune surveillance. Pathologies in humans associated with diminished intestinal barrier function include inflammatory bowel disease, ulcerative colitis (UC), Crohn's disease, cancers, and immune and neurological dysfunction. ⁶ It has been hypothesized that intestinal barrier dysfunction and alterations in microbiota contribute to inflammaging, the increase in aging-associated inflammation observed in many animals, including humans. ^{7 –9} Recent work suggests that alkaline phosphatases associated with the intestine play a key role in maintaining the intestinal barrier. ^10,11

Alkaline phosphatases are metalloenzymes that cleave phosphate from a variety of substrates. Humans express several isozymes classified as tissue nonspecific (e.g., bone, liver, and kidney) and tissue specific (e.g., intestine, placenta, and germ cells). Intestinal alkaline phosphatase (IAP) exhibits its biological activity in alkaline conditions, with highest activity at pH 9.7. IAP expressed in the apical microvilli of the brush border of enterocytes plays an important role in gut homeostasis. IAP is expressed throughout the gastrointestinal (GI) tract, with highest levels in the duodenum and lower levels in the jejunum, ileum, and colon. IAP is released into the gut lumen and can be detected in the stool. ¹²

A recent paper by Kuhn et al. demonstrates that IAP can slow the aging process by preserving gut barrier function and by reducing gut-mediated inflammaging. ¹⁰ Earlier human and animal data (reviewed in Fawley and Goulay ¹³ ) demonstrated that exogenous IAP can provide a protective effect versus intestinal and systemic inflammation in a variety of diseases. In fact, recombinant human IAP is being evaluated in clinical trials. This new paper meticulously dissects the mechanism by which IAP supports gut health and thereby plays a critical role in aging.

Age-Dependent Decline in IAP Activity Is Associated with Reduced Gut Barrier Function and Systemic Inflammation

Ileal IAP concentrations dropped by >50% from age 20 to age 80 in a cohort of 60 human volunteers. In mice, stool and ileal IAP concentrations decreased significantly (∼40–50%) from young (4 months old) to old individuals (21 months old). These changes were associated with an increase in the “leakiness” of the murine gut measured by uptake of FITC-dextran. This breach of barrier function was associated with an uptick in serum endotoxin assumed to be the stimulus for increased systemic levels of inflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin (IL)-6. To support a mechanism previously described by this group, ¹⁴ local intestinal expression of tight junction proteins occludin and ZO-1 were observed to drop significantly, suggesting that the integrity of the tight junctions was compromised. In each of these experiments, the direction of the age-related changes (i.e., tight junction dysfunction → gut leakiness → endotoxemia → cytokine levels → inflammation) were augmented in specific IAP knockout (KO) mice and supported the idea of administration of oral IAP as a “replacement therapy” for inflammaging. ¹⁰

Absence of IAP Enhances Aging-Related Liver Inflammation and Steatosis

The portal circulation bathes the liver in gut-derived blood. Portal endotoxin more than doubled with aging and doubled again in the IAP KO mice. Levels of endotoxin measured by the limulus amebocyte lysate test were up to 1,000-fold higher in the portal versus systemic circulation. It was not unexpected that markers of liver inflammation such as TNFα and IL-6 increased with aging and that the IAP KO mice exhibited even higher levels. These triggers of inflammation contributed to significant liver fat accumulation (steatosis) with age that was more severe in the IAP KO mice. ¹⁰ Such hallmarks of metabolic syndrome are characteristic of aging in many organisms.

Exogenous, Supplemental IAP Reverses Age-Induced Barrier Dysfunction and Extends Life-Span

Based on earlier studies demonstrating a key role for IAP in gut dysfunction with aging, ¹⁵ replacement therapy was next evaluated beginning at the age of 10 months (i.e., “middle-aged mice”). Mice were supplied with autoclaved water containing bovine IAP (100 IU/mL). The water bottles were refilled daily with 30 mL (3,000 IU) of IAP or vehicle. Stool samples analyzed to document the increased intraluminal activity showed around a fivefold increase in IAP activity over baseline in the treated animals. Importantly, there were no differences in weight gain or food consumption among the various experimental groups of mice. Wild-type (WT) C57BL/6J mice lived a median of 23 months compared to the significantly shorter life-span of the IAP KO mice at 19 months. IAP-supplemented WT mice exhibited a significantly longer median survival of 32 months. ¹⁰

The oral IAP supplementation reduced gut permeability as measured by FITC-dextran uptake and serum endotoxin levels. In addition, this replacement therapy reduced inflammatory cytokines IL-6, IL-1β, TNFα as well as fecal lipocalin, a marker of chronic intestinal inflammation.

IAP Supplementation Improves Metabolic Dysfunction Associated with Aging

As noted above, IAP KO mice exhibited the characteristic fatty liver of metabolic syndrome observed in aging. In contrast, the IAP-supplemented mice showed an improved metabolic profile, with lower liver enzymes (AST, ALT), higher HDL cholesterol, lower triglycerides and low-density lipoprotein cholesterol, and improved glucose tolerance.

The shorter or longer median mouse life-spans corresponding to lower or higher levels of intestinal IAP were also associated with increased (IAP KO) or decreased (IAP supplemented) frailty scores. ¹⁰

IAP Supplementation Prevents Aging-Associated Microbiome Dysbiosis

Sequential (prior to, 1 month after, and 6 months after treatment) fecal 16S ribosomal RNA sequencing and analysis was carried out to document age-associated changes in commensal bacterial populations of WT versus IAP-fed mice. Principal coordinate analysis (PCoA) of the relative abundance of bacterial phyla revealed significant changes during aging in the WT mice, consistent with the literature, in experimental models as well as humans. However, essentially no changes from the patterns found in the young mice were found among the IAP-treated animals as they aged. ¹⁰

IAP Supplementation Improves Physical Performance and Survival of Drosophilae

Drosophilae are an important model organism for aging research due to their short life-spans and easy genetic manipulation. Despite the fact that the lineages of humans and Drosophilae separated some 700 million years ago, more than 50% of fly genes have homologs in the human genome. ¹⁶ Kuhn et al. carried out elegant studies of Drosophilae showing that: (1) IAP activity decreases with age (young 3 weeks vs. old 7 weeks); (2) genetic knockdown of IAP reduces life-span, and this effect can be reversed with supplemental dietary IAP; (3) climbing activity of the flies (a measure of physical performance) is reduced during aging by 50% or more and can be reversed by dietary IAP. ¹⁰

Molecular Mechanisms

IAP exhibits many important activities that sum together to maintain gut health. At the molecular level, Liu et al. showed that IAP gene deletion in mouse embryonic fibroblasts resulted in significantly lower levels of tight junction proteins ZO-1, ZO-2, and Occludin compared to levels in WT control cells. ¹⁴ Furthermore, IAP overexpression in colon cancer Caco-2 and T84 cells, used to model what happens in the normal human intestine, resulted in approximately twofold increases in the mRNA expression of ZO-1 and ZO-2. ¹⁴ IAP treatment ameliorated lipopolysaccharide-induced permeability in a 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. ¹⁰

Gut lipopolysaccharide (LPS) plays a critical role in inflammaging. ¹⁷ Lumenal IAP has been documented to dephosphorylate LPS, which detoxifies LPS. ^18,19 Kuhn et al. showed that: (1) systemic and portal endotoxin levels increase with aging; (2) this increase stimulates many cytokine mediators of inflammation (e.g., IL-1β, IL6, and TNFα); and (3) oral supplementation with IAP results in reduction of endotoxin back to levels characteristic of young animals.

IAP can dephosphorylate a variety of other bacterial and host-derived ligands in addition to LPS. Among these are CpG DNA, flagellin, UDP, and so on, each of which works through a specific receptor (e.g., Toll-like receptors, pathogen-associated molecular patterns, damage-associated molecular patterns, etc.) to exert its inflammatory impact on target cells. ^{20 21} In addition, Malo et al. demonstrated that IAP promotes the growth of beneficial GI aerobic and anaerobic bacteria. ²² Compared to WT mice, IAP KO mice have more ATP in their GI luminal contents. Exogenous IAP promotes the growth of intestinal commensal bacteria by reducing the concentration of luminal ATP and other nucleotide triphosphates.

Medical Implications

The therapeutic potential of alkaline phosphatase has previously been investigated in some detail. For example, two double-blind, randomized, placebo-controlled Phase II clinical trials in septic patients with acute kidney injury (SA-AKI) demonstrated a beneficial renal effect of parenterally (intravenously) delivered bovine alkaline phosphatase. ^{23 –25} Seeking to reduce possible immunogenicity, this Dutch group has developed a recombinant chimeric human alkaline phosphatase (huAP). Protective effects have been confirmed in vitro and in vivo in a rat model of LPS-induced AKI. ²⁶ Safety and tolerability of the human enzyme was demonstrated in a Phase I study ²⁷ followed by the STOP-AKI trial, an international (53 recruiting sites), randomized, double-blind, placebo-controlled, dose-finding, adaptive Phase IIa/IIb study in 301 adult patients admitted to the intensive care unit with a diagnosis of SA-AKI. ²⁸ After initial dose-range finding studies, patients were randomized to receive 1.6 mg/kg (n = 82) compared to placebo (n = 86). Among patients who were critically ill with SA-AKI, huAP compared to placebo did not significantly improve short-term kidney function. However, Tang et al. carried out a systematic review and meta-analysis of the four randomized controlled trials involving huAP therapy of 392 patients with SA-AKI. In their analysis, huAP showed a relatively late protective effect by improving endogenous creatinine clearance (ECC) at days 7, 14, and 28. ECC levels improved when patients received a huAP dose of 0.212 mg/kg. Mortality improved at days 28 and 90, respectively, when patients received a huAP dose of 1.6 mg/kg. Based on these studies, huAP is being evaluated in a Pase III trial in SA-AKI sponsored by AM Pharma.

IAP expression in the gut is reduced in patients with inflammatory bowel disease, and exogenous administration of IAP has a beneficial therapeutic effect in rodent models of colitis. ²⁹ Results from Phase I and Phase II clinical trials, with administration of bovine AP by duodenal drip, showed potential as a tolerable and efficacious treatment for UC. AM-Pharma conducted an open-label, multicenter trial, administering bovine AP continuously for 7 days. The trial recruited 21 moderate-to-severe UC patients who were unresponsive to treatment with steroids and immunosuppressants. The Mayo score showed a significant decrease in patients receiving bovine AP versus baseline at day 21 and the MTWSI score at day 21 and day 63. In addition, the trial showed there were no clinically relevant adverse events causing withdrawal or considered serious, or laboratory abnormalities or antibody formation against the bovine AP. AM Pharma has developed an oral formulation of their recombinant huAP and plans additional human studies in inflammatory bowel disease.

The studies of Kuhn et al. support the replacement of IAP to support GI health and reduce several of the hallmarks of aging in humans. All studies described to date support the safety of both parenteral and oral administration of this important enzyme. Whether a less acute endpoint than SA-AKI or UC such a frailty can be addressed is certainly worth considering. Because AM-Pharma has raised a considerable amount of capital (∼$200 million) to support work to date, one can assume that if/when their improved huAP is approved, the cost will be substantial and unlikely to be affordable for long-term chronic use.

It would be of interest to understand if IAP itself is the only alkaline phosphatase that can inactivate key gut-disrupting substances such as LPS. Data indicate other alkaline phosphatases such as tissue nonspecific alkaline phosphatase (TNAP) can inactivate LPS in blood, for example. ³⁰ Since TNAP is highly expressed in the liver, there may be benefits to diets that include liver, depending on the extent of active enzyme remaining. Diet may have profound effects on IAP expression: omega-3 fatty acids have been reported to increase IAP, as well as favoring microbiota that are not pro-inflammatory in mice. ³¹ Moreover, supplementation with probiotics that induce IAP or secrete alkaline phosphatase capable of inactivating LPS may be another strategy for maintaining gut integrity.

Of interest is that fasting or malnutrition lowers IAP expression and increases gut barrier dysfunction in mice and humans. ³² However, this seems contradictory to the beneficial effects observed with caloric restriction (CR), which is protective of intestinal barrier integrity. ^33,34 A possible explanation is that data suggest reducing calories alters the microbiome in rats and humans, favoring microorganisms that are less pro-inflammatory, thereby lowering the concentration of substances such as LPS. However, long-term data on IAP expression during CR is needed, as the decreases observed in short-term fasting may not be present over longer periods of time.