Abstract
Physical exercise confers systemic immunomodulatory benefits, yet the molecular mediators linking metabolic stress to immune cell reprogramming remain incompletely defined. N-lactoyl-phenylalanine (Lac-Phe), a lactate-phenylalanine conjugate synthesized by carnosine dipeptidase 2 (CNDP2), has emerged as a critical exercise-derived metabolite that bridges peripheral energy metabolism with central and peripheral immune regulation. This review systematically summarizes the biosynthetic regulatory network of Lac-Phe, with emphasis on its immunomodulatory functions. And translational advances include biomimetic scaffolds enabling localized Lac-Phe release for tissue regeneration and the first human clinical trial (NCT06743009) evaluating its metabolic and immunomodulatory efficacy. Nevertheless, the unidentified direct membrane receptor on immune cells, unaddressed safety risks of long-term immune modulation, and immature immune-targeted delivery approaches impede clinical translation. Future priorities should focus on receptor identification through chemical proteomics and CRISPR screening, determination of the pharmacological safety window for chronic immunomodulation, and development of localized delivery strategies to accelerate clinical application in immune-mediated disorders.

Keywords
N-Lactoyl-Phenylalanine, Exerkine, Immunometabolism, CNDP2

Abbreviations
CNDP2: Carnosine dipeptidase 2; IBD: Inflammatory bowel disease; Lac-Phe: N-lactoyl-phenylalanine; MELAS: Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-Like Episodes; MGWAS: Metabolome-Wide Genome-Wide Association Study; SCI: Spinal Cord Injury; SGGT: Solution‑Gated Graphene Field‑Effect Transistor; SLC17A1/3: Solute Carrier Family 17 Members 1 and 3

Introduction
Physical exercise is a potent immunomodulatory intervention that regulates immune cell trafficking, inflammatory cytokine profiles, and tissue-resident macrophage polarization across multiple organ systems [1,2]. Regular physical activity reduces systemic low-grade inflammation, a hallmark of metabolic diseases, autoimmune disorders, and accelerated immunosenescence [3]. However, the circulating molecular mediators that transduce metabolic signals from contracting skeletal muscle to distal immune compartments have only begun to be elucidated.

N-lactoyl-phenylalanine (Lac-Phe) is a representative functional exercise-derived metabolite discovered through advances in metabolomics [4]. In 2022, it was identified as a core appetite-suppressing metabolite induced by exercise, rapidly becoming a research focus in the metabolic field [5]. While initially characterized as an anorexigenic metabolite suppressing feeding behavior through hypothalamic AgRP neuron inhibition [6], emerging evidence positions Lac-Phe as a pleiotropic immunometabolic signal. Lac-Phe modulates macrophage/microglial polarization states, suppresses NF-κB-driven inflammation in intestinal mucosa, and promotes AMPK-PGC1α-mediated metabolic reprogramming in neuroimmune contexts [7,8]. These findings establish Lac-Phe as a molecular effector linking exercise-induced metabolic stress to innate immune cell plasticity.

Despite rapid progress, several fundamental questions constrain the immunological understanding of Lac-Phe. First, the direct membrane receptor or immune cell-specific transducer of Lac-Phe remains unidentified [9]. Second, whether Lac-Phe functions as a hormone, a paracrine immunometabolic cue, or both, is unresolved. Third, the therapeutic window for Lac-Phe-mediated immunomodulation, particularly the threshold between physiological anti-inflammatory effects and potential immunosuppressive risks, has not been defined.

This review systematically integrates existing research progress centered on immunology, rather than mere metabolic regulation [10,11], to elaborate Lac-Phe’s biosynthetic features, core immunomodulatory mechanisms, pathological roles in diverse inflammatory diseases, and latest translational achievements, alongside pending research bottlenecks requiring further exploration.

Biosynthesis and Cellular Sources: An Immunometabolic Perspective
Enzymatic basis and tissue-specific expression
CNDP2 serves as the rate-limiting catalytic enzyme responsible for endogenous Lac-Phe biosynthesis via the condensation of lactate and L-phenylalanine [5,12]. Beyond its well-documented expression in skeletal muscle, intestinal epithelium and adipose tissues, abundant CNDP2 expression has been validated in innate immune cells including tissue macrophages and central nervous system-resident microglia, which endows these immune populations with autonomous Lac-Phe synthetic capacity [5,13,14].

Lac-Phe pools originating from exercise versus pharmacological induction possess disparate tissue origins and divergent systemic immunological effects [15]. A single bout of high‑intensity sprint interval training acutely elevates plasma Lac‑Phe from a resting concentration of approximately 0.1 μM to nearly 0.2 μM within 30-60 minutes post‑exercise [5]. Elevated Lac-Phe levels are positively correlated with exercise intensity: sprint exercise > resistance training > moderate-intensity endurance exercise [16]. Such intensity-dependent variation is presumably attributed to lactate serving as the rate-limiting substrate, since high-intensity exercise markedly enhances glycolytic flux and triggers massive lactate production [17]. Meanwhile, CNDP2 transcript abundance negatively correlates with slow-twitch skeletal muscle proportion [14] and Long-term endurance training also upregulates Lac-Phe levels in patients with type 1 diabetes [18], partially explaining variable Lac-Phe induction amplitude among individuals with divergent exercise responsiveness.

By contrast, metformin-triggered Lac-Phe biosynthesis primarily occurs within intestinal epithelial CNDP2⁺ cells [13,19]. In addition, diet also affects the level of Lac-Phe [19,20]. Metabolome-wide genome-wide association study (mGWAS) analyses indicate that Lac-Phe levels are predominantly influenced by dynamic metabolic or inflammatory states rather than by fixed genetic factors, further supporting the notion that its syn-thesis is highly regulated by environmental and physiological conditions [13].

Notably, detectable levels of circulating Lac-Phe persist in CNDP2 knockout mice [12], suggesting the existence of alternative synthetic pathways, implying that whether Lac-Phe derived from other tissues possesses distinct metabolic fates and biological functions remains entirely unknown.

The potential of Lac-Phe as an immune metabolic signal
Li, et al. identified solute carrier family 17 members 1 and 3 (SLC17A1/3) as the primary transporters mediating renal excretion of Lac-Phe [12]. SLC17A1/3 is highly expressed in renal tubular epithelial cells, and its overexpression significantly enhances transmembrane transport of Lac-Phe. Genetic ablation of SLC17A1/3 markedly reduces urinary Lac-Phe excretion without affecting plasma Lac-Phe levels, suggesting the existence of additional Lac-Phe metabolic mechanisms that remain to be elucidated⁸. Moreover, whether renal clearance of Lac-Phe is modulated by exercise, diet, or disease states remains completely unknown. This knowledge gap severely hampers the standardized application of urinary Lac-Phe as a clinical biomarker. Furthermore, plasma Lac-Phe levels are markedly elevated under severe stress conditions such as septic shock and are associated with poor prognosis, suggesting that clearance pathways may be suppressed under pathological stress [21].

Molecular Mechanisms of Lac-Phe-Mediated Immunomodulation
Spinal cord injury
Spinal cord injury (SCI) triggers secondary injury processes involving microglial/macrophage activation, inflammatory responses, and lipid metabolic dysregulation [22,23]. Lac-Phe modulates lipid metabolism in microglia/macrophages via the AMPK-PGC1α-PPARγ pathway, reducing lipid accumulation, suppressing inflammatory responses, and promoting functional recovery following SCI [8]. Lac-Phe activates the classical energy sensor AMPK, which in turn initiates the PGC1α-PPARγ transcriptional axis, a pathway typically associated with mitochondrial biogenesis and fatty acid oxidation [24], suggesting that Lac-Phe may achieve immunophenotypic switching by remodeling cellular energy metabolism. This study expands the potential application of Lac-Phe in central nervous system disorders, indicating that it may exert neuroprotective functions.

Inflammatory bowel disease
Serum Lac-Phe levels are decreased and colonic CNDP2 expression is downregulated in patients with inflammatory bowel disease (IBD), suggesting that Lac-Phe deficiency may contribute to the pathological process [25]. Yu, et al. demonstrated that Lac-Phe inhibits macrophage polarization toward the pro-inflammatory M1 phenotype while promoting the expression of anti-inflammatory M2 phenotype markers, an effect mediated through suppression of the NF-κB signaling pathway. In a dextran sulfate sodium‑induced mouse model of colitis, exogenous Lac-Phe administration significantly alleviated disease severity, reduced colonic mucosal inflammatory cell infiltration, protected epithelial barrier integrity, and lowered colonic tissue inflammation scores [7]. This immunomodulatory effect provides a novel molecular mechanism underlying the anti‑inflammatory benefits of exercise. Unlike the findings in spinal cord injury, where Lac‑Phe activates AMPK, Lac‑Phe primarily inhibits NF‑κB in IBD, suggesting that the tissue microenvironment dictates the preferential engagement of downstream signaling pathways by Lac‑Phe.

Hypertension
Preliminary studies have shown that Lac-Phe administration alleviates the elevation of both systolic and diastolic blood pressure in angiotensin II‑induced hypertensive mice [26]. However, this finding requires validation in independent large‑scale populations, and it remains unclear whether the hypotensive mechanism operates independently of the metabolic and immune effects of Lac-Phe. Given its anti‑inflammatory and metabolic regulatory actions, Lac-Phe may confer cardiovascular protection through mechanisms such as improving vascular endothelial function and reducing oxidative stress. Nevertheless, direct evidence is currently limited, and further animal studies and clinical investigations are warranted to substantiate the cardiovascular protective potential of Lac-Phe.

Others
Plasma Lac-Phe levels are significantly elevated in patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), and correlate with disease severity. Mechanistically, this elevation may be attributed to lactate accumulation resulting from mitochondrial respiratory chain dysfunction, which provides substrate for Lac-Phe synthesis [27]. Furthermore, studies have indicated a correlation between Lac-Phe levels and cognitive function [28], consistent with the well-established effect of exercise in improving cognitive performance, suggesting that Lac-Phe may mediate the beneficial effects of exercise on brain health [29]. However, given the cross-sectional design of this study, only an association rather than causality can be inferred [30]. Future longitudinal cohort studies or Mendelian randomization analyses are warranted to evaluate the value of Lac-Phe as a biomarker of cognitive decline.

Future Development
Clinical translation: From clinical trials to delivery strategies
In 2025, the first human clinical trial of Lac‑Phe (NCT06743009) was officially initiated. This study employs a double‑blind, randomized, crossover design and aims to evaluate the effects of intravenously administered Lac‑Phe on appetite, energy metabolism, and glucose homeostasis in individuals with obesity [9]. This clinical trial will provide critical evidence regarding the pharmacodynamic profile and safety of Lac‑Phe in humans, representing an important milestone in translating basic research on Lac‑Phe toward clinical applications. However, the intravenous route bypasses the local tissue production of Lac‑Phe (e.g., in skeletal muscle and intestine) that occurs during exercise, and may therefore not fully recapitulate the pharmacokinetics and paracrine effects under physiological conditions. Consequently, alternative administration strategies that more closely mimic the physiological pattern remain to be developed.

In the context of local delivery, Liu, et al. constructed a bilayer biomimetic scaffold that enables timed‑release by loading an anti‑senescence peptide in the upper layer and Lac‑Phe in the lower layer, synergistically modulating the immune and metabolic microenvironment and ultimately achieving simultaneous osteochondral tissue repair in an animal model [31]. This finding suggests that local application of Lac‑Phe holds unique potential in tissue engineering and regenerative medicine. For metabolic indications requiring systemic action (e.g., obesity and type 2 diabetes), intestinally restricted release systems or prodrugs targeting specific organs need to be designed to increase local effective concentrations while mitigating the potential risks associated with high systemic exposure.

It must be emphasized that Hedaya, et al. found in cell models that high concentrations of Lac‑Phe interfere with insulin signaling and impair mitochondrial respiration [32], serving as a caution that the pharmacological safety window of Lac‑Phe may be narrow. Future clinical trials must first establish this therapeutic window and systematically evaluate the metabolic toxicity of long‑term high‑dose exposure. To date, no studies have explored the use of prodrugs or intestinally targeted delivery to circumvent high systemic exposure; this should become an important direction for medicinal chemistry and formulation research.

Detection technologies and receptor identification
In the realm of detection technologies, Li, et al. successfully constructed a biosensor capable of detecting L‑lactate and L‑phenylalanine based on solution‑gated graphene field‑effect transistor (SGGT) technology, providing a novel technical approach for rapidly assessing individual responses to exercise [33]. Furthermore, a liquid chromatography‑tandem mass spectrometry method using dried blood spot samples enables quantitative measurement of Lac‑Phe concentrations at multiple time points before, during, and after exercise [34]. These technologies offer convenient tools for individualized exercise effect assessment and dynamic Lac‑Phe monitoring in clinical studies.

In contrast, the direct membrane receptor or molecular target of Lac‑Phe remains unidentified, representing the most critical bottleneck currently constraining mechanistic research and drug development. Although existing studies have shown that Lac‑Phe increases KATP channel current in a dose‑dependent manner [6], no evidence has demonstrated direct binding of Lac‑Phe to the subunits of this channel. Similarly, the metabolite genome‑wide association study (mGWAS) by Elashi, et al. did not identify significant genetic loci associated with Lac‑Phe levels [35], suggesting that Lac‑Phe may exert its functions through metabotropic receptors or transporters, a feature that further complicates target identification.

Based on these considerations, we propose the following priority research directions: (1) unbiased chemical proteomics approaches (e.g., photoaffinity probes, drug affinity responsive target stability [DARTS] technology) to directly capture Lac‑Phe‑binding proteins [36,37]; (2) CRISPR genome‑wide screening to identify essential genes mediating the cellular effects of Lac‑Phe [38]; and (3) upon target identification, structural biology studies to provide a foundation for subsequent drug design [39]. Prior to achieving these goals, any drug development efforts targeting Lac‑Phe mimetics or antagonists will face considerable uncertainty.

Conclusion
Within just three years since its initial identification in 2022 as an exercise‑induced appetite‑suppressing metabolite, Lac-Phe research has rapidly progressed from fundamental mechanistic exploration toward clinical translation. Lac‑Phe modulates macrophage/microglial polarization in immunoregulation, demonstrating therapeutic potential in conditions such as SCI and IBD. Nevertheless, the unidentified direct membrane receptor, the unclear safety window, and the lack of local delivery strategies represent three core obstacles limiting its clinical translation. Future priorities should therefore focus on target identification, determination of the therapeutic window, and optimization of delivery strategies.

Acknowledgments
Not applicable.

Funding Sources
This study was supported by the Guangxi Medical and Health Appropriate Technology Development and Application Project (Grant/Award Number: ‘S2021107’); Clinical Research “Climbing” Program of the First Affiliated Hospital of Guangxi Medical University (Grant/Award Number: ‘YYZS2021002’); and National Natural Science Foundation of China (Grant/Award Number: ‘82260367’, ‘82271371’).

Disclosures
The content is solely the responsibility of the authors and the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions
Lingduo Shao: Conceived and designed research, drafted manuscript.

Mengqi Yang: Edited and revised manuscript.

Hongyu Xu: Edited and revised manuscript.

Li Chen: Edited and revised manuscript, approved final version of manuscript.