Beneficial vascular effects of oral phosphatidylserine supplementation in type 2 diabetes

Neil J McMillan; Marc A Augenreich; Francisco I Ramirez-Perez; Gavin Power; Katherine Burr; Olubodun M Lateef; Manikandan Nagarajan; Christopher A Foote; Natnicha Imkaew; Thomas J Jurrissen; Yoskaly Lazo-Fernandez; Edgar E Betancourt-Cortes; Larissa Ferreira-Santos; Jesus H Beltran-Ornelas; Camila Manrique-Acevedo; Luis A Martinez-Lemus; Jaume Padilla

doi:10.1152/japplphysiol.00281.2026

. Author manuscript; available in PMC: 2026 Jul 28.

Published in final edited form as: J Appl Physiol (1985). 2026 Jun 18;141(1):130–148. doi: 10.1152/japplphysiol.00281.2026

Beneficial vascular effects of oral phosphatidylserine supplementation in type 2 diabetes

Neil J McMillan ^1,², Marc A Augenreich ^1,², Francisco I Ramirez-Perez ¹, Gavin Power ^1,², Katherine Burr ¹, Olubodun M Lateef ^1,³, Manikandan Nagarajan ^1,³, Christopher A Foote ^1,⁴, Natnicha Imkaew ¹, Thomas J Jurrissen ^1,², Yoskaly Lazo-Fernandez ¹, Edgar E Betancourt-Cortes ¹, Larissa Ferreira-Santos ^1,⁵, Jesus H Beltran-Ornelas ¹, Camila Manrique-Acevedo ^1,^6,⁷, Luis A Martinez-Lemus ^1,^3,^8,^*, Jaume Padilla ^1,^2,^7,^*

PMCID: PMC13404116 NIHMSID: NIHMS2191085 PMID: 42312814

Abstract

Endothelial insulin resistance is a characteristic of type 2 diabetes (T2D) that contributes to reduced nitric oxide bioavailability, impaired vasodilation, and arterial stiffening. We recently provided evidence that endothelial insulin resistance in T2D may be attributed to the shedding of insulin receptors by ADAM17. As prior work by others suggested that exogenous phosphatidylserine (PS) can competitively inhibit ADAM17, we hypothesized that oral PS supplementation would improve vascular function in diabetes. First, we corroborated the ability of PS to interact with and inhibit ADAM17 activity using in vitro approaches and experiments in isolated arteries. Next, we tested the vascular effects of PS in diabetic mice (db/db) and subsequently in individuals with T2D through a randomized, double-blind, placebo-controlled clinical trial. In a cell-free system, we found soluble PS binds to ADAM17 and blunts its activity, an effect also observed in cultured endothelial cells and isolated arteries, underscoring its inhibitory capacity. In diabetic mice, oral administration of PS (200mg/kg/day for 4wk) improved insulin-induced dilation in isolated resistance arteries and reduced ex vivo and in vivo indices of arterial stiffness. In individuals with T2D, PS supplementation (900mg/day for 4wk, delivering ~280mg/day PS) enhanced leg blood flow responses to an oral glucose load and reduced load-dependent aortic pulse wave velocity. Lastly, we observed that PS reduced vascular oxidative stress. This work supports the potential of oral PS as a therapeutic strategy to improve vascular function in T2D, and suggests that the beneficial effects of PS may be driven by its vascular insulin-sensitizing and antioxidant actions.

NEW & NOTEWORTHY

The sheddase activity of a disintegrin and matrix metalloproteinase 17 (ADAM17) is augmented in the setting of type 2 diabetes (T2D), likely contributing to the shedding of the extracellular domain of the insulin receptor on the arterial endothelial surface, leading to endothelial dysfunction. Findings from this work support the notion that exogenous phosphatidylserine can inhibit arterial ADAM17 activity and improve markers of vascular health in diabetic mice and humans with diabetes.

INTRODUCTION

A hallmark feature in the initiation and progression of cardiovascular disease in type 2 diabetes (T2D) is vascular insulin resistance, which can result from diminished insulin binding to endothelial insulin receptors and/or disrupted downstream intracellular insulin signaling (1–4). Impaired vascular insulin actions lead to blunted insulin-stimulated vasodilation and blood flow (BF), contributing to glycemic dysregulation and promoting overall vascular dysfunction and disease, including reduced nitric oxide bioavailability and arterial stiffening (5–13). Arterial stiffening in T2D reflects both functional and structural changes in the vascular wall, including altered vascular tone, reduced nitric oxide signaling, oxidative stress, inflammation, and extracellular matrix remodeling. Therefore, complementary assessments of stiffness can provide insight into both vascular function in vivo and intrinsic material properties of the vascular wall. Although insulin resistance in the vasculature is recognized as an early step and causal factor in the pathogenesis of vascular disease, therapeutic strategies targeting the molecular underpinnings of endothelial insulin resistance are lacking.

A key contributor to the proinflammatory and pro-oxidant state in T2D is the increased activity of a disintegrin and matrix metalloproteinase 17 (ADAM17), a membrane-bound enzyme that cleaves the ectodomain of transmembrane proteins (14–16). While the most well-known substrate of ADAM17 is tumor necrosis factor (TNF), emerging evidence indicates that ADAM17 has multiple membrane-anchored molecules as substrates (16). In this regard, we recently demonstrated that activation of ADAM17 in endothelial cells cleaves the insulin receptor-alpha (IRα) from the cell surface, thereby impairing insulin signaling and insulin-induced dilation (15,17). Notably, we also reported that ADAM17 activity is increased in the vascular wall and plasma samples of individuals with T2D and that this corresponded with a reduced presence of IRα and blunted insulin-induced vasodilation in isolated arteries (15,17).

Work from Sommer et al. elucidated that externalization of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is required for ADAM17 sheddase activity (18). When externalized, the anionic PS binds to cationic amino acid clusters of the PS-binding motif in the membrane-proximal domain (the so-called “hinge” region) of the enzyme, inducing a conformational change into an optimal “bent” position for sheddase function (18). Exogenous PS (i.e., non-membrane source) impairs ADAM17 sheddase activity, likely by inhibiting the conformational change into the “bent” position required for shedding (18). Here, sheddase activity refers to proteolytic cleavage of the extracellular domain of a membrane-anchored protein, releasing its soluble ectodomain and reducing its cell surface content. Importantly, in the current study, we report the ability of PS to bind to ADAM17 and inhibit its activity using in vitro approaches and experiments in isolated arteries. These observations, combined with the fact that PS is available as an oral supplement, prompted us to consider oral PS as a therapeutic approach to improve vascular function in T2D.

To that end, we hypothesized that oral PS supplementation would improve indices of vascular insulin sensitivity and overall vascular function in diabetes, and that these effects would be associated with preservation of IRα on the endothelial surface. We initially tested this hypothesis in a mouse model of diabetes (db/db mice) and conducted follow-up experiments in cultured endothelial cells to further substantiate the vascular antioxidant effects of PS observed in vivo. Finally, we translated the findings to individuals with T2D through a randomized, double-blind, placebo-controlled clinical trial.

METHODS

In Vitro and Ex Vivo Experiments

Surface plasmon resonance (SPR) was used to assess the direct molecular interaction between a soluble PS head group analog (sPS, O-phospho-L-serine; P0878, Sigma-Aldrich) and recombinant human ADAM17 (ADAM17-r; 5μg/mL concentration per sensor; 930-ABD, R&D Systems). SPR experiments were performed on an OpenSPR instrument (Nicoya, Canada) using high-capacity carboxyl sensor chips (Nicoya; SEN-AU-100–10-HC-COOH), following a protocol adapted from previous work (19). ADAM17-r was immobilized on the sensor surface by standard amine-coupling chemistry using sodium acetate buffer (pH 5.0). Degassed and filtered PBS-T (pH 7.4; Nicoya) was used as the running buffer throughout all experiments. Following immobilization, unreacted surface sites were blocked with ethanolamine (1M, pH 8.5; Nicoya). Sensor surfaces were regenerated between injections using glycine-HCl (10mM, pH 1.5; Nicoya) to restore baseline signal. After immobilization of the ligand ADAM17-r (baseline), increasing concentrations of the analyte sPS (1, 3, 10, 30, and 100mM) were sequentially injected, with complete regeneration to baseline performed between each concentration. All analyte injections were performed at a flow rate of 20μL/min, with an association phase of 300s followed by a dissociation phase of 600s. Experiments were repeated using independent sensor chips for each sPS concentration. As an additional validation-only negative control experiment, soluble phosphocholine chloride calcium salt tetrahydrate (sPC; P0378–5G, Sigma-Aldrich) was tested at the same molar concentrations as sPS (18).

To determine if PS can mitigate ADAM17 activity, an additional set of experiments was performed. First, in a 96-well plate, a cell-free assay was conducted wherein ADAM17-r (100ng/mL, 930-ABD, R&D Systems), sPS (O-phospho-L-serine, 10mM), or the ADAM17 inhibitor TAPI-0 (50μM, Abcam, ab141497) was added to the vehicle control (DMSO 0.5%). TAPI-0 is a hydroxamate-based broad-spectrum metalloproteinase inhibitor commonly used to inhibit Tumor Necrosis Factor-α Converting Enzyme (TACE)/ADAM17-mediated ectodomain shedding. After 1h of incubation, ADAM17 activity was measured using the Sensolyte 520 TACE fluorimetric activity assay kit (No. AS-72085, AnaSpec, Fremont, CA), as previously described (15,17). Fluorescence values are presented as the change from baseline (time zero). Negative change values, when observed, were reported as calculated.

To extend our findings that PS inhibits ADAM17 sheddase activity in a cell-free assay, passage-4 human umbilical vein endothelial cells (No.CC-2519, Lonza, Basel, Switzerland) were cultured in a 96-well plate with VascuLife EnGS culture media (2% fetal bovine serum, FBS) to 95% confluency, then serum starved (0.5% FBS) overnight. Cells were then pretreated for 1h with sPS (100μM) or TAPI-0 (50μM, Ab141497). To emulate the ADAM17 hyperactivity seen in T2D, cells were transduced with an adenovirus (MOI=100) expressing human ADAM17 (Ad.hADAM17, ADV-200349 No.2001, Vector Biosystems, Malvern, PA) or a control virus (Ad-CMV-null, No.1300, Vector Biosystems) for 24h in 96-well plates. Cells overexpressing ADAM17 (ADAM17-OE) were pretreated for 1h with sPS (100μM) or TAPI-0 (50μM), and ADAM17 activity was again assessed with the Sensolyte 520 TACE fluorimetric activity assay kit.

To corroborate results from in vitro experiments, mesenteric arteries from C57BL/6 mice were excised, cannulated, and pressurized using pressure myography (Living Systems Instrumentation, Burlington, VT), as described (20,21). ADAM17 activity (Sensolyte 520 TACE fluorimetric activity assay kit) was determined in three conditions: vehicle-treated vessels (DMSO), sPS-treated vessels (100μM), and TAPI-0-treated vessels (50μM). Treatments were applied 1h prior to application of, and in unison with, the fluorescently labeled substrate of ADAM17. Widefield epifluorescent images were acquired with a Leica DMi8 automated microscope, with THUNDER imaging technology (Leica Microsystems, Inc., Morrisville, NC) using a 20X/0.4 NA air objective. After subtraction of background intensity, the mean intensity was calculated (final-baseline) to quantify ADAM17 activity. To address whether PS exposure can improve insulin-induced dilation in isolated arteries from animals not previously exposed to PS, mesenteric arteries were isolated from C57BL/6 mice. Arteries were intraluminally incubated overnight with either vehicle or sPS (100μM), and the vasomotor responses to insulin and sodium nitroprusside (SNP) were assessed as described below.

To further substantiate the vascular antioxidant effects of PS observed in the preclinical mouse trial that is described below, a series of follow-up experiments was conducted in cultured endothelial cells. First, the ability of sPS to blunt ADAM17-induced shedding of TNF was tested. TNF was measured in the lysate and supernatant of ADAM17-OE endothelial cells in response to a 1-h sPS exposure (100μM). Western blotting was employed (primary antibody: ab6671, Abcam, 1:500; secondary: IgG anti-rabbit, BioRad, 1:2000) as previously described (17,22). After noting that sPS blunts the shedding of TNF in ADAM17-OE endothelial cells, experiments were conducted to examine the effects of ADAM17 overactivity and TNF signaling on reactive oxygen species (ROS), and its mitigation by sPS. Specifically, ADAM17-OE endothelial cells were incubated with vehicle or sPS (100μM), CellROX Green (Invitrogen, No.C10444, 5μM final well concentration in 10mM HEPES buffer), and Hoechst nuclear stain (Invitrogen, No.3342, 2μM final well concentration in HEPES buffer) for 1h. Cells were washed with PBS, fixed with 4% paraformaldehyde, washed with PBS, and fluorescence read on a plate reader (BioTeK Synergy H1, Agilent, Santa Clara, CA) at the recommended excitation/emission spectra (CellRox Green: 485/520nm, Hoechst nuclear stain: 360/460nm). After observing a robust effect of sPS in mitigating ADAM17-induced ROS, we sought to delineate the contribution of O₂⁻, and to determine whether this effect was mediated by TNF signaling. To this end, dihydroethidium (DHE) fluorescence was examined in ADAM17-OE endothelial cells after incubation with a TNF-neutralizing antibody (nTNF; mAb No.7321, Cell Signaling, 1μg/mL, 24h) or sPS (100μM) for 1h. DHE (Invitrogen, No.D11347, 10μM) and Hoechst nuclear stain were added to all wells with 30min remaining in the 1-h treatment. Fluorescence microscopy (Leica Microsystems, Inc. Morrisville, NC) was used to acquire the fluorescent signals (DHE 518/606 nm, Hoechst nuclear stain: 360/460 nm), and DHE intensity was quantified utilizing a region-of-interest (ROI) analysis (two 300×300μm regions). To examine the effect of sPS on TNF-induced O₂⁻ production, a separate experiment was conducted in which cells were treated for 1h with TNF (H8916, Sigma-Aldrich, 10ng/mL), with or without sPS (100μM) or with Tempol (No.581500, Calbiochem, 1mM), used as a positive control. Outcomes were normalized to nuclear intensity to account for differences in cell number.

Preclinical Trial in Diabetic Mice

Animal procedures were approved by the University of Missouri Animal Use Committee and performed in accordance with the National Institutes of Health guidelines. Wildtype C57BL/6 and leptin receptor-deficient male mice (db/db), obtained from the Jackson Laboratory, were fed a standard chow (5053-PicoLab Rodent Diet 20, LabDiet) and provided water ad libitum. Animals were housed under a 12-h:12-h dark/light cycle, and animal caretakers monitored their health. At 9wk of age, db/db mice were additionally fed 50±5mg of peanut butter daily for 1wk (for familiarization purposes). At 10wk of age, db/db mice were randomized into two groups and were fed either 1) daily peanut butter (n=10) or 2) daily peanut butter + 200mg/kg/day of soy phosphatidylserine (PS; 870336, Avanti Polar Lipids, Croda International Plc, n=10) for 4wk. Peanut butter was used as the vehicle because PS is a phospholipid that is not easily administered in aqueous solution, and peanut butter is highly palatable to rodents and, when mixed with PS, allowed accurate day-to-day dose delivery, and minimized stress during repeated dosing after a 1-wk familiarization period. Following the 4-wk feeding paradigm (at 14wk of age), animals were euthanized while under surgical plane anesthesia with 2% isoflurane (AKORN Animal Health) via inhalation with room air (250mL/min) followed by pneumothorax and exsanguination. Arteries were excised for mechanical and functional testing, and for proteomics analyses. Blood was collected for plasma analyses. Complementary hemodynamic, lipidomic, proteomic, and inflammatory measurements were included to contextualize the vascular effects of PS and to determine whether these effects were accompanied by broader vascular, cardiometabolic, or inflammatory changes. Each mouse represents one experimental unit. One mouse in the PS group died due to complications during blood pressure measurements, resulting in n=9 for PS-fed mice. A schematic of the preclinical trial is shown in Figure 1A. Individuals performing experiments in this preclinical trial were blinded to the condition of the animal during data collection and analysis.

FIGURE 1: — A) Preclinical trial design with phosphatidylserine (PS) supplement phospholipid composition (n=3). B) Randomized, double-blind, parallel arm human clinical trial design. PS supplement composition compared with the placebo capsules, with phospholipid species breakdown (the inset details the phospholipid composition of the human supplement, which is a soy-derived extract containing 31.1% phosphatidylserine, 19.5% phosphatidic acid, 10.6% lysophosphatidylethanolamine, and 38.8% from other components combined; n=3/group).

In Vivo Measures

Blood Pressure

Blood pressure was assessed using a CODA tail-cuff system (CODA-HT2; Kent Scientific, Torrington, CT) according to manufacturer guidelines and as previously described (23). Mice were acclimated to the restrainer and tail-cuff system for four consecutive days prior to data collection to minimize stress-induced variability. During each session, mice were placed in a temperature-controlled environment to ensure adequate tail BF. On the final day (the day before euthanasia), multiple measurements were recorded, and the average of at least 10 successful readings per mouse was used for analysis.

Aortic Pulse Wave Velocity

Arterial stiffness is influenced by both hemodynamic loading conditions and intrinsic vascular wall material properties. Measurement of pulse wave velocity (PWV) in vivo provides an index of load-dependent arterial stiffness. Aortic stiffness was assessed in vivo using a Vevo 3100 ultrasound system (Fujifilm, VisualSonics) as previously described (23,24). Image analyses were conducted using Vevo Vasc software. Briefly, mice were anesthetized with 1–2% isoflurane in 98–99% oxygen and placed supine on a temperature-controlled heating board (36.5–38.5°C) to maintain body temperature. Limb electrodes were secured for continuous ECG monitoring and high-resolution images were acquired in EKV mode at a frame rate of ≥3000fps. Images of the aortic wall and lumen along the longitudinal axis were collected for PWV measurements. The PWV value, reported in m/s, was derived using the transit time method, calculated as the difference in arrival times of pulse waves at two locations along the aorta divided by the distance between these points.

Ex Vivo Measures

Arterial Vascular Reactivity, Remodeling, and Mechanical Stiffness

Arteries were isolated to assess vascular reactivity (mesenteric, posterior cerebral, and aorta) and passive mechanical properties (femoral, mesenteric, and aorta), as previously described (25,26). For assessment of vascular reactivity, arteries were excised, cleaned of surrounding tissue, and mounted on a multi pin myograph system (DMT 620M; Danish Myo Technology A/S, Aarhus, Denmark) or cannulated and pressurized at 70mmHg on a pressure myography system (Living Systems). Vessels were preconstricted using either the thromboxane A₂ receptor agonist U46619 (aorta, 10⁻⁵M, via pin myography) or phenylephrine (mesenteric, cerebral, 10⁻⁵M, via pressure myography). To assess endothelium-dependent and independent vasodilatory responses, the vessels were exposed to increasing concentrations of insulin (10⁻⁹ to 10⁻⁵M), acetylcholine (ACh; 10⁻⁹ to 10⁻⁵M), or SNP (10⁻⁹ to 10⁻⁴M). To further determine vessel function, additional mesenterics were isolated, cannulated, and pressurized before preconstriction with phenylephrine (10⁻⁶M) and exposure to incremental increases in intraluminal flow (from 0.0–3.3mL/h, with 0.3mL/h increments every 2min), to assess flow-mediated dilation (FMD), as previously described (27).

To assess vascular remodeling and mechanical stiffness, smaller arteries (i.e., femoral and mesenteric) were cannulated, pressurized, and subjected to stepwise increases in intraluminal pressure (5 to 120mmHg) under passive conditions (i.e., calcium-free buffer). Intraluminal diameters and wall thicknesses were recorded at each step. The biomechanical properties of the smaller vessels were assessed, including the incremental modulus of elasticity (E_inc), and the calculated incremental PWV (cPWV_inc).

To determine the elastic properties of isolated mouse aortas, tensile strength was measured. A 2-mm ring from the proximal descending thoracic aorta was cleaned of surrounding tissue and mounted onto the pins of an automated tensometer (DMT 560TP-II; Danish Myo Technology A/S, Aarhus, Denmark). Following alignment and calibration, the pins were set to separate at a constant rate of 50μm/s to apply radial stress until tissue failure. Displacement and force were recorded continuously. Tissue length, for the determination of E_inc, was measured at an x10 magnification using an Amscope 10MP digital microscope camera (MA1000, Irvine, CA), and tissue length was calculated using ImageJ. A 1-mm ring adjacent to the test ring was fixed in 4% paraformaldehyde before paraffin embedding, transverse sectioning (5μm), and mounting on a microscopy slide to determine intraluminal diameter and wall thickness using a 40X/0.65 NA air objective (Olympus, BX43, Tokya, Japan). Intraluminal diameter and wall thickness were determined using a Python script. Strain and stress curves were generated based on measurements of intraluminal diameter and wall thickness. From these curves, E_inc was calculated using a paired stepwise approach (28).

Atomic Force Microscopy

To further evaluate aortic stiffness ex vivo, atomic force microscopy (AFM, JPK NanoWizard 4XP BioAFM, Bruker, Billerica, MA) was used to assess blood pressure-independent aortic wall biomechanics, as described previously (25,26). In other words, AFM was used to determine the contribution of intrinsic vascular wall material properties to arterial stiffness, independent of hemodynamic loading conditions. Briefly, thoracic aorta explants were longitudinally opened, and the adventitial surface of each explant was fastened to a plastic coverslip using Cell-Tak adhesive. Nanoindentation force-distance measurements were performed on the en face aortic explants using a triangular silicon nitride cantilever (type C; Bruker; MLCT) with an approximate tip radius of 20nm and a nominal spring constant of 0.01N/m. Repeated loading-unloading cycles were applied at defined locations. A minimum of 50 nanoindentation curves were performed on seven random locations within the 2mm² explants. A custom Python script was used to calculate force curves and Young’s modulus of elasticity from acquired indentation curves. This script followed the recommendations by Targosz-Korecka et al. and employed the Hertz model, shown below:

F = \frac{2}{π \tan (α)} \frac{E}{(1 - v^{2})} δ^{2}

where $F$ is the force (N), $α$ is the angle of opening for the tip (radians), $E$ is Young’s modulus of elasticity (kPa), $ν$ is the Poisson’s ratio of the sample (generally set to 0.5 arbitrary units for soft biological samples), and δ is the indentation depth (nm) (29). Any curves that deviated from the established mathematical model were discarded to ensure an unbiased approach. For overall clarity, stiffness-related outcomes were defined according to the measurement approach and species. In mice, in vivo PWV was used as an index of arterial stiffness under physiological loading conditions. Ex vivo E_inc and cPWV_inc indices were used to assess passive mechanical properties of isolated arteries, while AFM-derived aortic tissue stiffness was used as a local measure of intrinsic vascular wall material stiffness.

Lipidomic Analysis

To obtain a comprehensive lipid composition of the PS provided to mice (Avanti Polar Lipids No.870336) and the placebo and PS supplement provided to humans, samples were assessed by the Metabolomics Center at the University of Missouri. Briefly, 10mg was weighed out for each sample (n=3) for each condition (mouse PS, human placebo, human PS) and placed into 4mL glass vials. Methanol (375μL) and methyl tert-butyl ether (1250μL) were added, and samples were sonicated, shaken, and centrifuged at 3500xg for 10min. The upper layer containing the methyl tert-butyl ether and lipids was transferred to a new vial, dried with nitrogen, reconstituted with 1mL of CHCl₃/MeOH/H₂O (60:30:4.5), and analyzed on a Bruker Impact II mass spectrometer coupled to a Waters UPLC system. Liquid chromatography with tandem mass spectrometry was performed in negative ion mode. The data were processed using MS-DIAL with a matching score of 800 (out of 1000) for lipid identification.

Proteomic Analysis

To confirm the ability of PS to be detected in the plasma and arterial wall, a sub-study was performed. Mice (db/db) were fed peanut butter (n=5) or peanut butter + deuterium-labeled PS (labeled serine head, Cat No.0544, 1,2-dilinoleoyl-sn-glycero-3-phospho-L-serine-d3, Frankenstein BioReagents) (n=5) for 5 days. Following euthanasia, plasma and homogenized aorta samples were provided to the Charles W Gehrke Proteomics Center at the University of Missouri for quantification using mass spectrometry. The aorta samples and 25μL of the plasma samples were precipitated with three volumes of 100% acetone. The protein pellets were washed with 80% acetone in water and then resuspended in 25μL urea buffer (6M urea, 2M thiourea, 100mM ammonium bicarbonate, pH 8.0). The total protein content was determined using the 660nm assay, as per the manufacturer’s protocol (ThermoFisher, Pierce). An equal amount of protein (10μg) was digested with 0.5μg of trypsin overnight. Peptides were then desalted and concentrated using C18 tips (Cat No.87784, Lot No.TG271472) according to the manufacturer’s protocol (Pierce/Thermo Scientific). Detection of the deuterium label was conducted using mass spectrometry.

Aortic Ring Immunohistochemistry

Aortic rings (2mm) were isolated, fixed in 4% paraformaldehyde (PFA) for over 24h, and embedded in paraffin. Serial sections (5μm) were prepared for immunohistochemistry and treated with BLOXALL blocking solution (Vector SP-6000) for 10min at room temperature to quench endogenous peroxidase activity. To minimize non-specific binding, sections were subsequently blocked with 5% bovine serum albumin (BSA) in PBS for 1h at room temperature. Primary antibodies targeting nitrotyrosine (Anti-Nitrotyrosine, Merck AB5411, 1:200), and alpha smooth muscle actin (⍺-SMA, Anti-SMA, Invitrogen, PA5–85070, 1:1000) were applied overnight at 4°C. For signal detection, biotinylated secondary antibody (Goat Anti-Rabbit IgG, BA-1000–1.5) was incubated for 1h at room temperature, followed by the Vectastain Elite ABC HRP detection system (PK6100) for 30min. Immunoreactivity was visualized using 3,3’-diaminobenzidine (DAB) chromogen (ImmPACT DAB Substrate Kit, SK-4105) for 5min at room temperature, followed by counterstaining with hematoxylin (H-3404–100). Brightfield images were acquired using 20X and 40X air objectives in a Leica DM6 microscope. Three images obtained with the 40X objective per aortic ring were captured under identical exposure settings for quantitative analysis. Images were processed in ImageJ, where color deconvolution was performed using the H-DAB vector. The R-channel, corresponding specifically to the nitrotyrosine and α-SMA signals, was extracted for quantification.

Confocal Imaging

The amount of IRα on the surface of mesenteric arteries and filamentous actin (F-actin) content in mesenteric arteries were quantified using confocal imaging. Briefly, arteries were excised and fixed in 4% PFA. Following two washes with PBS and one with glycine (0.1M), vessels were permeabilized with 0.5% TX-100 for 15min. Subsequently, vessels were washed three times with PBS and blocked in a 1% BSA solution for 1h at room temperature. They were then intraluminally exposed to anti-IRα antibody (1:100 dilution, Abbiotec No. 200110) at 4°C overnight. Vessels were incubated in Alexa Fluor Plus 488 (1:200, Invitrogen, A32731) for 1h in the presence of 4′, 6-diamidino-2-phenylindole (DAPI; 0.5μg/mL), Alexa Fluor 633 Hydrazide (Molecular Probes, 0.2μM), and 200nM Alexa Fluor 546 phalloidin (Thermo Fisher, Cat No.A22283). Images of nuclei, IRα, and F-actin were acquired using a Stellaris 8 confocal microscope (Leica Microsystems, Inc., Morrisville, NC) with a 25X/0.95NA objective. To quantify IRα on the luminal surface of mesenteric arteries, confocal images were analyzed using a custom Python script. Using the internal elastic lamina as a mask, the custom script isolated the endothelial-specific IRα signal by quantifying positive voxels in an unbiased manner.

Clinical Trial in Individuals with T2D

Ethical Approval and Study Population

This study was registered at ClinicalTrials.gov (NCT04557228), approved by the University of Missouri Institutional Review Board (IRB, No.2025921), and conducted in accordance with the Declaration of Helsinki. Participants aged 45–64 years with a clinical diagnosis of T2D were recruited from Columbia, Missouri, and the surrounding areas. Prior to participation, all subjects provided written informed consent and completed a health history questionnaire. Exclusion criteria included a BMI >39 or <25kg/m², history of cardiovascular disease (myocardial infarction, stroke, coronary artery disease, or heart failure), hepatic or renal disease, hormone therapy (including estrogen and testosterone therapy), immunosuppressant therapy, autoimmune disease, active cancer, current tobacco use, excessive alcohol consumption (>14 drinks/wk for male and >7 drinks/wk for female), pregnancy, and body weight change ≥5% in the last 6 months. Participants were required to maintain a stable medication regimen prior to trial initiation. Participant characteristics are provided in Table 1. Participants using GLP-1 receptor agonist-based therapies were eligible if they met the body weight stability criteria mentioned above (Table 1). Menopausal status was recorded for female participants as pre- or post-menopausal and summarized descriptively in Table 1.

Table 1.

Subject characteristics, anthropometrics, and blood biomarkers before and after Placebo vs. Phosphatidylserine (PS) treatment in T2D.

	Placebo (n=18)		PS (n=16)
Variable	Baseline	Final	Baseline	Final

Age, yrs	53 ± 1		57 ± 1*
Sex, M/F	11/7		10/6
Menopausal Status, Pre/Post	3/4		0/6
Race, no. (%)
White	15 (88.9)		14 (87.5)
Black	1 (5.55)		2 (12.5)
Asian	1 (5.55)		0 (0)
Ethnicity, no. (%)
Non-Hispanic	17 (94.45)		16 (100)
Hispanic	1 (5.55)		0 (0)
Height, cm	175 ± 3		175 ± 2
Weight, kg	103.1 ± 4.3	104 ± 4.2	106.4 ± 3.0	106.3 ± 3.1
Body mass index, kg/m²	33.4 ± 0.8	33.5 ± 0.8	34.5 ± 0.9	34.4 ± 0.9
Lean Body Mass, kg	61.7 ± 3.2	61.5 ± 3.5	60.7 ± 2.5	60.8 ± 2.7
Fat Mass, kg	38.1 ± 2.2	38.3 ± 2.1	42.3 ± 2.5	42.0 ± 2.5
Visceral Adipose Tissue, g	2890 ± 307	3002 ± 328	2817 ± 310	2743 ± 294
Body Fat, %	37.6 ± 1.7	37.5 ± 1.6	40.6 ± 2.1	40.5 ± 2.1
Hemoglobin A1C, %	7.6 ± 0.5	7.5 ± 0.5	7.3 ± 0.3	7.2 ± 0.4
Fasted insulin, μIU/mL	11.7 ± 1.7	14.1 ± 1.7	14.6 ± 3.7	17.9 ± 4.4
Fasted blood glucose, mg/dL	134.1 ± 11.2	134.3 ± 13.6	126 ± 10.5	123 ± 11.7
HOMA-IR	4.0 ± 0.6	5.0 ± 0.9	4.9 ± 1.4	6.0 ± 1.7
Length of T2D diagnosis, yrs	10 ± 2		11 ± 2
SBP, mmHg	142 ± 4	138 ± 5	147 ± 5	141 ± 4
DBP, mmHg	75 ± 2	72 ± 2	71 ± 3	69 ± 2
MAP, mmHg	97 ± 3	93 ± 3	95 ± 3	91 ± 2
AST	28.9 ± 2.3	27.1 ± 2.1	34.6 ± 3.4	32.1 ± 2.7
ALT	29.0 ± 2.8	28.5 ± 2.5	33.8 ± 3.9	34.0 ± 4.0
Nitrite, nM	71.1 ± 8.3	87.4 ± 9.9	75.1 ± 10.3	73.5 ± 8.7
MDA, mmol/L	2.1 ± 0.2	1.8 ± 0.3	2.1 ± 0.2	1.6 ± 0.2
ADAM17 activity (A.U.)	0.70 ± 0.10	0.68 ± 0.10	1.06 ± 0.11	1.09 ± 0.12
Lipids, mg/dL
Triglycerides	169 ± 18	158 ± 17	163 ± 18	154 ± 16
Total cholesterol	151 ± 11	144 ± 12	156 ± 9	155 ± 11
HDL cholesterol	37 ± 2	36 ± 2	38 ± 3	38 ± 3
LDL cholesterol	84 ± 10	80 ± 11	87 ± 7	86 ± 8
Inflammatory Cytokines
hsCRP, mg/L	2.1 ± 0.4	1.9 ± 0.3	3.5 ± 0.8	4.1 ± 0.9
TNF, pg/mL	8.9 ± 0.7	8.9 ± 0.6	9.7 ± 0.3	10.4 ± 0.6
MCP-1, pg/mL	206.9 ± 21.4	210.0 ± 25.6	171.1 ± 4.9	182.5 ± 8.6
IL-6, pg/mL	3.8 ± 0.3	3.49 ± 0.4	3.3 ± 0.5	3.9 ± 0.6
IL-8, pg/mL	5.3 ± 0.6	5.1 ± 0.5	4.8 ± 0.3	5.3 ± 0.4
IL-10, pg/mL	2.3 ± 0.2	2.2 ± 0.2	2.5 ± 0.2	2.7 ± 0.3
Medications, no.
Hypoglycemic
Biguanide	10		11
Sulfonylurea	2		1
SGLT2 inhibitor	7		2
GLP-1R analogue	6		7
Insulin	6		7
Cardiovascular
ACE inhibitor	4		5
ARB	5		3
Diuretics	1		5
Beta blockers	3		3
Statins	11		8
CCB	1		3
Aspirin	3		3
Fibrates	0		4
Thyroid hormone therapy	1		3
Antidepressant	2		5

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Study Design

We employed a randomized, double-blind, placebo-controlled, parallel-arm intervention to test the hypothesis that exogenous PS supplementation would improve overall vascular function in individuals with T2D (Figure 1B). After meeting the study criteria, enrolled participants completed four study visits. In the week leading up to the baseline and final study visits, participants wore an ambulatory blood pressure (AMBP) monitor for 24h. After completing their baseline study visit, participants were randomized in a 1:1 fashion to either 4wk of PS supplementation (900mg/day; Bulk Supplements, Hendersen, NV) containing 31.1% pure PS (delivering ~280mg/day of PS; Figure 1B) or a matched inactive placebo. Importantly, PS has been utilized at varying doses (200–800mg/day) across numerous clinical trials to investigate its ergogenic properties and effects on cognition, with minimal to no side effects reported (30–37). The lipidomic profile of the PS supplement and compound is detailed in Figure 1B. The University of Missouri Investigational Drug Pharmacy randomized participants and allocated study medications to ensure blinding. Participants were not stratified by age or sex during randomization. To avoid a lag in dosage timing and study visit scheduling, two extra days of study medication was allocated per individual. Thirty-seven individuals were enrolled, and three individuals were ultimately removed from the intervention (n=1 due to unrelated illness, n=1 due to a previously unknown allergy to study medication; n=1 due to a previously undisclosed autoimmune condition), leaving a total of n=34 who completed the study (Placebo: n=18, PS: n=16) (Figure 2, Table 1).

FIGURE 2: — Diagram depicts the number of participants assessed for eligibility, excluded before randomization, and randomized to placebo or phosphatidylserine. Shown are participant allocation, follow-up, exclusions after randomization, and the numbers included in the final analyses for each treatment group.

Ambulatory Blood Pressure

Twenty-four-hour AMBP was monitored before the baseline and final study visits (ABPM 7100, Baxter International, Deerfield, IL). Importantly, this measure was taken at least 24hr before the baseline and final study visits but no more than 7 days prior. The participant’s arm circumference was measured, and an appropriately sized blood pressure cuff was fitted on the dominant arm. The unit was pre-programmed to measure blood pressure every half hour during waking hours and once per hour during non-waking hours. If a measurement failed, the unit was programmed to attempt another measurement 2min after the first. Participants were instructed to maintain their normal lifestyle patterns. Summary data were downloaded, analyzed (CardioPerfect WorkStation), and extracted for the 24-hr waking and non-waking periods (38).

ABPM data quality was assessed by the number and percentage of successful readings reported in the ABPM output for each participant. These values were summarized for the overall 24-h recording period and decomposed for waking and non-waking periods. The average number and percentage of successful measurements at the pre- and post-intervention measurement periods are reported in Table 2. The nocturnal systolic blood pressure (SBP) dipping was calculated as [(daytime SBP – nighttime SBP)/(daytime SBP)] × 100, as previously described (25).

Table 2.

Vascular and hemodynamic outcomes before and after Placebo vs. Phosphatidylserine (PS) treatment in T2D.

	Placebo (n=18)		PS (n=16)
Variable	Baseline	Final	Baseline	Final

Brachial Artery
Baseline diameter, cm	0.42 ± 0.02	0.42 ± 0.02	0.42 ± 0.03	0.42 ± 0.02
Peak diameter, cm	0.44 ± 0.02	0.44 ± 0.02	0.44 ± 03	0.44 ± 0.02
Absolute diameter change, cm	0.02 ± 0.003	0.02 ± 0.002	0.02 ± 0.002	0.02 ± 002
FMD, %	3.80 ± 0.58	4.33 ± 0.55	3.70 ± 0.50	4.11 ± 0.44
FMD/Shear AUC, au	0.032 ± 0.009	0.045 ± 0.004	0.045 ± 0.006	0.054 ± 0.007
Shear AUC, au	97884 ± 12239	104877 ± 12677	89534 ± 8877	85055 ± 8066
Time-to-peak dilation, s	74 ± 9	86 ± 7	74 ± 9	69 ± 7

Ambulatory BP
24-h SBP, mmHg	137 ± 4	134 ± 3	135 ± 2	133 ± 3
24-h DBP, mmHg	86 ± 3	84 ± 3	81 ± 2	80 ± 2
24-h MAP, mmHg	103 ± 3	101 ± 3	99 ± 2	98 ± 2
Daytime SBP, mmHg	140 ± 4	137 ± 4	136 ± 2	135 ± 3
Daytime DBP, mmHg	89 ± 3	86 ± 3	82 ± 2	81 ± 2
Daytime MAP, mmHg	106 ± 3	103 ± 3	100 ± 2	99 ± 2
Nighttime SBP, mmHg	126 ± 4	125 ± 3	129 ± 3	128 ± 3
Nighttime DBP, mmHg	79 ± 2	78 ± 2	73 ± 2	76 ± 2
Nighttime MAP, mmHg	95 ± 3	94 ± 2	92 ± 2	93 ± 3
Nocturnal Dipping, %	12.23 ± 3.98	10.60 ± 2.65	4.85 ± 2.5	4.91 ± 1.86
QC, successful readings
24-h valid readings	36.8 ± 1.7	35.8 ± 1.5	38.6 ± 1.1	39.0 ± 0.9
Daytime valid readings	28.4 ± 1.4	27.8 ± 0.9	29.4 ± 1.0	29.8 ± 0.8
Nighttime valid readings	9.0 ± 0.1	9.1 ± 0.2	9.2 ± 0.1	9.2 ± 0.3
24-h valid readings, %	69.5 ± 6.9	80.8 ± 3.6	77.3 ± 4.9	85.5 ± 3.8
Daytime valid readings, %	71.6 ± 6.2	79.3 ± 4.1	74.6 ± 5.4	83.6 ± 4.2
Nighttime valid readings, %	92.3 ± 4.1	92.8 ± 4.4	91.1 ± 4.3	95.4 ± 2.1
Passive Limb Movement (PLM)
Baseline
MAP, mmHg	101 ± 4	95 ± 4*	97 ± 3	95 ± 2
CO, L/min	3.5 ± 0.2	4.0 ± 0.3	4.5 ± 0.5	4.3 ± 0.3
SV, mL/beat	50.3 ± 1.8	58.9 ± 3.5	57.7 ± 4.7	56.5 ± 3.2
HR, bpm	69 ± 3	69 ± 3	77 ± 3	75 ± 2
BF, mL/min	805 ± 100	746 ± 41	849 ± 116	905 ± 106
LVC, mL/min/mmHg	8.3 ± 1.0	7.8 ± 0.5	9.0 ± 1.2	9.6 ± 1.1
PLM (Δpeak)
ΔMAP_peak, mmHg	−6 ± 1	−5 ± 1	−5 ± 1	−4 ± 1
ΔCO_peak, L/min	0.37 ± 0.07	0.29 ± 0.04	0.41 ± 0.11	0.43 ± 0.08
ΔSV_peak, mL/beat	5.5 ± 0.85	4.9 ± 0.80	5.7 ± 1.3	5.0 ± 0.96
ΔHR_peak, bpm	2 ± 1	2 ± 1	1 ± 1	2 ± 1
ΔBF_peak, mL/min	510 ± 86	439 ± 36	497 ± 97	521 ± 83
ΔLVC_peak, mL/min/mmHg	5.3 ± 0.9	4.5 ± 0.6	5.4 ± 1.0	5.6 ± 0.9

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Baseline and Final Study Visit Procedures

Experimental visits were performed at the Clinical and Translational Science Unit at the NextGen Precision Health Building at the University of Missouri campus in temperature-controlled rooms (~21°C). Participants were asked to refrain from vigorous physical activity or exercise for 24h and to avoid consuming alcohol and caffeine for at least 12h before the study visit. In preparation for the study visit, participants arrived following an overnight fast and having withheld any diabetes-specific medication that morning. Upon arrival (0800), anthropometric measures were taken, including height, weight, and dual-energy X-ray absorptiometry (DEXA; Lunar iDXA, GE Healthcare, Chicago, IL). The following experimental measures were then performed:

Brachial artery FMD

Brachial artery FMD was measured to reflect conduit artery endothelial function and assessed by a trained ultrasonographer, as previously documented and in accordance with established guidelines (39–42). Briefly, following 20min of supine rest in a quiet room, the brachial artery diameter and blood velocity were measured using 2D/Doppler ultrasound (GE Logiq P5). Images were acquired using an 11-MHz linear array transducer, velocity was obtained in duplex mode at a pulsed frequency of 5-MHz with an insonation angle of 60°, the sample volume was adjusted to encompass the lumen of the vessel without extending past the vessel walls, and the cursor was set midvessel parallel to the walls. Following 2min of baseline data acquisition, a cuff placed 3cm below the antecubital space was rapidly inflated to 250mmHg (E20, D.E. Hokanson, Bellevue, WA) for 5min. The cuff was then rapidly deflated, and continuous measurements of diameter and blood velocity were recorded for 3min. To ensure reproducibility within and between study visits, the probe placement was marked, the location was measured from the antecubital space and ultrasound settings and landmarks were recorded.

Passive Leg Movement

Passive leg movement (PLM) was performed according to published guidelines (43). Briefly, participants were seated in an upright posture in a chair with sufficient ground clearance for unimpeded leg movement and outfitted with a knee brace with a fixed 90° range of motion. The opposite leg was supported to ensure a relaxed state was maintained. Participants were then familiarized with the movement and given sufficient rest prior to the initiation of the first test (≥15min). The common femoral artery diameter and blood velocity were measured using Doppler ultrasound (GE Logiq P5) (42,43). After acquiring a 60-s baseline ultrasound measurement, trained research staff moved the participant’s leg through a 90° range of motion at a cadence of 1Hz for 60s, during which the leg passed through a cycle of 180°−90°−180° every second. Importantly, care was taken to ensure the movement was completely passive, and the participant did not assist with any form of muscle contraction. Measures were taken in duplicate, with at least a 10-min rest, to obtain PLM-induced hemodynamic outcomes. More rest was added between measures if baseline central and peripheral hemodynamics were not recovered. These measures closely reflect vascular function in the resistance arteries, and up to 80% of the BF response to PLM is mediated by nitric oxide (44–46). Herein, PLM complements FMD in the measurement of vascular function.

Leg BF in Response to an Oral Glucose Load

After 30min of supine rest, participants ingested a 75g oral glucose load (No. 100075, Azer Scientific, Morgantown, PA) within 2min. Superficial femoral diameter and velocity were assessed at baseline, and every 15min for 60min following ingestion. We have previously established that significant changes in leg BF occur within 60min following ingestion of 75g of glucose, and that this response is impaired in individuals with T2D (17,47).

Carotid-to-Femoral PWV

Aortic stiffness was assessed via carotid-to-femoral PWV (cfPWV; SphygmoCor XCEL: Cardiex, Sydney, AU) according to current recommendations and as previously described (25,48,49). The SphygmoCor XCEL device is a well-validated, non-invasive measure of aortic stiffness that simultaneously captures aortic (tonometer) and femoral (cuff) pulse waves. Transit times were calculated using the foot-to-foot method, with wave feet identified using intersecting tangent algorithms. The cfPWV value, reported as m/s, was calculated by dividing the distance traveled by the pulse wave transit time. Because PWV is influenced by both the intrinsic wall biomechanics (i.e., structural components) and the blood pressure (i.e., hemodynamic load) at the time of measurement, we decomposed human cfPWV into structural and load-dependent components. The structural and load-dependent arterial stiffness were calculated to determine the contribution of these mechanisms to any observed changes in cfPWV (50–52). Structural arterial stiffness was calculated as cfPWV adjusted to a reference mean arterial pressure (MAP) to minimize pressure dependence, while load-dependent stiffness represents the component of cfPWV driven by the individual’s blood pressure at the time of measurement. Briefly, structural stiffness was calculated utilizing the following equation:

S t r u c t u r a l c f P W V = \sqrt{\frac{A}{P r e f} \cdot {c f P W V}^{2} - \frac{A}{ρ} \cdot \ln (\frac{P r e f}{A})}

where $A$ is an arbitrary reference MAP for normotensive individuals (90mmHg), Pref is the measured MAP respective to each measurement, cfPWV is the unadjusted measured value, and $ρ$ is the density of blood (1050 kg/m³). Load-dependent stiffness was determined by taking the difference between the measured cfPWV and the calculated structural stiffness value.

Assessment of Biochemical Parameters

Prior to the oral glucose load, a nurse placed an intravenous catheter to obtain baseline (20mL) and serial blood draws (3mL; every 15min following drink ingestion for 60min) throughout the oral glucose load protocol to assess biochemical parameters. Venous samples were collected in EDTA vacutainer tubes and centrifuged for 15min at 1.4 rcf at 4°C. Plasma was sent to the University of Minnesota Advanced Research and Diagnostic Laboratory for assessment of baseline lipids (HDL, LDL, TC, TG), inflammatory cytokines (IL-6, IL-8, IL-10, MCP-1, TNF, CRP), and liver enzymes (AST, ALT). Whole blood samples were sent to the University of Missouri Diabetes Diagnostic Laboratory for assessment of hemoglobin A1c (HbA1c). Plasma insulin (ELISA, No. 80-INSHU-E10.1, ALPCO, Salem, NH) and glucose (colorimetric assay, No.1009582, Cayman Chemical, Ann Arbor, MI) responses to the oral glucose load were analyzed using commercially available kits per manufacturer’s guidelines. Plasma concentrations of lipid peroxidation (malondialdehyde) were assessed in-house using a commercially available kit (ab118970, Abcam). Pre- and post-assessment of ADAM17 was measured using the Sensolyte 520 TACE fluorimetric activity assay kit (No. AS-72085, AnaSpec, Fremont, CA), as previously described (15,17).

During blood acquisition, 3mL were collected separately into heparinized vacutainer tubes and immediately centrifuged for 6min at 3.0 rcf at 4°C. Plasma was transferred to microtubes that had been pretreated with 100mM NEM and 10mM DTPA to prevent S-nitrosothiol degradation to nitrite. Samples were then snap-frozen and stored at −80°C. Plasma nitrite was used as a surrogate for NO and assessed using the gold-standard method of ozone-based reductive chemiluminescence NO (CLD88, Eco Physics), according to the manufacturer’s guidelines and as previously described (21,25,27,53–55). Plasma samples (100μL) were injected in duplicate into a purge vessel containing glacial acetic acid and 0.5M ascorbic acid, which was then purged with pure nitrogen in line with the CLD88 gas-phase NO analyzer. The chemiluminescence signal was acquired (eDAQ ChartTM v5.5.27 software), and nitrite was quantified using the flow injection analysis (FIA) software extension (ADInstruments, Australia). The area under the curve for each sample peak was calculated and converted to a concentration using a calibrated standard curve of known sodium nitrite standards.

Analysis of Vascular Related Outcomes

Ultrasound recordings were obtained using real-time capture software (Elgato Video Capture, Elgato, CA) and analyzed offline using specialized edge-detection software (Cardiovascular Suite, Quipu srl, Pisa, Italy). For all ultrasound videos, BF was calculated from continuous diameter and mean blood velocity recordings of the respective artery using the following equation: 3.14 × [diameter (cm)/2]² × mean blood velocity (cm/s) × 60, and was reported in mL/min. Brachial artery FMD percent change was calculated as [(peak diameter – baseline diameter)/baseline diameter] × 100. The shear rate was calculated as [4 × (mean velocity/diameter)] to estimate shear stress without blood viscosity. As an index of reactive hyperemia, the postocclusion hyperemic shear rate area under the curve (AUC) was calculated up to 60s. During the PLM and oral glucose load measures, heart rate was assessed using a 3-electrode electrocardiogram configured in Lead II (Bio Amp, ADInstruments), and beat-to-beat blood pressure was acquired using finger photoplethysmography (Human NIBP; ADInstruments). Arterial pressure waveforms were calibrated to average upper arm blood pressure (Welch Allyn Connex Vital Signs Monitor, Baxter International Inc.), as previously described (25,56,57). Leg vascular conductance (LVC) was calculated as [BF (mL/min) ÷ MAP (mmHg)] × 100 and reported as mL/min/mmHg. Hemodynamic outcomes (stroke volume: SV, cardiac output: CO, and total peripheral resistance: TPR) were estimated from the calibrated arterial pressure waveforms using the Modelflow method (LabChart, ADInstruments), which accounts for both sex and age.

Common femoral BF acquired during the PLM tests was calculated as described above. For PLM measures, the baseline BF represents an average of 60s. The flow-related responses to PLM are reported at baseline (FBF, LVC), peak (Δ_peak), and %Δ (BF%Δ, LVC%Δ, AUC). As diameter has been shown to remain constant in the common femoral artery during movement, the baseline average diameter was used to calculate flow across the movement period, respectively (43). Central hemodynamic responses, HR, SV, CO, and MAP, are reported at baseline, and the change (Δ) is calculated as peak – rest. Reported responses reflect the average of duplicate measures.

Superficial femoral artery (SFA) BF is reported as an average of 4-min videos acquired during the oral glucose load challenge. Notably, the baseline values represent an average of two videos acquired within the 15min preceding drink ingestion. Following drinking ingestion, 4-min videos were recorded and analyzed every 15min for 60min. Postprandial glucose and insulin incremental AUC were calculated using the trapezoidal method over 1h. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as: [fasting glucose (mg/dL) × fasting insulin (μU/mL)]/405.

All ultrasound measurements were performed by a single trained operator. In the present study, intra-operator variability was calculated from repeated measurements and expressed as coefficients of variation (CVs). The intra-operator CV for PWV measurements in mice was <8%. In the clinical trial, the intra-operator CVs were <10% for FMD, <2% for arterial diameter measurements (FMD and SFA), <10% for SFA BF calculations, and <3% for cfPWV measurements. These values support the reproducibility of the non-invasive arterial assessments performed in this study.

Statistical Analysis

GraphPad Prism (v.10.4.1) was used for all statistical analyses. For all outcome variables, normality was assessed using the Shapiro-Wilk test. For the preclinical trial and the in vitro and ex vivo experiments, the Robust regression and outlier removal method (ROUT; with Q=5%) was used to identify outliers. Treatment-related differences were assessed using paired or unpaired t tests, Mann-Whitney U tests, or Wilcoxon matched-pairs signed-rank tests, as appropriate. For single time-point measures, group differences were evaluated using one-way analysis of variance (ANOVA) or the Kruskal-Wallis tests, followed by Dunnett’s or Dunn’s post hoc test, respectively. Repeated-measures curves (e.g., vasoreactivity responses and biomechanical characterization) were analyzed using two-way ANOVA with Bonferroni post hoc correction. For the human trial, we performed a priori sample size calculations using an α of 0.05 and 80% power. We first conducted a power analysis using vascular insulin-induced dilation data from the preclinical component of this study. The analysis used means’ responses of 79.40% in PS-treated db/db mice and 43.44% in vehicle-treated db/db mice, with a pooled SD of 18.92%, resulting in an estimated sample size of 16 subjects per group. We then performed a complementary power analysis based on previously published human data from our group for SFA BF during an oral glucose load (47) to detect a Treatment × Time interaction in a repeated-measures design with two groups studied at baseline and post-intervention. This analysis assumed an effect size of f=0.24 and a correlation among repeated measures of r=0.63. To account for possible lack of measurements in some outcomes due to technical issues, we increased our experimental units by 15%. Under these assumptions, we determined that 16 subjects per group would also be sufficient to detect differences in leg BF responses to an oral glucose load. For the main outcomes (with repeated measures), two-way repeated-measures ANOVA was used to assess treatment (PS vs. placebo) × time interactions. When appropriate, planned paired t tests were used to compare final vs. baseline measurements within each group. Missing values during the oral glucose load were accounted for by using nonlinear regression with a best-fit curve for each individual response. No sex differences were observed for the main outcome variables, so the data from subjects were pooled for analysis. Individual responses are included in the figures where appropriate, and data are presented as means±SEM. A P≤0.05 was considered significant for all statistical tests.

RESULTS

In Vitro and Ex Vivo Experiments:

PS inhibits ADAM17 activity and increases insulin-induced dilation

We found that sPS selectively binds to ADAM17-r in a concentration-dependent manner, as demonstrated by SPR (Figure 3, A and B). In contrast, sPC, used as a negative control, showed minimal binding to ADAM17-r across all concentrations used. These results validated the selectivity of the PS-ADAM17 interaction and supported the use of standard buffer controls in all subsequent experiments. Building on this observation, we next tested whether PS inhibits ADAM17 activity in a cell-free assay, cultured cells, and isolated vessels. We found ADAM17 activity was increased with ADAM17-r in a cell-free assay, which was abrogated with sPS or TAPI-0, an ADAM17 inhibitor (Figure 3C). Treatment of naive endothelial cells with sPS or TAPI-0 reduced ADAM17 activity (Figure 3D). Overexpression of ADAM17 in endothelial cells increased ADAM17 activity compared with control (control virus, Ad-CMV-null), an effect that was also blunted with the treatment of sPS or TAPI-0 (Figure 3E). Moreover, isolated mesenteric arteries from C57BL/6 mice exposed to sPS or TAPI-0 exhibited a reduction in ADAM17 activity (Figure 3F). Additionally, overnight incubation with sPS increased insulin-induced dilation, but not SNP-induced dilation, in isolated mesenteric arteries from C57BL/6 mice (Figure 3G).

Preclinical Trial in Diabetic Mice:

Oral administration of PS improves endothelial function and insulin-induced dilation in mesenteric and cerebral arteries

Lipidomic analysis revealed that the PS supplement provided to mice for 4wk was 99.20% PS (Figure 1A). In a small cohort of db/db mice fed deuterium-labeled PS for 5 days, proteomic analysis identified PS in their plasma and aortas (data not shown). Thus, the provided PS is highly pure and is entering the bloodstream and incorporating with the arterial wall. In our preclinical study in db/db mice, 4wk of PS supplementation did not alter body weight or epididymal fat weight, nor fasting blood glucose (Figure 4, A–C). Mesenteric arteries from db/db mice treated with PS demonstrated improved insulin and flow-induced dilation (Figure 4D), with no differences in SNP-induced dilation. Importantly, mesenteric arteries from PS-treated db/db mice exhibited more IRα content on the luminal surface (Figure 4D), approaching levels observed in wildtype mice. Insulin-induced dilation was also greater in the cerebral arteries of PS-treated mice (Figure 4E). PS in db/db mice also abrogated the impaired endothelium-dependent relaxation (i.e., response to ACh) in aortic rings, while endothelium-independent relaxation (i.e., response to SNP) remained unchanged (Figure 4F).

FIGURE 4: — **A-B)** Body and epididydimal weight across groups (wildtype, WT: n=10, *db/db*: n=10, *db/db* + phosphatidylserine (PS), n=9). Statistical analyses were performed using one-way ANOVA or the Kruskal-Wallis test, with Dunnett’s or Dunn’s post hoc tests, respectively. *P≤0.05. C) *db/db* (n=10) and *db/db* + PS (n=9) had higher fasting blood glucose values compared with WT (n=10). Statistical analysis was performed using Kruskal-Wallis test with Dunn’s test to control for multiple comparisons. *P≤0.05 vs. WT. D) Insulin and flow-induced dilation, but not SNP-induced dilation, are improved in mesenteric arteries from PS-treated *db/db* mice (n=8) compared with *db/db* mice (n=6–9). Statistical analysis was performed using two-way ANOVA with Bonferroni test to control for multiple comparisons. ^#P≤0.05 vs. *db/db*. *P≤0.05 vs. WT. The endothelial insulin receptor α (IRα) content is reduced in *db/db* mice (n=7) compared with WT (n=6), but is rescued with PS supplementation (n=6). In the representative confocal images of isolated mesenteric arteries, green=IRα and red=internal elastic lamina. Analysis was performed using one-way ANOVA with Dunnett’s post hoc test to control for multiple comparisons. ^#P≤0.05 vs. *db/db*. *P≤0.05 vs. WT. E) Insulin-induced dilation, but not SNP-induced dilation, is improved in posterior cerebral arteries following PS supplementation in *db/db* mice (n=5) compared with *db/db* (n=11). Statistical analyses were performed using two-way ANOVA with Bonferroni correction to control for multiple comparisons. ^#P≤0.05 vs. *db/db*. *P≤0.05 vs. WT. F) Acetylcholine-induced aortic relaxation but not that induced by SNP is improved following PS supplementation (n=8) compared with *db/db* mice (n=13). Statistical analyses were performed using two-way ANOVA with Bonferroni correction to control for multiple comparisons. ^#P≤0.05 vs. *db/db*. *P≤0.05 vs. WT. All data are reported as mean±SEM.

Oral administration of PS reduces blood pressure and arterial stiffness

The db/db mice treated with PS for 4wk exhibited lower blood pressure (Figure 5A). In addition, in vivo aortic PWV, ex vivo aortic stiffness by AFM, and aortic E_inc were lower in PS-treated mice (Figure 5, B–D), indicative of reduced aortic stiffness. Similarly, E_inc and cPWV_inc were lower in isolated femoral and mesenteric arteries from the PS-fed mice (Figure 5, E and F). These findings were accompanied by a decrease in α-SMA in the aorta that approached statistical significance (P=0.093, Figure 5G) and a reduction in F-actin (Figure 5H) in mesenteric arteries from PS-fed mice. Lastly, nitrotyrosine (a marker of oxidative stress) was reduced in aortic rings from PS-fed mice (Figure 5I).

PS inhibits ADAM17 activity and mitigates ADAM17-TNF-induced ROS

Expanding on the finding that PS supplementation reduced aortic nitrotyrosine staining in db/db mice, we tested whether sPS could mitigate ADAM17-induced shedding of TNF and subsequent oxidative stress in cultured endothelial cells. Indeed, sPS treatment for 1h attenuated shedding of TNF and ROS production in ADAM17-OE endothelial cells (Figure 6, A and B). Further corroborating these findings, ADAM17-OE endothelial cells demonstrated increased O₂⁻ production (DHE), and this increase was abrogated by sPS treatment or a TNF-neutralizing antibody (Figure 6C). Treatment of naive endothelial cells with human recombinant TNF increased ROS production, and this effect was prevented by sPS treatment or Tempol, used as a positive control (Figure 6D).

FIGURE 6: — A) Treating human umbilical venous endothelial cells with soluble phosphatidylserine (sPS, O-phospho-L-serine, 100 μM) for 1h reduces ADAM17-mediated tumor necrosis factor (TNF) shedding. B) Endothelial cells overexpressing ADAM17 (ADAM17-OE) display higher oxidative stress production, which is mitigated with 1-h treatment with sPS (100μM). C) Superoxide (O₂⁻) production in endothelial cells, as measured by DHE fold difference, is increased in ADAM17-OE, a finding that was mitigated with a 1-h treatment with sPS (100μM) or a neutralizing TNF antibody (1μg/mL). D) Treating endothelial cells with TNF augments O₂⁻ production as measured by DHE, a finding that is reduced with sPS (100μM) or Tempol (1mM). Statistical analyses were performed using one-way ANOVA or the Kruskal-Wallis test, with Dunnett’s or Dunn’s post hoc tests, respectively, to control for multiple comparisons. *P≤0.05 vs. corresponding control. ^#P≤0.05 vs. ADAM17-OE or TNF. All data are reported as mean±SEM.

Clinical Trial in Individuals with T2D:

Oral administration of PS improves leg BF responses to an oral glucose load, and reduces cfPWV

Lipidomic analysis demonstrated the PS supplement provided for 4wk was predominantly PS (31.1%), with a mix of other phospholipid species (phosphatidic acid: 19.5%, lysophosphatidylethanolamine: 10.6%, others combined: 38.8% (Figure 1B). Individuals in the placebo group averaged 4.04wk of medication consumption, whereas the PS group averaged 4.05wk of medication consumption, indicating exceptional compliance. Subject characteristics, baseline values, and response to supplementation of biochemical parameters are summarized in Table 1. It should be noted that the individuals randomized to the PS group were slightly older than the placebo group (Table 1). Blood glucose, plasma insulin, and hemodynamic responses to an oral glucose load are illustrated in Figure 7A. There was no effect of supplementation on blood glucose and plasma insulin responses to an oral glucose load in either the placebo or PS group (Figure 7A). There were also no changes observed in SFA BF%Δ or SFA Conductance %Δ in the placebo group. However, there was a significant increase in SFA BF%Δ (Treatment × Time interaction, P=0.06; main effect of Treatment, P≤0.05) and SFA Conductance %Δ (Treatment × Time interaction, P=0.10; main effect of Treatment, P≤0.05), and their respective AUC (P≤0.05 for both SFA BF and SFA Conductance), in response to PS supplementation (Figure 7A). In addition, there was a decrease in cfPWV following PS supplementation, approaching statistical significance (Treatment × Time interaction, P=0.31; main effect of Time, P=0.03; P=0.067 for the PS group (baseline vs final); P>0.05 for placebo) (Figure 7B). Further analysis demonstrated that load-dependent stiffness was reduced in the PS group, while structural-dependent stiffness was unaltered (Figure 7B). No differences were observed in structural or load-dependent stiffness in the placebo group (Figure 7B). There were no changes noted in either group in other cardiovascular outcomes, including brachial artery FMD, AMBP, or leg BF responses to PLM (Table 2). This suggests that the reduction in cfPWV with PS may have been driven by acute hemodynamic factors (e.g., lower blood pressure at the time of testing), rather than by permanent structural changes in the arterial wall over the 4-wk period. However, no significant differences in baseline blood pressure were observed. No differences were observed in plasma lipids, inflammatory cytokines, ADAM17 activity, or plasma nitrite in either group (Table 1).

FIGURE 7: — A) Glucose and insulin responses to an oral glucose load did not differ from baseline (BL) to final for placebo supplementation (n=15). Issues with IV access resulted in the loss of n=2, and n=1 had a baseline glucose was above our threshold of 200mg/dL and did not receive the oral glucose load. Participants without an IV were given the oral glucose load and followed with finger sticks to ensure safety. Also, the percent change in superficial femoral artery (SFA) blood flow (BF) and conductance in response to the oral glucose load did not differ from BL to final for placebo supplementation (top of panel A). Corresponding area under the curves (AUCs) were also not different (n=17). Similarly, glucose and insulin responses to an oral glucose load did not differ from BL to final for phosphatidylserine (PS) supplementation (n=13). Issues with IV access resulted in the loss of n=3 for glucose and insulin outcomes. These individuals were followed with finger stick to ensure patient safety. However, percent change in SFA BF and conductance (and corresponding AUCs) in response to the oral glucose load were increased following 4wk of PS supplementation (n=16) (bottom portion of panel A). Statistical analyses were performed using two-way ANOVA, the asterisk indicates a statistically significant main effect of PS. Paired t tests were used to determine differences in AUC. *P≤0.05 vs. BL. B) Carotid-femoral pulse wave velocity (cfPWV) was reduced in the PS group, approaching statistical significance (P=0.067; n=15), but remained unchanged in the placebo group (n=18). A significant reduction was observed in load-dependent cfPWV in the PS group, but not the placebo group. No differences were observed in structural-dependent cfPWV for either group. One participant was lost in the PS group due to insufficient cfPWV signal quality. Statistical analyses were performed using paired t test or Wilcoxon signed-rank test, as appropriate. *P≤0.05 vs. BL. All data are reported as mean±SEM.

DISCUSSION

The primary findings of this investigation are severalfold. First, we demonstrate that PS can bind to ADAM17 and inhibit its activity using in vitro models and ex vivo isolated resistance arteries, thereby enhancing insulin-induced vasodilation. Second, we show that 4wk of oral PS supplementation in diabetic mice improves insulin-induced dilation in isolated resistance arteries, an effect that is accompanied by preservation of endothelial IRα on the luminal surface. These effects of PS were accompanied by improvements in mesenteric FMD and reductions in blood pressure and indices of arterial stiffness, including aortic PWV. Next, in individuals with T2D, 4wk of PS supplementation resulted in increased BF responses to an oral glucose load, suggestive of enhanced vascular insulin sensitivity, and reduced load-dependent aortic stiffness. Lastly, follow-up experiments in cultured endothelial cells revealed that PS not only inhibits ADAM17 but also mitigates oxidative stress stemming from ADAM17-induced TNF signaling.

The rationale for our hypothesis that PS would restore vascular insulin sensitivity and improve vascular function was based on the work of Sommer et al., who demonstrated that the soluble form of PS competitively inhibits ADAM17 sheddase activity (18). To our knowledge, their study was the first, and only to date, to display that mechanism, and as such, the effects of exogenous PS on ADAM17 activity in the endothelium had not been previously investigated. Here, we extended upon these observations by demonstrating that sPS binds to ADAM17 and blunts its activity in a cell-free assay, in endothelial cells, and in isolated resistance arteries. Importantly, incubation of resistance arteries with sPS resulted in enhanced insulin-induced dilation. These findings, combined with the availability of PS as an oral supplement, urged us to consider oral PS as a therapeutic approach to improve vascular function in T2D, which we tested in mice and humans.

Trials examining the in vivo effect of PS began in the 1990s and have a long safety record in humans (30–34). To date, trials have primarily focused on PS as an ergogenic aid or as a tool to improve cognition and memory. Although two trials aimed at improving cognition reported modest reductions in blood pressure (58,59), ours is, to our knowledge, the first study to investigate the vascular effects of PS in humans. Our findings fill that gap by showing vascular benefits in people with T2D. The precise half-life of orally administered PS is not well established, and its pharmacokinetic profile in individuals with T2D remains unknown. Previously, in a cohort of fasted, healthy volunteers, kinetic analysis revealed that serum levels of PS peaked at 90min after oral ingestion of 500mg PS (with 400mg phosphatidic acid) and returned to near baseline levels by 180min (60). However, PS kinetics may differ in T2D because of altered lipid metabolism, adiposity, medication use, and insulin resistance. Moreover, because PS is a phospholipid, circulating half-life may not fully capture tissue distribution, membrane-associated actions, or downstream effects on ADAM17 activity. Thus, once-a-day dosing may produce intermittent PS exposure, and any benefits may be due to consistent, day-after-day consumption similar to supplementations such as the omega-3 fatty acids docosahexaenoic and eicosapentaenoic acids (61–63). Future studies should determine whether divided dosing improves PS availability, target engagement, and vascular efficacy in T2D. Although we did not assess the acute fate of PS ingestion in humans, we conducted a small sub-study in a separate cohort of db/db mice in which we provided either peanut butter (i.e., vehicle) or peanut butter with deuterium-labeled PS for 5 days and collected tissue ~24h following administration. Proteomics revealed the presence of the deuterium-labeled serine head in both plasma and the aortic walls, a finding not observed in the group fed unmodified peanut butter. Therefore, our observed vascular benefits are likely, at least in part, due to the direct actions of PS on the vasculature, including inhibition of ADAM17 sheddase activity. This is notable because, as mentioned above, prior work from our group indicates that ADAM17 sheds IRα from the surface of the endothelium, causing endothelial insulin resistance (15,17).

Resistance arteries isolated from db/db mice treated with PS for 4wk displayed greater insulin-induced dilation, relative to arteries from vehicle control db/db mice. Of note, these vascular insulin-sensitizing effects of PS coincided with greater preservation of IRα content on the endothelial surface. These effects of PS occurred despite no changes in blood glucose or body weight, reinforcing the possibility that ingested PS may exert direct vascular effects. Similarly, individuals with T2D who were randomized to PS supplementation demonstrated increased leg BF and vascular conductance in response to an oral glucose load. This suggests an improvement in vascular responsiveness to insulin, particularly when considering that endogenous insulin production following the oral glucose load was not affected by PS. In agreement with the preclinical data, there were no changes in glycemic control or body weight. However, unlike in the preclinical study, the improvement in vascular insulin sensitivity in humans occurred without changes in other indices of endothelial function. That is, while diabetic mice receiving PS showed improved ACh-induced relaxation in the aorta and improved FMD in mesenteric arteries, participants with T2D treated with PS did not exhibit changes in brachial artery FMD or leg BF responses to PLM.

Vascular insulin resistance is considered an early driver in the development and progression of vascular dysfunction, and it is also a defect that responds readily to therapeutic intervention. For example, in small and large animal models of diet-induced obesity, vascular insulin resistance develops early on in the disease process and even precedes the manifestation of other indices of endothelial dysfunction (4,8,9,64). Similarly, we previously showed that a 10-day obesogenic intervention in young adults was sufficient to reduce vascular insulin sensitivity without altering FMD, particularly in males (55). Conversely, we found that seven days of passive heat therapy improved leg BF responses to an oral glucose load in T2D without appreciable changes in FMD (47). It is possible that a longer PS intervention would have yielded more robust endothelial benefits in humans, mirroring the effects observed in the preclinical trial.

An important finding from both the preclinical and human trials was the reduction in indices of arterial stiffness following PS treatment. In diabetic mice, PS supplementation decreased arterial stiffness, as assessed in vivo via PWV and ex vivo via mechanical testing (i.e., using AFM, tensile stretch, and pressure myography). These changes were accompanied by decreased α-SMA expression in the aorta that approached statistical significance, along with a significant reduction in F-actin content. This suggests that PS may influence cytoskeletal remodeling, vascular smooth muscle contractile marker expression, or intrinsic vascular wall material properties in diabetic mice. Individuals with T2D who received oral PS exhibited a reduction in cfPWV, particularly when accounting for their blood pressure measurements. This finding is referred to as load-dependent arterial stiffness, which is primarily influenced by mechanical forces (i.e., intravascular pressure) engaging collagen fibers within the vascular wall. In contrast, structural-dependent stiffness, which estimates pressure-independent stiffness of the arterial wall, was unaffected by PS in the clinical trial. Therefore, although the preclinical data suggest potential effects of PS on intrinsic vascular wall stiffness and cytoskeletal remodeling, the human data did not provide evidence of detectable pressure-independent structural remodeling over the intervention period. As noted above, it is possible that a longer intervention would be required to produce more robust structural destiffening effects in humans. However, there is evidence in the literature that therapeutic interventions may have limited efficacy once substantial structural remodeling and severe arterial stiffness have developed, particularly in T2D (21,48,65).

Another intriguing finding was that aortas from PS-treated diabetic mice displayed reduced nitrotyrosine staining, a well-established marker of oxidative stress. We followed up on this observation with additional mechanistic experiments in cultured endothelial cells. In ADAM17-OE endothelial cells, sPS reduced TNF shedding and overall ROS production. Soluble PS also attenuated ADAM17-driven generation of radicals, and this dampening of oxidative stress was also achieved when cells were treated with a TNF-neutralizing antibody. This indicates that increased endothelial ADAM17 activity-induced ROS production is likely mediated by TNF signaling. Consistent with this, TNF exposure increased superoxide production, which was also mitigated by sPS, or Tempol (used as a positive control). In aggregate, these findings reinforce the antioxidant vascular effects of PS and suggest that, beyond inhibiting ADAM17 and enhancing vascular insulin sensitivity, PS buffers oxidative stress stemming from ADAM17-induced TNF signaling, thereby limiting the endothelial injury that accompanies vascular dysfunction.

Several aspects of this study warrant further consideration. First, the primary preclinical study (4-wk PS treatment) included only male mice. Yet, both sexes were used in the mechanistic ex vivo studies and prioritized in the clinical trial of middle-aged/older adults with T2D, underscoring efforts to ensure the generalizability of our findings and translational relevance. Nevertheless, differences in age, sex, and vascular phenotype may influence the extent to which the preclinical findings translate to humans. Therefore, our preclinical findings should be interpreted primarily as mechanistic support for the clinical observations rather than as direct quantitative predictors of human treatment response. Second, our sample demographics were largely representative of the population in central Missouri; therefore, future studies should aim to include a higher proportion of participants from diverse racial and ethnic backgrounds. Third, our clinical trial had a relatively short duration of 4wk to mirror the preclinical trial. Future trials should strive to increase the sample size and study duration to expand upon our encouraging findings. Fourth, the PS formulation differed between the preclinical and clinical trials. Mice received a relatively high dose of pure PS, whereas the human supplement contained predominantly PS along with other phospholipids (900mg/day of PS supplement delivering ~280 mg/day PS and ~176 mg/day phosphatidic acid), providing ~6-fold less PS than the equivalent dose used in mice. Fifth, while robust evidence is provided that sPS effectively lowers ADAM17 activity in vitro and ex vivo, to our knowledge, there are no available tools to capture tissular ADAM17 activity in response to in vivo PS treatment. As such, for the preclinical and clinical PS trials, the readouts were largely limited to functional outcomes. Lastly, many participants in this cohort were taking medications known to influence cardiovascular function, including SGLT2 inhibitors, GLP-1 receptor agonists, statins, and antihypertensives. Importantly, to minimize confounding, participants were required to maintain a stable medication regimen prior to trial initiation.

In conclusion, this work provides evidence supporting the potential of oral PS as a therapeutic strategy to improve vascular function in T2D, and suggests that the beneficial effects of PS may be driven by its vascular insulin-sensitizing and antioxidant actions (Figure 8). This evidence is based on the integration of cell-free experiments, studies in cultured endothelial cells and isolated arteries, a preclinical trial in diabetic mice, and a placebo-controlled clinical trial in individuals with T2D.

FIGURE 8: — Exogenous administration of phosphatidylserine (PS) provides beneficial vascular effects in type 2 diabetes, potentially through vascular insulin-sensitizing effects (left) and antioxidant actions (right).

ACKNOWLEDGMENTS

The authors appreciate the clinical study participants for their time and effort. We acknowledge the nurses and staff at the Clinical and Translational Research Unit at the University of Missouri. Furthermore, we acknowledge Olivia Burken, Avery Zerrer, Maya Burken, and Mariana Morales for their technical assistance.

GRANTS

This work was supported, in part, by National Institutes of Health Grant R01HL151384 (to L.A.M.-L. and J.P.) and grants from the American Heart Association (23PRE1020897 to G.P., 26PRE1559973 to O.M.L., 25DIVSUP1463861 to F.I.R.-P., and 24EIA1248820 to J.P.) and the São Paulo Research Foundation (FAPESP, 2024/09267–4 to L.F.-S.). N.J.M. and M.A.A. were supported by the Life Sciences Fellowship at the University of Missouri-Columbia.

Footnotes

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors. Jaume Padilla is an associate editor of the Journal of Applied Physiology and was not involved and did not have access to information regarding the peer-review process or final disposition of this article. An alternate editor oversaw the peer-review and decision-making process for this article.

REFERENCES

1.Padilla J, Manrique-Acevedo C, Martinez-Lemus LA. New insights into mechanisms of endothelial insulin resistance in type 2 diabetes. Am J Physiol-Heart Circ Physiol. 2022. Dec 1;323(6):H1231–8. doi: 10.1152/ajpheart.00537.2022 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Bansilal S, Farkouh ME, Fuster V. Role of Insulin Resistance and Hyperglycemia in the Development of Atherosclerosis. Am J Cardiol. 2007. Feb;99(4):6–14. doi: 10.1016/j.amjcard.2006.11.002 [DOI] [PubMed] [Google Scholar]
3.Vincent MA, Clerk LH, Lindner JR, Klibanov AL, Clark MG, Rattigan S, et al. Microvascular Recruitment Is an Early Insulin Effect That Regulates Skeletal Muscle Glucose Uptake In Vivo. Diabetes. 2004. Jun 1;53(6):1418–23. doi: 10.2337/diabetes.53.6.1418 [DOI] [PubMed] [Google Scholar]
4.Kim F, Pham M, Maloney E, Rizzo NO, Morton GJ, Wisse BE, et al. Vascular Inflammation, Insulin Resistance, and Reduced Nitric Oxide Production Precede the Onset of Peripheral Insulin Resistance. Arterioscler Thromb Vasc Biol. 2008. Nov;28(11):1982–8. doi: 10.1161/ATVBAHA.108.169722 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Laakso M, Edelman SV, Brechtel G, Baron AD. Impaired Insulin-Mediated Skeletal Muscle Blood Flow in Patients With NIDDM. Vol. 41. 1992;41. [DOI] [PubMed] [Google Scholar]
6.Baron AD, Laakso M, Brechtel G, Edelman SV. Mechanism of insulin resistance in insulin-dependent diabetes mellitus: a major role for reduced skeletal muscle blood flow. J Clin Endocrinol Metab. 1991. Sep;73(3):637–43. doi: 10.1210/jcem-73-3-637 [DOI] [PubMed] [Google Scholar]
7.Baron AD, Tarshoby M, Hook G, Lazaridis EN, Cronin J, Johnson A, et al. Interaction between insulin sensitivity and muscle perfusion on glucose uptake in human skeletal muscle: evidence for capillary recruitment. Diabetes. 2000. May;49(5):768–74. doi: 10.2337/diabetes.49.5.768 [DOI] [PubMed] [Google Scholar]
8.Olver TD, Grunewald ZI, Jurrissen TJ, MacPherson REK, LeBlanc PJ, Schnurbusch TR, et al. Microvascular insulin resistance in skeletal muscle and brain occurs early in the development of juvenile obesity in pigs. Am J Physiol-Regul Integr Comp Physiol. 2018. Feb 1;314(2):R252–64. doi: 10.1152/ajpregu.00213.2017 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Eringa EC, Stehouwer CDA, Roos MH, Westerhof N, Sipkema P. Selective resistance to vasoactive effects of insulin in muscle resistance arteries of obese Zucker ( fa / fa ) rats. Am J Physiol-Endocrinol Metab. 2007. Nov;293(5):E1134–9. doi: 10.1152/ajpendo.00516.2006 [DOI] [PubMed] [Google Scholar]
10.Loehr LR, Meyer ML, Poon AK, Selvin E, Palta P, Tanaka H, et al. Prediabetes and Diabetes Are Associated With Arterial Stiffness in Older Adults: The ARIC Study. Am J Hypertens. 2016. Sep;29(9):1038–45. doi: 10.1093/ajh/hpw036 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Zheng M, Zhang X, Chen S, Song Y, Zhao Q, Gao X, et al. Arterial Stiffness Preceding Diabetes: A Longitudinal Study. Circ Res. 2020. Dec 4;127(12):1491–8. doi: 10.1161/CIRCRESAHA.120.317950 [DOI] [PubMed] [Google Scholar]
12.Weisbrod RM, Shiang T, Al Sayah L, Fry JL, Bajpai S, Reinhart-King CA, et al. Arterial stiffening precedes systolic hypertension in diet-induced obesity. Hypertens Dallas Tex 1979. 2013. Dec;62(6):1105–10. doi: 10.1161/HYPERTENSIONAHA.113.01744 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Barrett EJ, Eggleston EM, Inyard AC, Wang H, Li G, Chai W, et al. The vascular actions of insulin control its delivery to muscle and regulate the rate-limiting step in skeletal muscle insulin action. Diabetologia. 2009. May 1;52(5):752–64. doi: 10.1007/s00125-009-1313-z [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Tang BY, Ge J, Wu Y, Wen J, Tang XH. The Role of ADAM17 in Inflammation-Related Atherosclerosis . J Cardiovasc Transl Res. 2022. Dec;15(6):1283–96. doi: 10.1007/s12265-022-10275-4 [DOI] [PubMed] [Google Scholar]
15.Ghiarone T, Castorena-Gonzalez JA, Foote CA, Ramirez-Perez FI, Ferreira-Santos L, Cabral-Amador FJ, et al. ADAM17 cleaves the insulin receptor ectodomain on endothelial cells and causes vascular insulin resistance. Am J Physiol-Heart Circ Physiol. 2022. Oct 1;323(4):H688–701. doi: 10.1152/ajpheart.00039.2022 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Kawai T, Elliott KJ, Scalia R, Eguchi S. Contribution of ADAM17 and related ADAMs in cardiovascular diseases. Cell Mol Life Sci. 2021. May;78(9):4161–87. doi: 10.1007/s00018-021-03779-w [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Ferreira-Santos L, Ramirez-Perez FI, Foote CA, Augenreich MA, McMillan NJ, Williams MB, et al. Neuraminidase-induced externalization of phosphatidylserine activates ADAM17 and impairs insulin signaling in endothelial cells. Am J Physiol-Heart Circ Physiol. 2024. Jan 1;326(1):H270–7. doi: 10.1152/ajpheart.00638.2023 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Sommer A, Kordowski F, Büch J, Maretzky T, Evers A, Andrä J, et al. Phosphatidylserine exposure is required for ADAM17 sheddase function. Nat Commun. 2016. May 10;7(1):11523. doi: 10.1038/ncomms11523 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Power G, Cho M, Beltran-Ornelas J, Augenreich M, Imkaew N, Lateef O, et al. Endothelial CD44 cleavage by ADAM17 impairs shear stress mechanotransduction. Function. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Pennington KA, Ramirez-Perez FI, Pollock KE, Talton OO, Foote CA, Reyes-Aldasoro CC, et al. Maternal Hyperleptinemia Is Associated with Male Offspring’s Altered Vascular Function and Structure in Mice. Su Y, editor. PLOS ONE. 2016. May 17;11(5):e0155377. doi: 10.1371/journal.pone.0155377 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Manrique-Acevedo C, Soares RN, Smith JA, Park LK, Burr K, Ramirez-Perez FI, et al. Impact of sex and diet-induced weight loss on vascular insulin sensitivity in type 2 diabetes. Am J Physiol Regul Integr Comp Physiol. 2023. Mar 1;324(3):R293–304. doi: 10.1152/ajpregu.00249.2022 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Power G, Lateef OM, Ramirez-Perez FI, Lazo-Fernandez Y, Augenreich MA, Ferreira-Santos L, et al. Reduced cofilin activity as a mechanism contributing to endothelial cell stiffening in type 2 diabetes. Am J Physiol-Heart Circ Physiol. 2025. Jan 1;328(1):H84–92. doi: 10.1152/ajpheart.00667.2024 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Sharma N, Sun Z, Hill MA, Hans CP. Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model. 2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Ramirez-Perez FI, Woodford ML, Morales-Quinones M, Grunewald ZI, Cabral-Amador FJ, Yoshida T, et al. Mutation of the 5’-untranslated region stem-loop mRNA structure reduces type I collagen deposition and arterial stiffness in male obese mice. Am J Physiol Heart Circ Physiol. 2021. Aug 1;321(2):H435–45. doi: 10.1152/ajpheart.00076.2021 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Smith JA, Ramirez-Perez FI, Burr K, Gonzalez-Vallejo JD, Morales-Quinones M, McMillan NJ, et al. Impact of dietary supplementation of glycocalyx precursors on vascular function in type 2 diabetes. J Appl Physiol. 2024. Dec;137(6):1592–603. doi: 10.1152/japplphysiol.00651.2024 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Ramirez-Perez FI, Cabral-Amador FJ, Whaley-Connell AT, Aroor AR, Morales-Quinones M, Woodford ML, et al. Cystamine reduces vascular stiffness in Western diet-fed female mice. Am J Physiol-Heart Circ Physiol. 2022. Feb 1;322(2):H167–80. doi: 10.1152/ajpheart.00431.2021 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Foote CA, Ramirez-Perez FI, Smith JA, Ghiarone T, Morales-Quinones M, McMillan NJ, et al. Neuraminidase inhibition improves endothelial function in diabetic mice. Am J Physiol Heart Circ Physiol. 2023. Dec 1;325(6):H1337–53. doi: 10.1152/ajpheart.00337.2023 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Steppan J, Bergman Y, Viegas K, Armstrong D, Tan S, Wang H, et al. Tissue Transglutaminase Modulates Vascular Stiffness and Function Through Crosslinking‐Dependent and Crosslinking-Independent Functions. J Am Heart Assoc. 2017. Feb 2;6(2):e004161. doi: 10.1161/JAHA.116.004161 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Targosz-Korecka M, Jaglarz M, Malek-Zietek KE, Gregorius A, Zakrzewska A, Sitek B, et al. AFM-based detection of glycocalyx degradation and endothelial stiffening in the db/db mouse model of diabetes. Sci Rep. 2017. Nov 21;7(1):15951. doi: 10.1038/s41598-017-16179-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Cenacchi T, Bertoldin T, Farina C, Fiori MG, Crepaldi G, Azzini CF, et al. Cognitive decline in the elderly: A double- blind, placebo- controlled multicenter study on efficacy of phosphatidylserine administration. Aging Clin Exp Res. 1993. Apr;5(2):123–33. doi: 10.1007/BF03324139 [DOI] [PubMed] [Google Scholar]
31.Monteleone P, Maj M, Beinat L, Natale M, Kemali D. Blunting by chronic phosphatidylserine administration of the stress-induced activation of the hypothalamo-pituitary-adrenal axis in healthy men. Eur J Clin Pharmacol. 1992;42(4):385–8. doi: 10.1007/BF00280123. [DOI] [PubMed] [Google Scholar]
32.Jorissen BL, Brouns F, Van Boxtel MPJ, Riedel WJ. Safety of Soy-derived Phosphatidylserine in Elderly People. Nutr Neurosci. 2002. Jan;5(5):337–43. doi: 10.1080/1028415021000033802 [DOI] [PubMed] [Google Scholar]
33.Kingsley M Effects of Phosphatidylserine Supplementation on Exercising Humans: Sports Med. 2006;36(8):657–69. doi: 10.2165/00007256-200636080-00003 [DOI] [PubMed] [Google Scholar]
34.Kingsley MI, Wadsworth D, Kilduff LP, Mceneny J, Benton D. Effects of Phosphatidylserine on Oxidative Stress following Intermittent Running. Med Sci Sports Exerc. 2005. Aug;37(8):1300–6. doi: 10.1249/01.mss.0000175306.05465.7e [DOI] [PubMed] [Google Scholar]
35.Starks MA, Starks SL, Kingsley M, Purpura M, Jäger R. The effects of phosphatidylserine on endocrine response to moderate intensity exercise. J Int Soc Sports Nutr. 2008. Jul 28;5:11. doi: 10.1186/1550-2783-5-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Kingsley MI, Miller M, Kilduff LP, McENENY J, Benton D. Effects of Phosphatidylserine on Exercise Capacity during Cycling in Active Males. Med Sci Sports Exerc. 2006. Jan;38(1):64–71. doi: 10.1249/01.mss.0000183195.10867.d0 [DOI] [PubMed] [Google Scholar]
37.Kingsley MI, Kilduff LP, Mceneny J, Dietzig RE, Benton D. Phosphatidylserine Supplementation and Recovery following Downhill Running. Med Sci Sports Exerc. 2006. Sep;38(9):1617–25. doi: 10.1249/01.mss.0000229459.11452.a0 [DOI] [PubMed] [Google Scholar]
38.Zhu X, Hwu I, Jacabacci S, Wu S. The Utility of 24-h Ambulatory Blood Pressure Monitoring for the Diagnosis and Management of White Coat Hypertension in a Primary Care Setting. J Prim Care Community Health. 2022. Jan;13:21501319221123457. doi: 10.1177/21501319221123457 [DOI] [Google Scholar]
39.Harris RA, Nishiyama SK, Wray DW, Richardson RS. Ultrasound Assessment of Flow-Mediated Dilation. Hypertension. 2010. May 1;55(5):1075–85. doi: 10.1161/HYPERTENSIONAHA.110.150821 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Thijssen DHJ, Black MA, Pyke KE, Padilla J, Atkinson G, Harris RA, et al. Assessment of flow-mediated dilation in humans: a methodological and physiological guideline. Am J Physiol Heart Circ Physiol. 2011. Jan;300(1):H2–12. doi: 10.1152/ajpheart.00471.2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Pyke KE, Tschakovsky ME. The relationship between shear stress and flow-mediated dilatation: implications for the assessment of endothelial function: The shear stress stimulus-flow-mediated dilatation relationship. J Physiol. 2005. Oct;568(2):357–69. doi: 10.1113/jphysiol.2005.089755 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Limberg JK, Casey DP, Trinity JD, Nicholson WT, Wray DW, Tschakovsky ME, et al. Assessment of resistance vessel function in human skeletal muscle: guidelines for experimental design, Doppler ultrasound, and pharmacology. Am J Physiol Heart Circ Physiol. 2020. Feb 1;318(2):H301–25. doi: 10.1152/ajpheart.00649.2019 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Gifford JR, Richardson RS. CORP: Ultrasound assessment of vascular function with the passive leg movement technique. J Appl Physiol. 2017. Dec 1;123(6):1708–20. doi: 10.1152/japplphysiol.00557.2017 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Trinity JD, Groot HJ, Layec G, Rossman MJ, Ives SJ, Runnels S, et al. Nitric oxide and passive limb movement: a new approach to assess vascular function. J Physiol. 2012. Mar;590(6):1413–25. doi: 10.1113/jphysiol.2011.224741 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Broxterman RM, Trinity JD, Gifford JR, Kwon OS, Kithas AC, Hydren JR, et al. Single passive leg movement assessment of vascular function: contribution of nitric oxide. J Appl Physiol. 2017. Dec 1;123(6):1468–76. doi: 10.1152/japplphysiol.00533.2017 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Shields KL, Broxterman RM, Jarrett CL, Bisconti AV, Park SH, Richardson RS. The passive leg movement technique for assessing vascular function: defining the distribution of blood flow and the impact of occluding the lower leg. Exp Physiol. 2019. Oct;104(10):1575–84. doi: 10.1113/EP087845 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Pettit-Mee RJ, Power G, Cabral-Amador FJ, Ramirez-Perez FI, Soares RN, Sharma N, et al. Endothelial HSP72 is not reduced in type 2 diabetes nor is it a key determinant of endothelial insulin sensitivity. Am J Physiol-Regul Integr Comp Physiol. 2022. Jul 1;323(1):R43–58. doi: 10.1152/ajpregu.00006.2022 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Park LK, Parks EJ, Pettit-Mee RJ, Woodford ML, Ghiarone T, Smith JA, et al. Skeletal muscle microvascular insulin resistance in type 2 diabetes is not improved by eight weeks of regular walking. J Appl Physiol Bethesda Md 1985. 2020. Aug 1;129(2):283–96. doi: 10.1152/japplphysiol.00174.2020 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Townsend RR, Wilkinson IB, Schiffrin EL, Avolio AP, Chirinos JA, Cockcroft JR, et al. Recommendations for Improving and Standardizing Vascular Research on Arterial Stiffness: A Scientific Statement From the American Heart Association. Hypertension. 2015. Sep;66(3):698–722. doi: 10.1161/HYP.0000000000000033 [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Davis KG, Armstrong MK, Nuckols VR, Smith MN, Pewowaruk R, Gimblet CJ, et al. Load-dependent mechanisms contribute to increased aortic stiffness among women with a history of preeclampsia: relation with cardiovagal baroreflex sensitivity. Am J Physiol-Heart Circ Physiol. 2024. Dec 1;327(6):H1406–12. doi: 10.1152/ajpheart.00556.2024 [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Armstrong MK, Jain S, Nuckols V, Pewowaruk R, Zhang X, DuBose L, et al. The association of structural versus load-dependent large artery stiffness mechanisms with cerebrovascular damage and cortical atrophy in humans. GeroScience. 2024. Jun 20;46(6):5587–97. doi: 10.1007/s11357-024-01254-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Pewowaruk RJ, Korcarz C, Tedla Y, Burke G, Greenland P, Wu C, et al. Carotid Artery Stiffness Mechanisms Associated With Cardiovascular Disease Events and Incident Hypertension: the Multi-Ethnic Study of Atherosclerosis (MESA). Hypertension. 2022. Mar;79(3):659–66. doi: 10.1161/HYPERTENSIONAHA.121.18772 [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Nagababu E, Rifkind JM. Measurement of Plasma Nitrite by Chemiluminescence. In: Uppu RM, Murthy SN, Pryor WA, Parinandi NL, editors. Free Radicals and Antioxidant Protocols [Internet] Totowa, NJ: Humana Press; 2010. [cited 2024 Nov 26]. p. 41–9. (Methods in Molecular Biology). Available from: http://link.springer.com/10.1007/978-1-60327-029-8_3 doi: 10.1007/978-1-60327-029-8_3 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Nagababu E, Rifkind JM. Measurement of Plasma Nitrite by Chemiluminescence without Interference of S-, N-nitroso and Nitrated Species. Free Radic Biol Med. 2007. Apr 15;42(8):1146–54. doi: 10.1016/j.freeradbiomed.2006.12.029 [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Smith JA, Soares RN, McMillan NJ, Jurrissen TJ, Martinez-Lemus LA, Padilla J, et al. Young Women Are Protected Against Vascular Insulin Resistance Induced by Adoption of an Obesogenic Lifestyle. Endocrinology. 2022. Oct 11;163(11):bqac137. doi: 10.1210/endocr/bqac137 [DOI] [PMC free article] [PubMed] [Google Scholar]
56.McMillan NJ, Soares RN, Harper JL, Shariffi B, Moreno-Cabañas A, Curry TB, et al. Role of the arterial baroreflex in the sympathetic response to hyperinsulinemia in adult humans. Am J Physiol-Endocrinol Metab. 2022. Apr 1;322(4):E355–65. doi: 10.1152/ajpendo.00391.2021 [DOI] [PMC free article] [PubMed] [Google Scholar]
57.McMillan NJ, Jacob DW, Shariffi B, Harper JL, Foster GE, Manrique-Acevedo C, et al. Effect of acute intranasal insulin administration on muscle sympathetic nerve activity in healthy young adults. Am J Physiol Heart Circ Physiol. 2024. Jul 1;327(1):H000. doi: 10.1152/ajpheart.00253.2024 [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Vakhapova V, Richter Y, Cohen T, Herzog Y, Korczyn AD. Safety of phosphatidylserine containing omega-3 fatty acids in non-demented elderly: a double-blind placebo-controlled trial followed by an open-label extension. BMC Neurol. 2011. Dec;11(1):79. doi: 10.1186/1471-2377-11-79 [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Richter Y, Herzog Y, Lifshitz, Yael, Hayun, Rami, Zchut S. The effect of soybean-derived phosphatidylserine on cognitive performance in elderly with subjective memory complaints: a pilot study. Clin Interv Aging. 2013. May;557. doi: 10.2147/CIA.S40348 [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Moré MI, Freitas U, Rutenberg D. Positive Effects of Soy Lecithin-Derived Phosphatidylserine plus Phosphatidic Acid on Memory, Cognition, Daily Functioning, and Mood in Elderly Patients with Alzheimer’s Disease and Dementia. Adv Ther. 2014. Dec;31(12):1247–62. doi: 10.1007/s12325-014-0165-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Skulas-Ray AC, Wilson PWF, Harris WS, Brinton EA, Kris-Etherton PM, Richter CK, et al. Omega-3 Fatty Acids for the Management of Hypertriglyceridemia: A Science Advisory From the American Heart Association. Circulation. 2019. Sep 17;140(12). doi: 10.1161/CIR.0000000000000709 [DOI] [PubMed] [Google Scholar]
62.Del Gobbo LC, Imamura F, Aslibekyan S, Marklund M, Virtanen JK, Wennberg M, et al. ω-3 Polyunsaturated Fatty Acid Biomarkers and Coronary Heart Disease: Pooling Project of 19 Cohort Studies. JAMA Intern Med. 2016. Aug 1;176(8):1155. doi: 10.1001/jamainternmed.2016.2925 [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Morris MC, Sacks F, Rosner B. Does fish oil lower blood pressure? A meta-analysis of controlled trials. Circulation. 1993. Aug;88(2):523–33. doi: 10.1161/01.CIR.88.2.523 [DOI] [PubMed] [Google Scholar]
64.Katakam PVG, Tulbert CD, Snipes JA, Erdös B, Miller AW, Busija DW. Impaired insulin-induced vasodilation in small coronary arteries of Zucker obese rats is mediated by reactive oxygen species. Am J Physiol-Heart Circ Physiol. 2005. Feb;288(2):H854–60. doi: 10.1152/ajpheart.00715.2004 [DOI] [PubMed] [Google Scholar]
65.Headley S, Germain M, Wood R, Joubert J, Milch C, Evans E, et al. Short-term Aerobic Exercise and Vascular Function in CKD Stage 3: A Randomized Controlled Trial. Am J Kidney Dis. 2014. Aug;64(2):222–9. doi: 10.1053/j.ajkd.2014.02.022 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R1] 1.Padilla J, Manrique-Acevedo C, Martinez-Lemus LA. New insights into mechanisms of endothelial insulin resistance in type 2 diabetes. Am J Physiol-Heart Circ Physiol. 2022. Dec 1;323(6):H1231–8. doi: 10.1152/ajpheart.00537.2022 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R2] 2.Bansilal S, Farkouh ME, Fuster V. Role of Insulin Resistance and Hyperglycemia in the Development of Atherosclerosis. Am J Cardiol. 2007. Feb;99(4):6–14. doi: 10.1016/j.amjcard.2006.11.002 [DOI] [PubMed] [Google Scholar]

[R3] 3.Vincent MA, Clerk LH, Lindner JR, Klibanov AL, Clark MG, Rattigan S, et al. Microvascular Recruitment Is an Early Insulin Effect That Regulates Skeletal Muscle Glucose Uptake In Vivo. Diabetes. 2004. Jun 1;53(6):1418–23. doi: 10.2337/diabetes.53.6.1418 [DOI] [PubMed] [Google Scholar]

[R4] 4.Kim F, Pham M, Maloney E, Rizzo NO, Morton GJ, Wisse BE, et al. Vascular Inflammation, Insulin Resistance, and Reduced Nitric Oxide Production Precede the Onset of Peripheral Insulin Resistance. Arterioscler Thromb Vasc Biol. 2008. Nov;28(11):1982–8. doi: 10.1161/ATVBAHA.108.169722 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] 5.Laakso M, Edelman SV, Brechtel G, Baron AD. Impaired Insulin-Mediated Skeletal Muscle Blood Flow in Patients With NIDDM. Vol. 41. 1992;41. [DOI] [PubMed] [Google Scholar]

[R6] 6.Baron AD, Laakso M, Brechtel G, Edelman SV. Mechanism of insulin resistance in insulin-dependent diabetes mellitus: a major role for reduced skeletal muscle blood flow. J Clin Endocrinol Metab. 1991. Sep;73(3):637–43. doi: 10.1210/jcem-73-3-637 [DOI] [PubMed] [Google Scholar]

[R7] 7.Baron AD, Tarshoby M, Hook G, Lazaridis EN, Cronin J, Johnson A, et al. Interaction between insulin sensitivity and muscle perfusion on glucose uptake in human skeletal muscle: evidence for capillary recruitment. Diabetes. 2000. May;49(5):768–74. doi: 10.2337/diabetes.49.5.768 [DOI] [PubMed] [Google Scholar]

[R8] 8.Olver TD, Grunewald ZI, Jurrissen TJ, MacPherson REK, LeBlanc PJ, Schnurbusch TR, et al. Microvascular insulin resistance in skeletal muscle and brain occurs early in the development of juvenile obesity in pigs. Am J Physiol-Regul Integr Comp Physiol. 2018. Feb 1;314(2):R252–64. doi: 10.1152/ajpregu.00213.2017 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R9] 9.Eringa EC, Stehouwer CDA, Roos MH, Westerhof N, Sipkema P. Selective resistance to vasoactive effects of insulin in muscle resistance arteries of obese Zucker ( fa / fa ) rats. Am J Physiol-Endocrinol Metab. 2007. Nov;293(5):E1134–9. doi: 10.1152/ajpendo.00516.2006 [DOI] [PubMed] [Google Scholar]

[R10] 10.Loehr LR, Meyer ML, Poon AK, Selvin E, Palta P, Tanaka H, et al. Prediabetes and Diabetes Are Associated With Arterial Stiffness in Older Adults: The ARIC Study. Am J Hypertens. 2016. Sep;29(9):1038–45. doi: 10.1093/ajh/hpw036 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] 11.Zheng M, Zhang X, Chen S, Song Y, Zhao Q, Gao X, et al. Arterial Stiffness Preceding Diabetes: A Longitudinal Study. Circ Res. 2020. Dec 4;127(12):1491–8. doi: 10.1161/CIRCRESAHA.120.317950 [DOI] [PubMed] [Google Scholar]

[R12] 12.Weisbrod RM, Shiang T, Al Sayah L, Fry JL, Bajpai S, Reinhart-King CA, et al. Arterial stiffening precedes systolic hypertension in diet-induced obesity. Hypertens Dallas Tex 1979. 2013. Dec;62(6):1105–10. doi: 10.1161/HYPERTENSIONAHA.113.01744 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] 13.Barrett EJ, Eggleston EM, Inyard AC, Wang H, Li G, Chai W, et al. The vascular actions of insulin control its delivery to muscle and regulate the rate-limiting step in skeletal muscle insulin action. Diabetologia. 2009. May 1;52(5):752–64. doi: 10.1007/s00125-009-1313-z [DOI] [PMC free article] [PubMed] [Google Scholar]

[R14] 14.Tang BY, Ge J, Wu Y, Wen J, Tang XH. The Role of ADAM17 in Inflammation-Related Atherosclerosis . J Cardiovasc Transl Res. 2022. Dec;15(6):1283–96. doi: 10.1007/s12265-022-10275-4 [DOI] [PubMed] [Google Scholar]

[R15] 15.Ghiarone T, Castorena-Gonzalez JA, Foote CA, Ramirez-Perez FI, Ferreira-Santos L, Cabral-Amador FJ, et al. ADAM17 cleaves the insulin receptor ectodomain on endothelial cells and causes vascular insulin resistance. Am J Physiol-Heart Circ Physiol. 2022. Oct 1;323(4):H688–701. doi: 10.1152/ajpheart.00039.2022 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] 16.Kawai T, Elliott KJ, Scalia R, Eguchi S. Contribution of ADAM17 and related ADAMs in cardiovascular diseases. Cell Mol Life Sci. 2021. May;78(9):4161–87. doi: 10.1007/s00018-021-03779-w [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] 17.Ferreira-Santos L, Ramirez-Perez FI, Foote CA, Augenreich MA, McMillan NJ, Williams MB, et al. Neuraminidase-induced externalization of phosphatidylserine activates ADAM17 and impairs insulin signaling in endothelial cells. Am J Physiol-Heart Circ Physiol. 2024. Jan 1;326(1):H270–7. doi: 10.1152/ajpheart.00638.2023 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] 18.Sommer A, Kordowski F, Büch J, Maretzky T, Evers A, Andrä J, et al. Phosphatidylserine exposure is required for ADAM17 sheddase function. Nat Commun. 2016. May 10;7(1):11523. doi: 10.1038/ncomms11523 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] 19.Power G, Cho M, Beltran-Ornelas J, Augenreich M, Imkaew N, Lateef O, et al. Endothelial CD44 cleavage by ADAM17 impairs shear stress mechanotransduction. Function. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] 20.Pennington KA, Ramirez-Perez FI, Pollock KE, Talton OO, Foote CA, Reyes-Aldasoro CC, et al. Maternal Hyperleptinemia Is Associated with Male Offspring’s Altered Vascular Function and Structure in Mice. Su Y, editor. PLOS ONE. 2016. May 17;11(5):e0155377. doi: 10.1371/journal.pone.0155377 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] 21.Manrique-Acevedo C, Soares RN, Smith JA, Park LK, Burr K, Ramirez-Perez FI, et al. Impact of sex and diet-induced weight loss on vascular insulin sensitivity in type 2 diabetes. Am J Physiol Regul Integr Comp Physiol. 2023. Mar 1;324(3):R293–304. doi: 10.1152/ajpregu.00249.2022 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Power G, Lateef OM, Ramirez-Perez FI, Lazo-Fernandez Y, Augenreich MA, Ferreira-Santos L, et al. Reduced cofilin activity as a mechanism contributing to endothelial cell stiffening in type 2 diabetes. Am J Physiol-Heart Circ Physiol. 2025. Jan 1;328(1):H84–92. doi: 10.1152/ajpheart.00667.2024 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Sharma N, Sun Z, Hill MA, Hans CP. Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model. 2021. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] 24.Ramirez-Perez FI, Woodford ML, Morales-Quinones M, Grunewald ZI, Cabral-Amador FJ, Yoshida T, et al. Mutation of the 5’-untranslated region stem-loop mRNA structure reduces type I collagen deposition and arterial stiffness in male obese mice. Am J Physiol Heart Circ Physiol. 2021. Aug 1;321(2):H435–45. doi: 10.1152/ajpheart.00076.2021 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] 25.Smith JA, Ramirez-Perez FI, Burr K, Gonzalez-Vallejo JD, Morales-Quinones M, McMillan NJ, et al. Impact of dietary supplementation of glycocalyx precursors on vascular function in type 2 diabetes. J Appl Physiol. 2024. Dec;137(6):1592–603. doi: 10.1152/japplphysiol.00651.2024 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] 26.Ramirez-Perez FI, Cabral-Amador FJ, Whaley-Connell AT, Aroor AR, Morales-Quinones M, Woodford ML, et al. Cystamine reduces vascular stiffness in Western diet-fed female mice. Am J Physiol-Heart Circ Physiol. 2022. Feb 1;322(2):H167–80. doi: 10.1152/ajpheart.00431.2021 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R27] 27.Foote CA, Ramirez-Perez FI, Smith JA, Ghiarone T, Morales-Quinones M, McMillan NJ, et al. Neuraminidase inhibition improves endothelial function in diabetic mice. Am J Physiol Heart Circ Physiol. 2023. Dec 1;325(6):H1337–53. doi: 10.1152/ajpheart.00337.2023 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R28] 28.Steppan J, Bergman Y, Viegas K, Armstrong D, Tan S, Wang H, et al. Tissue Transglutaminase Modulates Vascular Stiffness and Function Through Crosslinking‐Dependent and Crosslinking-Independent Functions. J Am Heart Assoc. 2017. Feb 2;6(2):e004161. doi: 10.1161/JAHA.116.004161 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R29] 29.Targosz-Korecka M, Jaglarz M, Malek-Zietek KE, Gregorius A, Zakrzewska A, Sitek B, et al. AFM-based detection of glycocalyx degradation and endothelial stiffening in the db/db mouse model of diabetes. Sci Rep. 2017. Nov 21;7(1):15951. doi: 10.1038/s41598-017-16179-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] 30.Cenacchi T, Bertoldin T, Farina C, Fiori MG, Crepaldi G, Azzini CF, et al. Cognitive decline in the elderly: A double- blind, placebo- controlled multicenter study on efficacy of phosphatidylserine administration. Aging Clin Exp Res. 1993. Apr;5(2):123–33. doi: 10.1007/BF03324139 [DOI] [PubMed] [Google Scholar]

[R31] 31.Monteleone P, Maj M, Beinat L, Natale M, Kemali D. Blunting by chronic phosphatidylserine administration of the stress-induced activation of the hypothalamo-pituitary-adrenal axis in healthy men. Eur J Clin Pharmacol. 1992;42(4):385–8. doi: 10.1007/BF00280123. [DOI] [PubMed] [Google Scholar]

[R32] 32.Jorissen BL, Brouns F, Van Boxtel MPJ, Riedel WJ. Safety of Soy-derived Phosphatidylserine in Elderly People. Nutr Neurosci. 2002. Jan;5(5):337–43. doi: 10.1080/1028415021000033802 [DOI] [PubMed] [Google Scholar]

[R33] 33.Kingsley M Effects of Phosphatidylserine Supplementation on Exercising Humans: Sports Med. 2006;36(8):657–69. doi: 10.2165/00007256-200636080-00003 [DOI] [PubMed] [Google Scholar]

[R34] 34.Kingsley MI, Wadsworth D, Kilduff LP, Mceneny J, Benton D. Effects of Phosphatidylserine on Oxidative Stress following Intermittent Running. Med Sci Sports Exerc. 2005. Aug;37(8):1300–6. doi: 10.1249/01.mss.0000175306.05465.7e [DOI] [PubMed] [Google Scholar]

[R35] 35.Starks MA, Starks SL, Kingsley M, Purpura M, Jäger R. The effects of phosphatidylserine on endocrine response to moderate intensity exercise. J Int Soc Sports Nutr. 2008. Jul 28;5:11. doi: 10.1186/1550-2783-5-11 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R36] 36.Kingsley MI, Miller M, Kilduff LP, McENENY J, Benton D. Effects of Phosphatidylserine on Exercise Capacity during Cycling in Active Males. Med Sci Sports Exerc. 2006. Jan;38(1):64–71. doi: 10.1249/01.mss.0000183195.10867.d0 [DOI] [PubMed] [Google Scholar]

[R37] 37.Kingsley MI, Kilduff LP, Mceneny J, Dietzig RE, Benton D. Phosphatidylserine Supplementation and Recovery following Downhill Running. Med Sci Sports Exerc. 2006. Sep;38(9):1617–25. doi: 10.1249/01.mss.0000229459.11452.a0 [DOI] [PubMed] [Google Scholar]

[R38] 38.Zhu X, Hwu I, Jacabacci S, Wu S. The Utility of 24-h Ambulatory Blood Pressure Monitoring for the Diagnosis and Management of White Coat Hypertension in a Primary Care Setting. J Prim Care Community Health. 2022. Jan;13:21501319221123457. doi: 10.1177/21501319221123457 [DOI] [Google Scholar]

[R39] 39.Harris RA, Nishiyama SK, Wray DW, Richardson RS. Ultrasound Assessment of Flow-Mediated Dilation. Hypertension. 2010. May 1;55(5):1075–85. doi: 10.1161/HYPERTENSIONAHA.110.150821 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R40] 40.Thijssen DHJ, Black MA, Pyke KE, Padilla J, Atkinson G, Harris RA, et al. Assessment of flow-mediated dilation in humans: a methodological and physiological guideline. Am J Physiol Heart Circ Physiol. 2011. Jan;300(1):H2–12. doi: 10.1152/ajpheart.00471.2010 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R41] 41.Pyke KE, Tschakovsky ME. The relationship between shear stress and flow-mediated dilatation: implications for the assessment of endothelial function: The shear stress stimulus-flow-mediated dilatation relationship. J Physiol. 2005. Oct;568(2):357–69. doi: 10.1113/jphysiol.2005.089755 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R42] 42.Limberg JK, Casey DP, Trinity JD, Nicholson WT, Wray DW, Tschakovsky ME, et al. Assessment of resistance vessel function in human skeletal muscle: guidelines for experimental design, Doppler ultrasound, and pharmacology. Am J Physiol Heart Circ Physiol. 2020. Feb 1;318(2):H301–25. doi: 10.1152/ajpheart.00649.2019 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R43] 43.Gifford JR, Richardson RS. CORP: Ultrasound assessment of vascular function with the passive leg movement technique. J Appl Physiol. 2017. Dec 1;123(6):1708–20. doi: 10.1152/japplphysiol.00557.2017 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R44] 44.Trinity JD, Groot HJ, Layec G, Rossman MJ, Ives SJ, Runnels S, et al. Nitric oxide and passive limb movement: a new approach to assess vascular function. J Physiol. 2012. Mar;590(6):1413–25. doi: 10.1113/jphysiol.2011.224741 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R45] 45.Broxterman RM, Trinity JD, Gifford JR, Kwon OS, Kithas AC, Hydren JR, et al. Single passive leg movement assessment of vascular function: contribution of nitric oxide. J Appl Physiol. 2017. Dec 1;123(6):1468–76. doi: 10.1152/japplphysiol.00533.2017 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R46] 46.Shields KL, Broxterman RM, Jarrett CL, Bisconti AV, Park SH, Richardson RS. The passive leg movement technique for assessing vascular function: defining the distribution of blood flow and the impact of occluding the lower leg. Exp Physiol. 2019. Oct;104(10):1575–84. doi: 10.1113/EP087845 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R47] 47.Pettit-Mee RJ, Power G, Cabral-Amador FJ, Ramirez-Perez FI, Soares RN, Sharma N, et al. Endothelial HSP72 is not reduced in type 2 diabetes nor is it a key determinant of endothelial insulin sensitivity. Am J Physiol-Regul Integr Comp Physiol. 2022. Jul 1;323(1):R43–58. doi: 10.1152/ajpregu.00006.2022 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R48] 48.Park LK, Parks EJ, Pettit-Mee RJ, Woodford ML, Ghiarone T, Smith JA, et al. Skeletal muscle microvascular insulin resistance in type 2 diabetes is not improved by eight weeks of regular walking. J Appl Physiol Bethesda Md 1985. 2020. Aug 1;129(2):283–96. doi: 10.1152/japplphysiol.00174.2020 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R49] 49.Townsend RR, Wilkinson IB, Schiffrin EL, Avolio AP, Chirinos JA, Cockcroft JR, et al. Recommendations for Improving and Standardizing Vascular Research on Arterial Stiffness: A Scientific Statement From the American Heart Association. Hypertension. 2015. Sep;66(3):698–722. doi: 10.1161/HYP.0000000000000033 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R50] 50.Davis KG, Armstrong MK, Nuckols VR, Smith MN, Pewowaruk R, Gimblet CJ, et al. Load-dependent mechanisms contribute to increased aortic stiffness among women with a history of preeclampsia: relation with cardiovagal baroreflex sensitivity. Am J Physiol-Heart Circ Physiol. 2024. Dec 1;327(6):H1406–12. doi: 10.1152/ajpheart.00556.2024 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R51] 51.Armstrong MK, Jain S, Nuckols V, Pewowaruk R, Zhang X, DuBose L, et al. The association of structural versus load-dependent large artery stiffness mechanisms with cerebrovascular damage and cortical atrophy in humans. GeroScience. 2024. Jun 20;46(6):5587–97. doi: 10.1007/s11357-024-01254-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R52] 52.Pewowaruk RJ, Korcarz C, Tedla Y, Burke G, Greenland P, Wu C, et al. Carotid Artery Stiffness Mechanisms Associated With Cardiovascular Disease Events and Incident Hypertension: the Multi-Ethnic Study of Atherosclerosis (MESA). Hypertension. 2022. Mar;79(3):659–66. doi: 10.1161/HYPERTENSIONAHA.121.18772 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R53] 53.Nagababu E, Rifkind JM. Measurement of Plasma Nitrite by Chemiluminescence. In: Uppu RM, Murthy SN, Pryor WA, Parinandi NL, editors. Free Radicals and Antioxidant Protocols [Internet] Totowa, NJ: Humana Press; 2010. [cited 2024 Nov 26]. p. 41–9. (Methods in Molecular Biology). Available from: http://link.springer.com/10.1007/978-1-60327-029-8_3 doi: 10.1007/978-1-60327-029-8_3 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R54] 54.Nagababu E, Rifkind JM. Measurement of Plasma Nitrite by Chemiluminescence without Interference of S-, N-nitroso and Nitrated Species. Free Radic Biol Med. 2007. Apr 15;42(8):1146–54. doi: 10.1016/j.freeradbiomed.2006.12.029 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R55] 55.Smith JA, Soares RN, McMillan NJ, Jurrissen TJ, Martinez-Lemus LA, Padilla J, et al. Young Women Are Protected Against Vascular Insulin Resistance Induced by Adoption of an Obesogenic Lifestyle. Endocrinology. 2022. Oct 11;163(11):bqac137. doi: 10.1210/endocr/bqac137 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R56] 56.McMillan NJ, Soares RN, Harper JL, Shariffi B, Moreno-Cabañas A, Curry TB, et al. Role of the arterial baroreflex in the sympathetic response to hyperinsulinemia in adult humans. Am J Physiol-Endocrinol Metab. 2022. Apr 1;322(4):E355–65. doi: 10.1152/ajpendo.00391.2021 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R57] 57.McMillan NJ, Jacob DW, Shariffi B, Harper JL, Foster GE, Manrique-Acevedo C, et al. Effect of acute intranasal insulin administration on muscle sympathetic nerve activity in healthy young adults. Am J Physiol Heart Circ Physiol. 2024. Jul 1;327(1):H000. doi: 10.1152/ajpheart.00253.2024 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R58] 58.Vakhapova V, Richter Y, Cohen T, Herzog Y, Korczyn AD. Safety of phosphatidylserine containing omega-3 fatty acids in non-demented elderly: a double-blind placebo-controlled trial followed by an open-label extension. BMC Neurol. 2011. Dec;11(1):79. doi: 10.1186/1471-2377-11-79 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R59] 59.Richter Y, Herzog Y, Lifshitz, Yael, Hayun, Rami, Zchut S. The effect of soybean-derived phosphatidylserine on cognitive performance in elderly with subjective memory complaints: a pilot study. Clin Interv Aging. 2013. May;557. doi: 10.2147/CIA.S40348 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R60] 60.Moré MI, Freitas U, Rutenberg D. Positive Effects of Soy Lecithin-Derived Phosphatidylserine plus Phosphatidic Acid on Memory, Cognition, Daily Functioning, and Mood in Elderly Patients with Alzheimer’s Disease and Dementia. Adv Ther. 2014. Dec;31(12):1247–62. doi: 10.1007/s12325-014-0165-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R61] 61.Skulas-Ray AC, Wilson PWF, Harris WS, Brinton EA, Kris-Etherton PM, Richter CK, et al. Omega-3 Fatty Acids for the Management of Hypertriglyceridemia: A Science Advisory From the American Heart Association. Circulation. 2019. Sep 17;140(12). doi: 10.1161/CIR.0000000000000709 [DOI] [PubMed] [Google Scholar]

[R62] 62.Del Gobbo LC, Imamura F, Aslibekyan S, Marklund M, Virtanen JK, Wennberg M, et al. ω-3 Polyunsaturated Fatty Acid Biomarkers and Coronary Heart Disease: Pooling Project of 19 Cohort Studies. JAMA Intern Med. 2016. Aug 1;176(8):1155. doi: 10.1001/jamainternmed.2016.2925 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R63] 63.Morris MC, Sacks F, Rosner B. Does fish oil lower blood pressure? A meta-analysis of controlled trials. Circulation. 1993. Aug;88(2):523–33. doi: 10.1161/01.CIR.88.2.523 [DOI] [PubMed] [Google Scholar]

[R64] 64.Katakam PVG, Tulbert CD, Snipes JA, Erdös B, Miller AW, Busija DW. Impaired insulin-induced vasodilation in small coronary arteries of Zucker obese rats is mediated by reactive oxygen species. Am J Physiol-Heart Circ Physiol. 2005. Feb;288(2):H854–60. doi: 10.1152/ajpheart.00715.2004 [DOI] [PubMed] [Google Scholar]

[R65] 65.Headley S, Germain M, Wood R, Joubert J, Milch C, Evans E, et al. Short-term Aerobic Exercise and Vascular Function in CKD Stage 3: A Randomized Controlled Trial. Am J Kidney Dis. 2014. Aug;64(2):222–9. doi: 10.1053/j.ajkd.2014.02.022 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Beneficial vascular effects of oral phosphatidylserine supplementation in type 2 diabetes

Neil J McMillan

Marc A Augenreich

Francisco I Ramirez-Perez

Gavin Power

Katherine Burr

Olubodun M Lateef

Manikandan Nagarajan

Christopher A Foote

Natnicha Imkaew

Thomas J Jurrissen

Yoskaly Lazo-Fernandez

Edgar E Betancourt-Cortes

Larissa Ferreira-Santos

Jesus H Beltran-Ornelas

Camila Manrique-Acevedo

Luis A Martinez-Lemus

Jaume Padilla

Abstract

NEW & NOTEWORTHY

INTRODUCTION

METHODS

In Vitro and Ex Vivo Experiments

Preclinical Trial in Diabetic Mice

FIGURE 1: Trial design and supplement compositional breakdown.

In Vivo Measures

Blood Pressure

Aortic Pulse Wave Velocity

Ex Vivo Measures

Arterial Vascular Reactivity, Remodeling, and Mechanical Stiffness

Atomic Force Microscopy

Lipidomic Analysis

Proteomic Analysis

Aortic Ring Immunohistochemistry

Confocal Imaging

Clinical Trial in Individuals with T2D

Ethical Approval and Study Population

Table 1.

Study Design

FIGURE 2: CONSORT flow diagram for the clinical trial.

Ambulatory Blood Pressure

Table 2.

Baseline and Final Study Visit Procedures

Brachial artery FMD

Passive Leg Movement

Leg BF in Response to an Oral Glucose Load

Carotid-to-Femoral PWV

Assessment of Biochemical Parameters

Analysis of Vascular Related Outcomes

Statistical Analysis

RESULTS

In Vitro and Ex Vivo Experiments:

PS inhibits ADAM17 activity and increases insulin-induced dilation

FIGURE 3: Phosphatidylserine inhibits ADAM17 activity and increases insulin-induced dilation.

Preclinical Trial in Diabetic Mice:

Oral administration of PS improves endothelial function and insulin-induced dilation in mesenteric and cerebral arteries

FIGURE 4: Phosphatidylserine supplementation in diabetic mice improves cerebral and mesenteric artery endothelial function and insulin-induced dilation.

Oral administration of PS reduces blood pressure and arterial stiffness

FIGURE 5: Phosphatidylserine supplementation in diabetic mice reduces blood pressure and arterial stiffness.

PS inhibits ADAM17 activity and mitigates ADAM17-TNF-induced ROS

FIGURE 6: Phosphatidylserine inhibits ADAM17 activity and mitigates ADAM17-TNF-induced ROS.

Clinical Trial in Individuals with T2D:

Oral administration of PS improves leg BF responses to an oral glucose load, and reduces cfPWV

FIGURE 7: Phosphatidylserine improves leg blood flow and vascular conductance in response to an oral glucose load and reduces PWV in individuals with T2D.

DISCUSSION

FIGURE 8: Summary figure.

ACKNOWLEDGMENTS

GRANTS

Footnotes

REFERENCES

ACTIONS

PERMALINK

RESOURCES

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