RESUMEN
OBJECTIVE: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. CONCLUSIONS: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.
Asunto(s)
Fibrinógeno/metabolismo , Fragmentos de Péptidos/sangre , Glicoproteínas de Membrana Plaquetaria/metabolismo , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Productos de Degradación de Fibrina-Fibrinógeno/química , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Fibrinógeno/química , Humanos , Técnicas In Vitro , Ratones , Microscopía de Fuerza Atómica , Fragmentos de Péptidos/química , Péptidos/química , Péptidos/metabolismo , Agregación Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/química , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Transducción de Señal , Resonancia por Plasmón de SuperficieRESUMEN
Although polyphenols inhibit glucose absorption and transport in vitro, it is uncertain whether this activity is sufficient to attenuate glycaemic response in vivo. We examined this using orange juice, which contains high levels of hesperidin. We first used a combination of in vitro assays to evaluate the potential effect of hesperidin and other orange juice components on intestinal sugar absorption and then tested whether this translated to an effect in healthy volunteers. Hesperidin attenuated transfer of 14C-labelled glucose across differentiated Caco-2/TC7 cell monolayers. The involvement of the sugar transporter GLUT2 was demonstrated by experiments carried out in the absence of Na to exclude the contribution of sodium-glucose linked transporter 1 and further explored by the use of Xenopus laevis oocytes expressing human GLUT2 or GLUT5. Fructose transport was also affected by hesperidin partly by inhibition of GLUT5, while hesperidin, even at high concentration, did not inhibit rat intestinal sucrase activity. We conducted three separate crossover interventions, each on ten healthy volunteers using orange juice with different amounts of added hesperidin and water. The biggest difference in postprandial blood glucose between orange juice and control, containing equivalent amounts of glucose, fructose, sucrose, citric acid and ascorbate, was when the juice was diluted (ΔC max=-0·5 mm, P=0·0146). The effect was less pronounced when the juice was given at regular strength. Our data indicate that hesperidin can modulate postprandial glycaemic response of orange juice by partial inhibition of intestinal GLUT, but this depends on sugar and hesperidin concentrations.
Asunto(s)
Glucemia/metabolismo , Citrus sinensis , Fructosa/metabolismo , Jugos de Frutas y Vegetales/análisis , Hesperidina/farmacología , Sacarasa/metabolismo , Adulto , Estudios Cruzados , Femenino , Transportador de Glucosa de Tipo 2/metabolismo , Transportador de Glucosa de Tipo 5/metabolismo , Índice Glucémico , Voluntarios Sanos , Humanos , Absorción Intestinal/efectos de los fármacos , Masculino , Adulto JovenRESUMEN
PURPOSE: The secoiridoid oleuropein, as found in olives and olive leaves, modulates some biomarkers of diabetes risk in vivo. A possible mechanism may be to attenuate sugar digestion and absorption. METHODS: We explored the potential of oleuropein, prepared from olive leaves in a water soluble form (OLE), to inhibit digestive enzymes (α-amylase, maltase, sucrase), and lower [14C(U)]-glucose uptake in Xenopus oocytes expressing human GLUT2 and [14C(U)]-glucose transport across differentiated Caco-2 cell monolayers. We conducted 7 separate crossover, controlled, randomised intervention studies on healthy volunteers (double-blinded and placebo-controlled for the OLE supplement) to assess the effect of OLE on post-prandial blood glucose after consumption of bread, glucose or sucrose. RESULTS: OLE inhibited intestinal maltase, human sucrase, glucose transport across Caco-2 monolayers, and uptake of glucose by GLUT2 in Xenopus oocytes, but was a weak inhibitor of human α-amylase. OLE, in capsules, in solution or as naturally present in olives, did not affect post-prandial glucose derived from bread, while OLE in solution attenuated post-prandial blood glucose after consumption of 25 g sucrose, but had no effect when consumed with 50 g of sucrose or glucose. CONCLUSION: The combined inhibition of sucrase activity and of glucose transport observed in vitro was sufficient to modify digestion of low doses of sucrose in healthy volunteers. In comparison, the weak inhibition of α-amylase by OLE was not enough to modify blood sugar when consumed with a starch-rich food, suggesting that a threshold potency is required for inhibition of digestive enzymes in order to translate into in vivo effects.
Asunto(s)
Glucemia/metabolismo , Iridoides/metabolismo , Olea/metabolismo , Periodo Posprandial , Sacarosa/metabolismo , Azúcares/metabolismo , Adolescente , Adulto , Anciano , Animales , Transporte Biológico , Técnicas de Cultivo de Célula , Estudios Cruzados , Método Doble Ciego , Femenino , Humanos , Hidrólisis , Técnicas In Vitro , Glucósidos Iridoides , Masculino , Persona de Mediana Edad , Modelos Animales , Ratas , Valores de Referencia , Adulto JovenRESUMEN
GPVI is the major signalling receptor for collagen on platelets. Dimerization of GPVI is required for collagen binding and initiation of signalling through the associated FcR-γ chain. Recently, fibrin and fibrinogen have been identified as ligands for GPVI and shown to induce signalling in support of thrombus formation and stabilization. Contrasting observations have been reported on whether fibrin binds to monomeric or dimeric GPVI, or to neither form. In this article, we discuss reasons for the contradictory results and how to reconcile these. We conclude that a lack of structural knowledge regarding the GPVI constructs that are being used, along with the use of non-standardized reagents, might be the main cause of the discrepant results. This article aims to highlight some of the key areas that need to be addressed.
Asunto(s)
Plaquetas/metabolismo , Fibrina/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Humanos , Unión ProteicaRESUMEN
ABSTRACT: Glycoprotein VI (GPVI) plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVI-binding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22, and D18 bound GPVI with the highest affinities (dissociation constant (KD) in the nanomolar range). These Affimers inhibited GPVI-collagen-related peptide (CRP)-XL/collagen interactions, CRP-XL/collagen-induced platelet aggregation, and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and adenosine 5'-diphosphate. D22 but not M17/D18 displaced nanobody 2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1 domain, whereas M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody fragment antigen-binding fragment. D18 targets a new region on the D2 domain. We found that D18 is a stable noncovalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2 domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2 domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.
Asunto(s)
Plaquetas , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria , Unión Proteica , Multimerización de Proteína , Glicoproteínas de Membrana Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/química , Humanos , Plaquetas/metabolismo , Ligandos , Agregación Plaquetaria/efectos de los fármacos , Sitios de Unión , Colágeno/metabolismo , Colágeno/química , Proteínas Portadoras , PéptidosRESUMEN
The 2023 annual meeting of the British Society for Haemostasis and Thrombosis (BSHT) was held in Birmingham, United Kingdom. The theme of this year's meeting was novel therapeutics and emerging technology. Here, the exciting research presented at the meeting is discussed.
RESUMEN
BACKGROUND: The glycoprotein VI (GPVI) signaling pathway was previously reported to direct procoagulant platelet activity through collagen binding. However, the impact of GPVI-fibrin interaction on procoagulant platelet development and how it modulates the clot structure are unknown. OBJECTIVES: To determine the effect of GPVI-fibrin interaction on the platelet phenotype and its impact on the clot structure. METHODS: Procoagulant platelets in platelet-rich plasma clots were determined by scanning electron microscopy (wild-type and GPVI-deficient murine samples) and confocal microscopy. Procoagulant platelet number, clot density, clot porosity, and clot retraction were determined in platelet-rich plasma or whole blood clots of healthy volunteers in the presence of tyrosine kinase inhibitors (PRT-060318, ibrutinib, and dasatinib) and eptifibatide. RESULTS: GPVI-deficient clots showed a higher nonprocoagulant vs procoagulant platelet ratio than wild-type clots. The fiber density and the procoagulant platelet number decreased in the presence of Affimer proteins, inhibiting GPVI-fibrin(ogen) interaction and the tyrosine kinase inhibitors. The effect of GPVI signaling inhibitors on the procoagulant platelet number was exacerbated by eptifibatide. The tyrosine kinase inhibitors led to an increase in clot porosity; however, no differences were observed in the final clot weight, following clot retraction with the tyrosine kinase inhibitors, except for ibrutinib. In the presence of eptifibatide, clot retraction was impaired. CONCLUSION: Our findings showed that GPVI-fibrin interaction significantly contributes to the development of procoagulant platelets and that inhibition of GPVI signaling increases clot porosity. Clot contractibility was impaired by the integrin αIIbß3 and Btk pathway inhibition. Thus, inhibition of GPVI-fibrin interactions can alleviate structural characteristics that contribute to a prothrombotic clot phenotype, having potential important implications for novel antithrombotic interventions.
Asunto(s)
Fibrina , Trombosis , Animales , Ratones , Plaquetas/metabolismo , Eptifibatida/farmacología , Fibrina/química , Glicoproteínas de Membrana Plaquetaria/metabolismoRESUMEN
Thromboinflammation has become a topic of key interest in cardiovascular disease and the prevention of diabetes complications because of the interplay between thrombosis and inflammation in diabetes. Specifically, the significant risk of vascular thrombotic disease in diabetes highlights the need for new and better therapeutic targets to help manage and prevent vascular thrombo-occlusive disease in this condition. Similarly, the prominent role of inflammation in diabetes has sparked interest in anti-inflammatory agents to better prevent and control vascular disease. Investigations on the effects of anticoagulation and antiplatelet interventions in patients with diabetes and cardiovascular disease show a potential role for these agents in decreasing morbidity and mortality. Neutrophils and platelets are key players in inflammation and wound-healing response, respectively. The interaction between neutrophils and platelets is thought to be an important driver of thromboinflammation. Therefore, this review describes the mechanisms involved in platelet-neutrophil interactions that contribute to the development or exacerbation of thromboinflammation in the context of diabetes and its associated comorbidities. The effects observed by the antithrombotic/antidiabetic treatments and physical activity/dietary interventions on attenuating thromboinflammation are discussed. These data suggest that mechanisms involved in platelet-neutrophil interaction, platelet activation/aggregation, and the recruitment of neutrophils have a promising potential to become therapeutic targets to decrease thromboinflammation in patients with diabetes.
Asunto(s)
Enfermedades Cardiovasculares , Diabetes Mellitus , Trombosis , Humanos , Neutrófilos , Inflamación/prevención & control , Trombosis/prevención & control , Tromboinflamación , Fibrinolíticos/farmacología , Plaquetas , Diabetes Mellitus/tratamiento farmacológico , Anticoagulantes , Hipoglucemiantes/farmacologíaRESUMEN
The GPVI platelet receptor was recently validated as a safe antiplatelet target for the treatment of thrombosis using several peptidic modulators. In contrast, few weakly potent small-molecule GPVI antagonists have been reported. Those that have been published often lack evidence for target engagement, and their biological efficacy cannot be compared because of the natural donor variability associated with the assays implemented. Herein, we present the first side-by-side assessment of the reported GPVI small-molecule modulators. We have characterized their functional activities on platelet activation and aggregation using flow cytometry as well as light transmission and electrical impedance aggregometry. We also utilized microscale thermophoresis (MST) and saturation transfer difference (STD) NMR to validate GPVI binding and have used this along with molecular modeling to suggest potential binding interactions. We conclude that of the compounds examined, losartan and compound 5 are currently the most viable GPVI modulators.
RESUMEN
BACKGROUND: Abnormal clot structure has been identified in patients with thrombotic disorders. Anticoagulant therapy offers clear benefits for thrombosis prevention and treatment by reducing blood clot formation and size; nevertheless, there are limited data on the effects of different anticoagulants, where clotting is initiated with different triggers, on clot structure. OBJECTIVES: Our aim was to investigate the effects of vitamin K antagonists and factor Xa inhibitors on clot structure. METHODS: Clots from pooled plasma spiked with rivaroxaban, apixaban, or enoxaparin, as well as plasma from patients on warfarin, were compared to plasma without anticoagulation. The kinetic profile of polymerizing clots was obtained by turbidity, fiber density was determined by confocal microscopy, clot pore size was investigated by permeation, and fiber size was analyzed using scanning electron microscopy. Clotting agonist was either tissue factor or thrombin. RESULTS: Following clotting with tissue factor, all anticoagulated clots had a significantly increased lag time, with the exception of enoxaparin. Rivaroxaban additionally led to significantly less dense and more permeable clots, with thicker fibers. In contrast, turbidity analysis following initiation with thrombin showed few effects of anticoagulation, with only enoxaparin leading to a prolonged lag time. Enoxaparin clots made with thrombin were less dense and more permeable. CONCLUSION: Our results show that anticoagulants modulate clot structure particularly when induced by tissue factor, most likely due to reduction of thrombin generation. We propose that the effects of different anticoagulants could be assessed with a global clot structure measurement such as permeation or turbidity, providing information on clot phenotype.
RESUMEN
BACKGROUND: Fibrin polymerization, following fibrinopeptides A and B (FpA, FpB) cleavage, relies on newly exposed α- and ß-chains N-termini (GPR, GHR; A-, B-knobs, respectively) engaging preexistent a and b pockets in other fibrin(ogen) molecules' γ- and (B)ß-chains C-terminal regions. A role for mostly disordered (A)α-chains C-terminal regions "bridging" between fibrin molecules/fibrils has been proposed. OBJECTIVES: Fibrinogen Detroit is a clinically observed mutation (AαR19 â S) with nonengaging GPS A-knobs. By analogy, a similar Bß-chain mutation, BßR17 â S, should produce nonengaging GHS B-knobs. A homozygous "Double-Detroit" mutant (AαR19 â S, BßR17 â S; DD-FG) was developed: with A-a and B-b engagements endogenously blocked, other interactions would become apparent. METHODS: DD-FG, wild-type recombinant (WT-FG), and human plasma (hp-FG) fibrinogen self-association was studied by turbidimetry coupled with fibrinopeptides release high-performance liquid chromatography (HPLC)/mass spectrometry analyses, and by light-scattering following size-exclusion chromatography (SE-HPLC). RESULTS: In contrast to WT-FG and hp-FG, DD-FG produced no turbidity increase, irrespective of thrombin concentration. The SE-HPLC profile of concentrated DD-FG was unaffected by thrombin treatment, and light-scattering, at lower concentration, showed no intensity and hydrodynamic radius changes. Compared with hp-FG, both WT-FG and DD-FG showed no FpA cleavage difference, while ~50% FpB was not recovered. Correspondingly, SDS-PAGE/Western-blots revealed partial Bß-chain N-terminal and Aα-chain C-terminal degradation. Nevertheless, ~70% DD-FG molecules bearing (A)αC-regions potentially able to associate were available. Higher-concentration, nearly intact hp-FG with 500-fold molar excess GPRP-NH2 /GHRP-NH2 knobs-mimics experiments confirmed these no-association findings. CONCLUSIONS: (A)αC-regions interactions appear too weak to assist native fibrin polymerization, at least without knobs engagement. Their role in all stages should be carefully reconsidered.
Asunto(s)
Fibrina , Fibrinógeno , Fibrinógeno/genética , Humanos , Fragmentos de Péptidos , Polimerizacion , TrombinaRESUMEN
Only limited data are available on the inhibition of the sugar transporter GLUT5 by flavonoids or other classes of bioactives. Intestinal GLUT7 is poorly characterised and no information exists concerning its inhibition. We aimed to study the expression of GLUT7 in Caco-2/TC7 intestinal cells, and evaluate inhibition of glucose transport by GLUT2 and GLUT7, and of fructose transport by GLUT2, GLUT5 and GLUT7, by flavonoids. Differentiated Caco-2/TC7 cell monolayers were used to investigate GLUT7 expression, as well as biotinylation and immunofluorescence to assess GLUT7 location. For mechanistic sugar transport studies, X. laevis oocytes were injected with individual mRNA, and GLUT protein expression on oocyte membranes was confirmed. Oocytes were incubated with D-[14C(U)]-glucose or D-[14C(U)]-fructose in the presence of flavonoids, and uptake was estimated by liquid scintilation counting. In differentiated Caco-2/TC7 cell monolayers, GLUT7 was mostly expressed apically. When applied apically, or to both compartments, sorbitol, galactose, L-glucose or sucrose did not affect GLUT7 mRNA expression. Fructose applied to both sides increased GLUT7 mRNA (13%, pâ¯≤â¯0.001) and total GLUT7 protein (2.7-fold, pâ¯≤â¯0.05), while the ratio between apical, basolateral and total GLUT7 protein was unchanged. In the X. laevis oocyte model, GLUT2-mediated glucose and fructose transport were inhibited by quercetin, (-)-epigallocatechin gallate (EGCG) and apigenin, GLUT5-mediated fructose transport was inhibited by apigenin and EGCG, but not by quercetin, and GLUT7-mediated uptake of both glucose and fructose was inhibited by apigenin, but not by quercetin nor EGCG. Expression of GLUT7 was increased by fructose, but only when applied to Caco-2/TC7 cells both apically and basolaterally. Since GLUT2, GLUT5 and GLUT7 show different patterns of inhibition by the tested flavonoids, we suggest that they have the potential to be used as investigational tools to distinguish sugar transporter activity in different biological settings.
Asunto(s)
Flavonoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Transportador de Glucosa de Tipo 2/metabolismo , Transportador de Glucosa de Tipo 5/metabolismo , Animales , Células CACO-2 , Fructosa/metabolismo , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/antagonistas & inhibidores , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Transportador de Glucosa de Tipo 2/antagonistas & inhibidores , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 5/antagonistas & inhibidores , Transportador de Glucosa de Tipo 5/genética , Humanos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Xenopus laevisRESUMEN
SCOPE: High glycaemic sugars result in blood-glucose spikes, while large doses of post-prandial fructose inundate the liver, causing an imbalance in energy metabolism, both leading to increased risk of metabolic malfunction and type 2 diabetes. Acarbose, used for diabetes management, reduces post-prandial hyperglycaemia by delaying carbohydrate digestion. METHODS AND RESULTS: Chamomile and green teas both inhibited digestive enzymes (α-amylase and maltase) related to intestinal sugar release, as already established for acarbose. However, acarbose had no effect on uptake of sugars using both differentiated human Caco-2 cell monolayers and Xenopus oocytes expressing human glucose transporter-2 (GLUT2) and GLUT5. Both teas effectively inhibited transport of fructose and glucose through GLUT2 inhibition, while chamomile tea also inhibited GLUT5. Long term incubation of Caco-2/TC7 cells with chamomile tea for 16 h or 4 days did not enhance the observed effects, indicating that inhibition is acute. Sucrase activity was directly inhibited by green tea and acarbose, but not chamomile. CONCLUSION: These findings show that chamomile and green teas are potential tools to manage absorption and metabolism of sugars with efficacy against high sugar bolus stress inflicted, for example, by high fructose syrups, where the drug acarbose would be ineffective.
Asunto(s)
Acarbosa/farmacología , Manzanilla/química , Glucosa/metabolismo , Té/química , Tés de Hierbas , Animales , Transporte Biológico/efectos de los fármacos , Células CACO-2 , Diferenciación Celular/efectos de los fármacos , Fructosa/metabolismo , Transportador de Glucosa de Tipo 2/antagonistas & inhibidores , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 2/metabolismo , Transportador de Glucosa de Tipo 5/antagonistas & inhibidores , Transportador de Glucosa de Tipo 5/genética , Transportador de Glucosa de Tipo 5/metabolismo , Humanos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Sacarosa/metabolismo , Xenopus , alfa-Amilasas/antagonistas & inhibidores , alfa-Amilasas/metabolismoRESUMEN
Background: Low-glycemic index diets have demonstrated health benefits associated with a reduced risk of developing type 2 diabetes.Objectives: We tested whether pomegranate polyphenols could lower the glycemic response of a high-glycemic index food when consumed together and the mechanism by which this might occur.Design: We compared the acute effect of a pomegranate juice and a polyphenol-rich extract from pomegranate (supplement) on the bread-derived postprandial blood glucose concentration in 2 randomized, crossover, controlled studies (double-blinded for the supplements), each on 16 healthy volunteers. An additional randomized, crossover, controlled study on 16 volunteers consuming constituent fruit acids in a pH-balanced solution (same pH as pomegranate) and bread was conducted to determine any contributions to postprandial responses caused by acidic beverages.Results: As primary outcome, the incremental area under the curve for bread-derived blood glucose (-33.1% ± 18.1%, P = 0.000005) and peak blood glucose (25.4% ± 19.3%, P = 0.0004) were attenuated by pomegranate juice, compared with a control solution containing the equivalent amount of sugars. In contrast, the pomegranate supplement, or a solution containing the malic and citric acid components of the juice, was ineffective. The pomegranate polyphenol punicalagin was a very effective inhibitor of human α-amylase in vitro, comparable to the drug acarbose. Neither the pomegranate extract nor the individual component polyphenols inhibited 14C-D-glucose transport across differentiated Caco-2/TC7 cell monolayers, but they inhibited uptake of 14C-glucose into Xenopus oocytes expressing the human glucose transporter type 2. Further, some of the predicted pomegranate gut microbiota metabolites modulated 14C-D-glucose and 14C-deoxy-D-glucose uptake into hepatic HepG2 cells.Conclusions: These data indicate that pomegranate polyphenols, when present in a beverage but not in a supplement, can reduce the postprandial glycemic response of bread, whereas microbial metabolites from pomegranate polyphenols exhibit the potential to further modulate sugar metabolism much later in the postprandial period. This trial was registered at clinicaltrials.gov as NCT02486978, NCT02624609, and NCT03242876.