ABSTRACT
The gut microbiome supplies essential metabolites such as short‐chain fatty acids to skeletal muscle mitochondria, and the composition and activity of the microbiota is in turn affected by ...muscle fitness. To further our understanding of the complex interactions between the gut microbiome and muscle, we examined the effect of microbiota‐derived phenolic metabolites on the ability of human muscle cells to take up and metabolize glucose. As a model, we used the differentiated human skeletal muscle myoblast line, LHCN‐M2, which expresses typical muscle phenotypic markers. We initially tested a selected panel of parent phenolic compounds and microbial metabolites, and their respective phenolic conjugates, as found in blood. Several of the tested compounds increased glucose uptake and metabolism, notably in high glucose—and insulin‐treated myotubes. One of the most effective was isovanillic acid 3‐O‐sulfate (IVAS), a metabolite from the microbiome found in the blood, primarily derived from consumed cyanidin 3‐O‐glucoside, a major compound in berry fruits. IVAS stimulated a dose‐dependent increase in glucose transport through glucose transporter GLUT4‐ and PI3K‐dependent mechanisms. IVAS also up‐regulated GLUT1, GLUT4, and PI3K p85α protein, and increased phosphorylation of Akt. The stimulation of glucose uptake and metabolism by a unique microbiome metabolite provides a novel link among diet, gut microbiota, and skeletal muscle energy source utilization.—Houghton, M. J., Kerimi, A., Mouly, V., Tumova, S., Williamson, G. Gut microbiome catabolites as novel modulators of muscle cell glucose metabolism. FASEB J. 33, 1887–1898 (2019). www.fasebj.org
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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%, p ≤ 0.001) and total GLUT7 protein (2.7-fold, p ≤ 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.
Glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are potent survival factors for dopaminergic neurons and motoneurons with therapeutic potential for Parkinson's disease. ...Soluble GFLs bind to a ligand-specific glycosylphosphatidylinositol-anchored coreceptor (GDNF family receptor α) and signal through the receptor tyrosine kinase RET. In this paper, we show that all immobilized matrix-bound GFLs, except persephin, use a fundamentally different receptor. They interact with syndecan-3, a transmembrane heparan sulfate (HS) proteoglycan, by binding to its HS chains with high affinity. GFL-syndecan-3 interaction mediates both cell spreading and neurite outgrowth with the involvement of Src kinase activation. GDNF promotes migration of cortical neurons in a syndecan-3-dependent manner, and in agreement, mice lacking syndecan-3 or GDNF have a reduced number of cortical γ-aminobutyric acid-releasing neurons, suggesting a central role for the two molecules in cortical development. Collectively, syndecan-3 may directly transduce GFL signals or serve as a coreceptor, presenting GFLs to the signaling receptor RET.
Orai1 and the associated calcium release-activated calcium (CRAC) channel were discovered in the immune system. Existence also in endothelial cells has been suggested, but the relevance to ...endothelial biology is mostly unknown.
The aim of this study was to investigate the relevance of Orai1 and CRAC channels to vascular endothelial growth factor (VEGF) and endothelial tube formation.
In human umbilical vein endothelial cells, Orai1 disruption by short-interfering RNA or dominant-negative mutant Orai1 inhibited calcium release-activated (store-operated) calcium entry, VEGF-evoked calcium entry, cell migration, and in vitro tube formation. Expression of exogenous wild-type Orai1 rescued the tube formation. VEGF receptor-2 and Orai1 partially colocalized. Orai1 disruption also inhibited calcium entry and tube formation in endothelial progenitor cells from human blood. A known blocker of the immune cell CRAC channel (3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide) was a strong blocker of store-operated calcium entry in endothelial cells and inhibited calcium entry evoked by VEGF in 3 types of human endothelial cell. The compound lacked effect on VEGF-evoked calcium-release, STIM1 clustering, and 2 types of transient receptor potential channels, TRPC6 and TRPV4. Without effect on cell viability, the compound inhibited human endothelial cell migration and tube formation in vitro and suppressed angiogenesis in vivo in the chick chorioallantoic membrane. The compound showed 100-fold greater potency for endothelial compared with immune cell calcium entry.
The data suggest positive roles for Orai1 and CRAC channels in VEGF-evoked calcium entry and new opportunity for chemical modulation of angiogenesis.
Juvenile hormone (JH) signalling provides vital regulatory functions during insect development via transcriptional regulation of genes critical for the progression of metamorphosis and oogenesis. ...Despite the importance of JH signalling, the underlying molecular mechanisms remain largely unknown. Our current understanding of the pathway depends on static end‐point information and suffers from the lack of time‐resolved data. Here, we have addressed the dynamic aspect of JH signalling by monitoring in real time the interactions of insect JH receptor proteins. Use of two tags that reconstitute a functional luciferase when in proximity enabled us to follow the rapid assembly of a JH receptor heterodimer from basic helix–loop–helix/Per‐Arnt‐SIM (bHLH‐PAS) proteins, methoprene‐tolerant (Met) and taiman (Tai), upon specific JH binding to Met. On a similar timescale (minutes), the dissociation of Met‐Met complexes occurred, again strictly dependent on Met interaction with specific agonist ligands. To resolve questions regarding the regulatory role of the chaperone Hsp90/83 in the JHR complex formation, we used the same technique to demonstrate that the Met‐Hsp83 complex persisted in the agonist absence but readily dissociated upon specific binding of JH to Met. Preincubation with the Hsp90 inhibitor geldanamycin showed that the chaperone interaction protected Met from degradation and was critical for Met to produce the active signalling dimer with Tai. Thus, the JH receptor functions appear to be governed by principles similar to those regulating the aryl hydrocarbon receptor, the closest vertebrate homologue of the arthropod JH receptor.
The insect juvenile hormone receptor (JHR) engages in an actively signalling heterodimer after dissociating from stabilising protein complexes formed in the absence of the hormone. The rapid transition, which strictly depends on the agonist‐receptor binding, was monitored in real time using a split luciferase assay in live cells. These data present primary time‐resolved information on JHR function.
N-syndecan (syndecan-3) is a transmembrane proteoglycan that is abundantly expressed in the major axonal pathways and in the migratory routes of the developing brain. When ligated by heparin-binding ...(HB) growth-associated molecule (GAM; pleiotrophin), N-syndecan mediates cortactin-Src kinase-dependent neurite outgrowth. However, the functional role of N-syndecan in brain development remains unexplored. In this study, we show that N-syndecan deficiency perturbs the laminar structure of the cerebral cortex as a result of impaired radial migration. In addition, neural migration in the rostral migratory stream is impaired in the N-syndecan-null mice. We suggest that the migration defect depends on impaired HB-GAM-induced Src kinase activation and haptotactic migration. Furthermore, we show that N-syndecan interacts with EGF receptor (EGFR) at the plasma membrane and is required in EGFR-induced neuronal migration.
Consumption of dietary bioactives is an avenue to enhancing the effective healthiness of diets by attenuating the glycaemic response. The intestinal brush border enzyme sucrase-isomaltase (SI) is the ...sole enzyme hydrolysing consumed sucrose, and we previously showed the acute effects of olive leaf extract (OLE) on sucrase activity when given together with sugars both in vitro and in vivo. Here we tested whether OLE could affect sucrase expression when pre-incubated chronically, a "priming" effect not dependent on competitive interaction with SI, in both a cell model and a human intervention. Using differentiated Caco-2/TC7 cells, long-term pre-treatment with oleuropein-rich olive leaf extract (OLE) lowered SI mRNA, surface protein and activity, and attenuated subsequent sucrose hydrolysis. Based on these results, a randomised, double-blinded, placebo-controlled, crossover pilot study was conducted. OLE (50 mg oleuropein) was consumed in capsule form 3 times a day for 1 week by 11 healthy young women followed by an oral sucrose tolerance test in the absence of OLE. However this treatment, compared to placebo, did not induce a change in post-prandial blood glucose maximum concentration (Glc
), time to reach Glc
and incremental area under the curve. These results indicate that changes in SI mRNA, protein and activity in an intestinal cell model by OLE are not sufficient under these conditions to induce a functional effect in vivo in healthy volunteers.
Two prominent concepts for the sensing of shear stress by endothelium are the PIEZO1 channel as a mediator of mechanically activated calcium ion entry and the PECAM1 cell adhesion molecule as the ...apex of a triad with CDH5 and VGFR2. Here, we investigated if there is a relationship. By inserting a non-disruptive tag in native PIEZO1 of mice, we reveal in situ overlap of PIEZO1 with PECAM1. Through reconstitution and high resolution microscopy studies we show that PECAM1 interacts with PIEZO1 and directs it to cell-cell junctions. PECAM1 extracellular N-terminus is critical in this, but a C-terminal intracellular domain linked to shear stress also contributes. CDH5 similarly drives PIEZO1 to junctions but unlike PECAM1 its interaction with PIEZO1 is dynamic, increasing with shear stress. PIEZO1 does not interact with VGFR2. PIEZO1 is required in Ca
-dependent formation of adherens junctions and associated cytoskeleton, consistent with it conferring force-dependent Ca
entry for junctional remodelling. The data suggest a pool of PIEZO1 at cell junctions, the coming together of PIEZO1 and PECAM1 mechanisms and intimate cooperation of PIEZO1 and adhesion molecules in tailoring junctional structure to mechanical requirement.
Heparan sulfate proteoglycans are complex molecules composed of a core protein with covalently attached glycosaminoglycan chains. While the protein part determines localization of the proteoglycan on ...the cell surfaces or in the extracellular matrix, the glycosaminoglycan component, heparan sulfate, mediates interactions with a variety of extracellular ligands such as growth factors and adhesion molecules. Through these interactions, heparan sulfate proteoglycans participate in many events during cell adhesion, migration, proliferation and differentiation. We are determining the multitude of proteoglycan functions, as their intricate roles in many pathways are revealed. They act as coreceptors for growth factors, participate in signalling during cell adhesion, modulate the activity of a broad range of molecules, and partake in many developmental and pathological processes, including tumorigenesis and wound repair. This review concentrates on biological roles of cell surface heparan sulfate proteoglycans, namely syndecans and glypicans, and outlines the progress achieved during the last decade in unraveling the molecular interactions behind proteoglycan functions.
Vascular endothelial growth factor (VEGF) acts, in part, by triggering calcium ion (Ca(2+)) entry. Here, we sought understanding of a Synta66-resistant Ca(2+) entry pathway activated by VEGF.
...Measurement of intracellular Ca(2+) in human umbilical vein endothelial cells detected a Synta66-resistant component of VEGF-activated Ca(2+) entry that occurred within 2 minutes after VEGF exposure. Knockdown of the channel-forming protein Orai3 suppressed this Ca(2+) entry. Similar effects occurred in 3 further types of human endothelial cell. Orai3 knockdown was inhibitory for VEGF-dependent endothelial tube formation in Matrigel in vitro and in vivo in the mouse. Unexpectedly, immunofluorescence and biotinylation experiments showed that Orai3 was not at the surface membrane unless VEGF was applied, after which it accumulated in the membrane within 2 minutes. The signaling pathway coupling VEGF to the effect on Orai3 involved activation of phospholipase Cγ1, Ca(2+) release, cytosolic group IV phospholipase A2α, arachidonic acid production, and, in part, microsomal glutathione S-transferase 2, an enzyme which catalyses the formation of leukotriene C4 from arachidonic acid. Shear stress reduced microsomal glutathione S-transferase 2 expression while inducing expression of leukotriene C4 synthase, suggesting reciprocal regulation of leukotriene C4-synthesizing enzymes and greater role of microsomal glutathione S-transferase 2 in low shear stress.
VEGF signaling via arachidonic acid and arachidonic acid metabolism causes Orai3 to accumulate at the cell surface to mediate Ca(2+) entry and downstream endothelial cell remodeling.
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