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
•α-Amylase inhibition was studied using a chromatographic HPAE-PAD method.•Maltoheptaoside (Mal-7) was used as the substrate.•Phenolics inhibit α-amylase through direct inhibition and by complexing ...with starch.•Conventional α-amylase methods detect both direct and indirect inhibition.•Short maltooligosaccharides can be used to assess direct α-amylase inhibition.
The aim was to determine inhibition of human α-amylase activity by (poly)phenols using maltoheptaoside as substrate with direct chromatographic product quantification, compared to hydrolysis of amylose and amylopectin estimated using 3,5-dinitrosalicylic acid. Acarbose exhibited similar IC50 values (50% inhibition) with maltoheptaoside, amylopectin or amylose as substrates (2.37 ± 0.11, 3.71 ± 0.12 and 2.08 ± 0.01 µM respectively). Epigallocatechin gallate, quercetagetin and punicalagin were weaker inhibitors of hydrolysis of maltoheptaoside (<50% inhibition) than amylose (IC50: epigallocatechin gallate = 20.41 ± 0.25 µM, quercetagetin = 30.15 ± 2.05 µM) or amylopectin. Interference using 3,5-dinitrosalicylic acid was in the order punicalagin > epigallocatechin gallate > quercetagetin, with minimal interference using maltoheptaoside as substrate. The main inhibition mechanism of epigallocatechin gallate and punicalagin was through complexation with starch, especially amylose, whereas only quercetagetin additionally binds to the α-amylase active site. Interference is minimised using maltoheptaoside as substrate with product detection by chromatography, potentially allowing assessment of direct enzyme inhibition by almost any compound.
Certain flavonoids can influence glucose metabolism by inhibiting enzymes involved in carbohydrate digestion and suppressing intestinal glucose absorption. In this study, four structurally-related ...flavonols (quercetin, kaempferol, quercetagetin and galangin) were evaluated individually for their ability to inhibit human α-glucosidases (sucrase, maltase and isomaltase), and were compared with the antidiabetic drug acarbose and the flavan-3-ol(−)-epigallocatechin-3-gallate (EGCG). Cell-free extracts from human intestinal Caco-2/TC7 cells were used as the enzyme source and products were quantified chromatographically with high accuracy, precision and sensitivity. Acarbose inhibited sucrase, maltase and isomaltase with IC50 values of 1.65, 13.9 and 39.1 µM, respectively. A similar inhibition pattern, but with comparatively higher values, was observed with EGCG. Of the flavonols, quercetagetin was the strongest inhibitor of α-glucosidases, with inhibition constants approaching those of acarbose, followed by galangin and kaempferol, while the weakest were quercetin and EGCG. The varied inhibitory effects of flavonols against human α-glucosidases depend on their structures, the enzyme source and substrates employed. The flavonols were more effective than EGCG, but less so than acarbose, and so may be useful in regulating sugar digestion and postprandial glycaemia without the side effects associated with acarbose treatment.
Hyperglycemia augments formation of intracellular reactive oxygen species (ROS) with associated mitochondrial damage and increased risk of insulin resistance in type 2 diabetes. We examined whether ...quercetin could reverse chronic high glucose-induced oxidative stress and mitochondrial dysfunction. Following long-term high glucose treatment, complex I activity was significantly decreased in isolated mitochondria from HepG2 cells. Quercetin dose-dependently recovered complex I activity and lowered cellular ROS generation under both high and normal glucose conditions. Respirometry studies showed that quercetin could counteract the detrimental increase in inner mitochondrial membrane proton leakage resulting from high glucose while it increased oxidative respiration, despite a decrease in electron transfer system (ETS) capacity, and lower non-ETS oxygen consumption. A quercetin-stimulated increase in cellular NAD+/NADH was evident within 2 h and a two-fold increase in PGC-1α mRNA within 6 h, in both normal and high glucose conditions. A similar pattern was also found for the mRNA expression of the repulsive guidance molecule b (RGMB) and its long non-coding RNA (lncRNA) RGMB-AS1 with quercetin, indicating a potential change of the glycolytic phenotype and suppression of aberrant cellular growth which is characteristic of the HepG2 cells. Direct effects of quercetin on PGC-1α activity were minimal, as quercetin only weakly enhanced PGC-1α binding to PPARα in vitro at higher concentrations. Our results suggest that quercetin may protect mitochondrial function from high glucose-induced stress by increasing cellular NAD+/NADH and activation of PGC-1α-mediated pathways. Lower ROS in combination with improved complex I activity and ETS coupling efficiency under conditions of amplified oxidative stress could reinforce mitochondrial integrity and improve redox status, beneficial in certain metabolic diseases.
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•Quercetin protects mitochondrial function from high glucose-induced stress.•Through increased RGMB mRNA and long non-coding RNA, quercetin may initiate a change of the HepG2 cell glycolytic phenotype.•Quercetin reversed chronic high glucose-induced oxidative stress and mitochondrial dysfunction.•Quercetin induced lower ROS in combination with improved complex I activity and ETS coupling efficiency.
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•Potent inhibition of human salivary and pancreatic α-amylases by walnut (poly)phenols.•Significant inhibition of human brush border sucrase-isomaltase by walnut ...(poly)phenols.•Significant inhibition of glucose transport across human intestinal monolayers by walnut (poly)phenols.•Potential of walnuts to lower postprandial glucose spikes may contribute to their proposed health benefits.
One approach to controlling type 2 diabetes (T2D) is to lower postprandialglucose spikesby slowing down the digestion of carbohydrates and the absorption of glucose in the small intestine. The consumption of walnuts is associated with a reduced risk of chronic diseases such as T2D, suggested to be partly due to the high content of (poly)phenols. This study evaluated, for the first time, the inhibitory effect of a (poly)phenol-rich walnut extract on human carbohydrate digesting enzymes (salivary and pancreatic α-amylases, brush border sucrase-isomaltase) and on glucose transport across fully differentiated human intestinal Caco-2/TC7 monolayers. The walnut extract was rich in multiple (poly)phenols (70 % w/w) as analysed by Folin-Ciocalteau and by LCMS. It exhibited potent inhibition of both human salivary (IC50: 32.2 ± 2.5 µg walnut (poly)phenols (WP)/mL) and pancreatic (IC50: 56.7 ± 1.7 µg WP/mL) α-amylases, with weaker effects on human sucrase (IC50: 990 ± 20 µg WP/mL), maltase (IC50: 1300 ± 80 µg WP/mL), and isomaltase (IC25: 830 ± 60 µg WP/mL) activities. Selected individual walnut (poly)phenols inhibited human salivary α-amylase in the order: 1,3,4,6-tetragalloylglucose > ellagic acid pentoside > 1,2,6-tri-O-galloyl-β-D-glucopyranose, with no inhibition by ellagic acid, gallic acid and 4-O-methylgallic acid. The (poly)phenol-rich walnut extract also attenuated (up to 59 %) the transfer of 2-deoxy-D-glucose across differentiated Caco-2/TC7 cell monolayers. This is the first report on the effect of (poly)phenol-rich extracts from any commonly-consumed nut kernel on any human starch-digesting enzyme, and suggests a mechanism through which walnut consumption may lower postprandial glucose spikes and contribute to their proposed health benefits.
Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of ...melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.
Abstract
The aim of this set of randomised cross‐over studies was to determine the impact of progressive heat exposure and carbohydrate or protein feeding during exertional stress on small intestine ...permeability using a dual sugar test. In our previous work, and typically in the field, recovery of lactulose and
l
‐rhamnose is measured cumulatively in urine. This follow‐up study exploits our novel high‐performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD) protocol to accurately quantify the sugars in plasma. Endurance‐trained participants completed experimental trial A (ET‐A;
n
= 8), consisting of 2 h running at 60% in temperate, warm and hot ambient conditions, and/or experimental trial B (ET‐B;
n
= 9), consisting of 2 h running at 60% in the heat while consuming water, carbohydrate or protein. Blood samples were collected and plasma lactulose (L) and
l
‐rhamnose (R) appearance, after dual sugar solution ingestion at 90 min of exercise, was quantified by HPAEC‐PAD to measure plasma L/R and reveal new information about intestinal permeability immediately post‐exercise and during recovery. In ET‐A, plasma L/R increased immediately post‐exercise in hot compared with temperate and warm conditions, while, in ET‐B, carbohydrate alleviated this, and this information was otherwise missed when measuring urine L/R. Consuming carbohydrate or protein before and during exercise attenuated small intestine permeability throughout recovery from exertional heat stress. We recommend using the dual sugar test with quantification of plasma sugars by HPAEC‐PAD at intervals to maximise intestinal permeability data collection in exercise gastroenterology research, as this gives additional information compared to urinary measurements.
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Key points
Intestinal permeability is typically assessed using a dual sugar test, by administering a drink containing non‐metabolisable sugars (e.g. lactulose (L) and
l
‐rhamnose (R)) that can enter the circulation by paracellular translocation when the epithelium is compromised, and are subsequently measured in urine.
We demonstrate that our recently developed ion chromatography protocol can be used to accurately quantify the L/R ratio in plasma, and that measuring L/R in plasma collected at intervals during the post‐exercise recovery period reveals novel acute response information compared to measuring 5‐h cumulative urine L/R.
We confirm that exercising in hot ambient conditions increases intestinal epithelial permeability immediately after exercise, while consuming carbohydrate or protein immediately before and during exercise attenuates this.
We recommend using our dual sugar absorption test protocol to maximise intestinal epithelial permeability data collection in exercise gastroenterology research and beyond.
Circadian clocks regulate metabolic homeostasis. Disruption to our circadian clocks, by lifestyle behaviors such as timing of eating and sleeping, has been linked to increased rates of metabolic ...disorders. There is now considerable evidence that selected dietary (poly)phenols, including flavonoids, phenolic acids and tannins, may modulate metabolic and circadian processes. This review evaluates the effects of (poly)phenols on circadian clock genes and linked metabolic homeostasis in vitro, and potential mechanisms of action, by critically evaluating the literature on mammalian cells. A systematic search was conducted to ensure full coverage of the literature and identified 43 relevant studies addressing the effects of (poly)phenols on cellular circadian processes. Nobiletin and tangeretin, found in citrus, (–)-epigallocatechin-3-gallate from green tea, urolithin A, a gut microbial metabolite from ellagitannins in fruit, curcumin, bavachalcone, cinnamic acid, and resveratrol at low micromolar concentrations all affect circadian molecular processes in multiple types of synchronized cells. Nobiletin emerges as a putative retinoic acid–related orphan receptor (RORα/γ) agonist, leading to induction of the circadian regulator brain and muscle ARNT-like 1 (BMAL1), and increased period circadian regulator 2 (PER2) amplitude and period. These effects are clear despite substantial variations in the protocols employed, and this review suggests a methodological framework to help future study design in this emerging area of research.
P-glycoprotein (P-gp) plays a critical role in protection of the intestinal epithelia by mediating efflux of drugs/xenobiotics from the intestinal mucosa into the gut lumen. Recent studies bring to ...light that P-gp also confers a critical link in communication between intestinal mucosal barrier function and the innate immune system. Yet, despite knowledge for over 10 years that P-gp plays a central role in gastrointestinal homeostasis, the precise molecular mechanism that controls its functional expression and regulation remains unclear. Here, we assessed how the intestinal microbiome drives P-gp expression and function.
We have identified a "functional core" microbiome of the intestinal gut community, specifically genera within the Clostridia and Bacilli classes, that is necessary and sufficient for P-gp induction in the intestinal epithelium in mouse models. Metagenomic analysis of this core microbial community revealed that short-chain fatty acid and secondary bile acid production positively associate with P-gp expression. We have further shown these two classes of microbiota-derived metabolites synergistically upregulate P-gp expression and function in vitro and in vivo. Moreover, in patients suffering from ulcerative colitis (UC), we find diminished P-gp expression coupled to the reduction of epithelial-derived anti-inflammatory endocannabinoids and luminal content (e.g., microbes or their metabolites) with a reduced capability to induce P-gp expression.
Overall, by means of both in vitro and in vivo studies as well as human subject sample analysis, we identify a mechanistic link between cooperative functional outputs of the complex microbial community and modulation of P-gp, an epithelial component, that functions to suppress overactive inflammation to maintain intestinal homeostasis. Hence, our data support a new cross-talk paradigm in microbiome regulation of mucosal inflammation. Video abstract.
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