Plasma metabolic signatures of healthy overweight subjects challenged with an oral glucose tolerance test

Insulin secretion following ingestion of a carbohydrate load affects a multitude of metabolic pathways that simultaneously change direction and quantity of interorgan fluxes of sugars, lipids and amino acids. In the present study, we aimed at identifying markers associated with differential responses to an OGTT a population of healthy adults. By use of three metabolite profiling platforms, we assessed these postprandial responses of a total of 202 metabolites in plasma of 72 healthy volunteers undergoing comprehensive phenotyping and of which half enrolled into a weight-loss program over a three-month period. A standard oral glucose tolerance test (OGTT) served as dietary challenge test to identify changes in postprandial metabolite profiles. Despite classified as healthy according to WHO criteria, two discrete clusters (A and B) were identified based on the postprandial glucose profiles with a balanced distribution of volunteers based on gender and other measures. Cluster A individuals displayed 26% higher postprandial glucose levels, delayed glucose clearance and increased fasting plasma concentrations of more than 20 known biomarkers of insulin resistance and diabetes previously identified in large cohort studies. The volunteers identified by canonical postprandial responses that form cluster A may be called pre-pre-diabetics and defined as “at risk” for development of insulin resistance. Moreover, postprandial changes in selected fatty acids and complex lipids, bile acids, amino acids, acylcarnitines and sugars like mannose revealed marked differences in the responses seen in cluster A and cluster B individuals that sustained over the entire challenge test period of 240 min. Almost all metabolites, including glucose and insulin, returned to baseline values within this timeframe, except a variety of amino acids and here those that have been linked to diabetes development. Analysis of the corresponding metabolite profile in a fasting blood sample may therefore allow for early identification of these subjects at risk for insulin resistance without the need to undergo an OGTT

Association between diet-quality scores, adiposity, total cholesterol and markers of nutritional status in European adults: findings from the Food4Me study

Diet-quality scores (DQS), which are developed across the globe, are used to define adherence to specific eating patterns and have been associated with risk of coronary heart disease and type-II diabetes. We explored the association between five diet-quality scores (Healthy Eating Index, HEI; Alternate Healthy Eating Index, AHEI; MedDietScore, MDS; PREDIMED Mediterranean Diet Score, P-MDS; Dutch Healthy Diet-Index, DHDI) and markers of metabolic health (anthropometry, objective physical activity levels (PAL), and dried blood spot total cholesterol (TC), total carotenoids, and omega-3 index) in the Food4Me cohort, using regression analysis. Dietary intake was assessed using a validated Food Frequency Questionnaire. Participants (n = 1480) were adults recruited from seven European Union (EU) countries. Overall, women had higher HEI and AHEI than men (p < 0.05), and scores varied significantly between countries. For all DQS, higher scores were associated with lower body mass index, lower waist-to-height ratio and waist circumference, and higher total carotenoids and omega-3-index (p trends < 0.05). Higher HEI, AHEI, DHDI, and P-MDS scores were associated with increased daily PAL, moderate and vigorous activity, and reduced sedentary behaviour (p trend < 0.05). We observed no association between DQS and TC. To conclude, higher DQS, which reflect better dietary patterns, were associated with markers of better nutritional status and metabolic health

Mediterranean Diet Adherence and Genetic Background Roles within a Web-Based Nutritional Intervention: The Food4Me Study

Mediterranean Diet (MedDiet) adherence has been proven to produce numerous health benefits. In addition, nutrigenetic studies have explained some individual variations in the response to specific dietary patterns. The present research aimed to explore associations and potential interactions between MedDiet adherence and genetic background throughout the Food4Me web-based nutritional intervention. Dietary, anthropometrical and biochemical data from volunteers of the Food4Me study were collected at baseline and after 6 months. Several genetic variants related to metabolic risk features were also analysed. A Genetic Risk Score (GRS) was derived from risk alleles and a Mediterranean Diet Score (MDS), based on validated food intake data, was estimated. At baseline, there were no interactions between GRS and MDS categories for metabolic traits. Linear mixed model repeated measures analyses showed a significantly greater decrease in total cholesterol in participants with a low GRS after a 6-month period, compared to those with a high GRS. Meanwhile, a high baseline MDS was associated with greater decreases in Body Mass Index (BMI), waist circumference and glucose. There also was a significant interaction between GRS and the MedDiet after the follow-up period. Among subjects with a high GRS, those with a high MDS evidenced a highly significant reduction in total carotenoids, while among those with a low GRS, there was no difference associated with MDS levels. These results suggest that a higher MedDiet adherence induces beneficial effects on metabolic outcomes, which can be affected by the genetic background in some specific markers

Quantitative and qualitative analysis of retinoids in <i>Artemia</i> and copepods by HPLC and diode array detection

Different concentrations of retinoids have been reported in Artemia. The levels vary from 0-362 µg g-1 Artemia (dry wt.). The aim of this study was to investigate the actual retinoid concentration in Artemia and compare it with levels in copepods. Samples of both DC-DHA Selco (INVE, Ghent, Belgium) from the bottle and after 12 h in the enrichment tank in addition to enriched and unenriched Artemia and copepods from a pond system were collected and analysed for vitamin A components. The analyses were conducted using two different HPLC (high pressure liquid chromatography) methods to ensure the justness of the results. The DC-DHA Selco contained 132.7 µg vitamin A g-1. After 12 h in the enrichment tank we could not detect any vitamin A. None of the following retinoids were observed in Artemia or copepods with any of the methods: all-trans-retinol (at-ROH), 9-cis-ROH or at-3,4-didehydro-ROH (at-3,4-dd-ROH). Method 2 detects several other retinoids when present, but none of the following were found in the two live prey organisms: cis or trans 3,4-dd-RA, cis or trans 3,4-dd-ROH, cis or trans 3,4-dd-RAL, cis or trans ROH or cis or trans RA. This implies that larvae feeding on either copepods or Artemia must meet their retinoid requirement from other sources than retinoids

T cell homing to tumors detected by 3D-coordinated positron emission tomography and magnetic resonance imaging

A general hindrance to progress in adoptive cellular therapy is the lack of detailed knowledge of the fate of transferred cells in the body of the recipient. In this study, we present a novel technique for tracking of I-labeled cells in situ, which combines the high spatial resolution of magnetic resonance imaging with the high sensitivity and spatial accuracy of positron emission tomography. We have used this technique, together with determination of tissue radioactivity, flow cytometry, and microscopy, to characterize and quantitate the specific accumulation of transferred CD8 T cells in tumor tissue in a mouse model. Transgenic CD8 T cells, specific for the ovalbumin peptide SIINFEKL, were adoptively transferred to recipients carrying a subcutaneous tumor of the ovalbumin-expressing malignant melanoma cell line B16-OVA. The number of SIINFEKL-specific CD8 cells in the tumor tissue was determined by flow cytometry each day for 8 consecutive days after adoptive transfer. From low levels 1 day after injection, their number gradually increased until day 5 when an average of 3.3x10 SIINFEKL-specific cells per gram tumor tissue was found. By applying the combined positron emission tomography/magnetic resonance imaging technique we were able to determine the position of the transferred, I-labeled SIINFEKL-specific T cells in 3 dimensions in recipient mice, and could demonstrate a highly significant accumulation of the I label in and around the subcutaneous B16-OVA tumors compared with normal tissue. Accumulation of I was significantly higher in B16-OVA than in B16 tumors not expressing the OVA antigen

Diet-induced weight loss and phenotypic flexibility among healthy overweight adults: a randomized trial

Background: The capacity of an individual to respond to change in food intake so that postprandial metabolic perturbations are resolved, and metabolism returns to its pre-prandial state, is called phenotypic flexibility. This ability may relate to health status better than metabolic markers in a fasting state. Aims: In this parallel randomized controlled trial study, an energy-restricted healthy diet and two dietary challenges were used to assess the effect of weight loss on phenotypic flexibility. Methods: 72 overweight volunteers underwent a 12-week dietary intervention. The participants were randomized to a weight loss group (WLG) with 20% less energy or a weight maintenance group (WMG). At weeks 1 and 12, participants were assessed for body composition by magnetic resonance imaging (MRI) and quantification of markers of metabolism and insulin sensitivity by analyses of the plasma metabolome during two different dietary challenges - an oral glucose tolerance test (OGTT), and a mixed meal tolerance test (MMTT). Results: Intended weight loss was achieved in the WLG (-5.6 kg, p<0.0001) and induced significant reduction in total and regional adipose tissue as well as ectopic fat in the liver. Amino acid-based markers of insulin action and resistance such as leucine and glutamate, were reduced in the post prandial phase of the OGTT in the WLG by 11.5% and 28%, respectively, after body weight reduction. Weight loss correlated with the magnitude of changes in metabolic responses to the dietary challenges. Large inter-individual variation in metabolic responses to the weight loss was observed. Conclusion: Application of dietary challenges increased sensitivity to detect metabolic response to weight loss intervention. Large inter-individual variation was observed across a wide range of measurements allowing the identification of distinct responses to the weight loss intervention, and mechanistic insight into the metabolic response to weight loss

Export from Pericentriolar Endocytic Recycling Compartment to Cell Surface Depends on Stable, Detyrosinated (Glu) Microtubules and Kinesin

A significant fraction of internalized transferrin (Tf) concentrates in the endocytic recycling compartment (ERC), which is near the microtubule-organizing center in many cell types. Tf then recycles back to the cell surface. The mechanisms controlling the localization, morphology, and function of the ERC are not fully understood. We examined the relationship of Tf trafficking with microtubules (MTs), specifically the subset of stable, detyrosinated Glu MTs. We found some correlation between the level of stable Glu MTs and the distribution of the ERC; in cells with low levels of Glu MTs concentrated near to the centriole, the ERC was often tightly clustered, whereas in cells with higher levels of Glu MTs throughout the cell, the ERC was more dispersed. The clustered ERC in Chinese hamster ovary cells became dispersed when the level of Glu MTs was increased with taxol treatment. Furthermore, in a temperature-sensitive Chinese hamster ovary cell line (B104-5), the cells had more Glu MTs when the ERC became dispersed at elevated temperature. Microinjecting purified anti-Glu tubulin antibody into B104-5 cells at elevated temperature induced the redistribution of the ERC to a tight cluster. Microinjection of anti-Glu tubulin antibody slowed recycling of Tf to the cell surface without affecting Tf internalization or delivery to the ERC. Similar inhibition of Tf recycling was caused by microinjecting anti-kinesin antibody. These results suggest that stable Glu MTs and kinesin play a role in the organization of the ERC and in facilitating movement of vesicles from the ERC to the cell surface

Microtubule Asymmetry during Neutrophil Polarization and Migration

The development of cell polarity in response to chemoattractant stimulation in human polymorphonuclear neutrophils (PMNs) is characterized by the rapid conversion from round to polarized morphology with a leading lamellipod at the front and a uropod at the rear. During PMN polarization, the microtubule (MT) array undergoes a dramatic reorientation toward the uropod that is maintained during motility and does not require large-scale MT disassembly or cell adhesion to the substratum. MTs are excluded from the leading lamella during polarization and motility, but treatment with a myosin light chain kinase inhibitor (ML-7) or the actin-disrupting drug cytochalasin D causes an expansion of the MT array and penetration of MTs into the lamellipod. Depolymerization of the MT array before stimulation caused 10% of the cells to lose their polarity by extending two opposing lateral lamellipodia. These multipolar cells showed altered localization of a leading lamella-specific marker, talin, and a uropod-specific marker, CD44. In summary, these results indicate that F-actin– and myosin II-dependent forces lead to the development and maintenance of MT asymmetry that may act to reinforce cell polarity during PMN migration

Centrosome Reorientation in Wound-Edge Cells Is Cell Type Specific

The reorientation of the microtubule organizing center during cell migration into a wound in the monolayer was directly observed in living wound-edge cells expressing γ-tubulin tagged with green fluorescent protein. Our results demonstrate that in CHO cells, the centrosome reorients to a position in front of the nucleus, toward the wound edge, whereas in PtK cells, the centrosome lags behind the nucleus during migration into the wound. In CHO cells, the average rate of centrosome motion was faster than that of the nucleus; the converse was true in PtK cells. In both cell lines, centrosome motion was stochastic, with periods of rapid motion interspersed with periods of slower motion. Centrosome reorientation in CHO cells required dynamic microtubules and cytoplasmic dynein/dynactin activity and could be prevented by altering cell-to-cell or cell-to-substrate adhesion. Microtubule marking experiments using photoactivation of caged tubulin demonstrate that microtubules are transported in the direction of cell motility in both cell lines but that in PtK cells, microtubules move individually, whereas their movement is more coherent in CHO cells. Our data demonstrate that centrosome reorientation is not required for directed migration and that diverse cells use distinct mechanisms for remodeling the microtubule array during directed migration