Effect of Different Nutritional Supplements on Glucose Response of Complete Meals in Two Crossover Studies.

Postprandial hyperglycemia is an important risk factor in the development and progression of type-2 diabetes and cardiometabolic diseases. Therefore, maintaining a low postprandial glucose response is key in preventing these diseases. Carbohydrate-rich meals are the main drivers of excessive glycemic excursions during the day. The consumption of whey protein premeals or mulberry leaf extract was reported to reduce postprandial glycemia through different mechanisms of action. The efficacy of these interventions was shown to be affected by the timing of the consumption or product characteristics. Two randomised crossover studies were performed, aiming to identify the optimal conditions to improve the efficacy of these nutritional supplements in reducing a glycemic response. The acute postprandial glycemic response was monitored with a continuous glucose monitoring device. The first study revealed that a preparation featuring 10 g of whey protein microgel reduced the postprandial glucose response by up to 30% (p = 0.001) and was more efficient than the whey protein isolates, independently of whether the preparation was ingested 30 or 10 min before a complete 320 kcal breakfast. The second study revealed that a preparation featuring 250 mg mulberry leaf extract was more efficient if it was taken together with a complete 510 kcal meal (−34%, p < 0.001) rather than ingested 5 min before (−26%, p = 0.002). These findings demonstrate that the efficacy of whey proteins premeal and mulberry leaf extracts can be optimised to provide potential nutritional solutions to lower the risk of type-2 diabetes or its complications.

Proteomics reveals unique plasma signatures in constitutional thinness.

PURPOSE: Studying the plasma proteome of control versus constitutionally thin (CT) individuals, exposed to overfeeding, may give insights into weight-gain management, providing relevant information to the clinical entity of weight-gain resistant CT, and discovering new markers for the condition. EXPERIMENTAL DESIGN: Untargeted protein relative quantification of 63 CT and normal-weight individuals was obtained in blood plasma at baseline, during and after an overfeeding challenge using mass spectrometry-based proteomics. RESULTS: The plasma proteome of CT subjects presented limited specificity with respect to controls at baseline. Yet, CT showed lower levels of inflammatory C-reactive protein and larger levels of protective insulin-like growth factor-binding protein 2. Differences were more marked during and after overfeeding. CT plasma proteome showed larger magnitude and significance in response, suggesting enhanced "resilience" and more rapid adaptation to changes. Four proteins behaved similarly between CT and controls, while five were regulated in opposite fashion. Ten proteins were differential during overfeeding in CT only (including increased fatty acid-binding protein and glyceraldehyde-3-phosphate dehydrogenase, and decreased apolipoprotein C-II and transferrin receptor protein 1). CONCLUSIONS AND CLINICAL RELEVANCE: This first proteomic profiling of a CT cohort reveals different plasma proteomes between CT subjects and controls in a longitudinal clinical trial. Our molecular observations further support that the resistance to weight gain in CT subjects appears predominantly biological. CLINICALTRIALS: gov Identifier: NCT02004821.

Identification of ALK in Thinness.

There is considerable inter-individual variability in susceptibility to weight gain despite an equally obesogenic environment in large parts of the world. Whereas many studies have focused on identifying the genetic susceptibility to obesity, we performed a GWAS on metabolically healthy thin individuals (lowest 6th percentile of the population-wide BMI spectrum) in a uniquely phenotyped Estonian cohort. We discovered anaplastic lymphoma kinase (ALK) as a candidate thinness gene. In Drosophila, RNAi mediated knockdown of Alk led to decreased triglyceride levels. In mice, genetic deletion of Alk resulted in thin animals with marked resistance to diet- and leptin-mutation-induced obesity. Mechanistically, we found that ALK expression in hypothalamic neurons controls energy expenditure via sympathetic control of adipose tissue lipolysis. Our genetic and mechanistic experiments identify ALK as a thinness gene, which is involved in the resistance to weight gain.

Resistance to lean mass gain in constitutional thinness in free-living conditions is not overpassed by overfeeding.

BACKGROUND: Constitutional thinness (CT), a non-malnourished underweight state with no eating disorders, is characterized by weight gain resistance to high fat diet. Data issued from muscle biopsies suggested blunted anabolic mechanisms in free-living state. Weight and metabolic responses to protein caloric supplementation has not been yet explored in CT. METHODS: A 2 week overfeeding (additional 600 kcal, 30 g protein, 72 g carbohydrate, and 21 g fat) was performed to compare two groups of CTs (12 women and 11 men) to normal-weight controls (12 women and 10 men). Bodyweight, food intake, energy expenditure, body composition, nitrogen balance, appetite hormones profiles, and urine metabolome were monitored before and after overfeeding. RESULTS: Before overfeeding, positive energy gap was found in both CT genders (309 ± 370 kcal in CT-F and 332 ± 709 kcal in CT-M) associated with higher relative protein intake per kilo (1.74 ± 0.32 g/kg/day in CT-F vs. 1.16 ± 0.23 in C-F, P < 0.0001; 1.56 ± 0.36 in CT-M vs. 1.22 ± 0.32 in C-M, P = 0.03), lower nitrogen (7.26 ± 2.36 g/day in CT-F vs. 11.41 ± 3.64 in C-F, P = 0.003; 9.70 ± 3.85 in CT-M vs. 14.14 ± 4.19 in C-M, P = 0.02), but higher essential amino acids urinary excretion (CT/C fold change of 1.13 for leucine and 1.14 for arginine) in free-living conditions. After overfeeding, CTs presented an accentuated positive energy gap, still higher than in controls (675 ± 540 in CTs vs. 379 ± 427 in C, P = 0.04). Increase in lean mass was induced in both controls genders but not in CTs (a trend was noticed in CT women), despite a similar nitrogen balance after overfeeding (5.06 ± 4.33 g/day in CTs vs. 4.28 ± 3.15 in controls, P = 0.49). Higher anorectic gut hormones' tone, glucagon-like peptide 1 and peptide tyrosine tyrosine, during test meal and higher snacking frequency were noticed before and after overfeeding in CTs. CONCLUSIONS: The blunted muscle energy mechanism, previously described in CTs in free-living state, is associated with basal saturated protein turn over suggested by the concordance of positive nitrogen balance and an increased urine excretion of several essential amino acids. This saturation cannot be overpassed by increasing this spontaneous high-protein intake suggesting a resistance to lean mass gain in CT phenotype.

Persistent low body weight in humans is associated with higher mitochondrial activity in white adipose tissue.

BACKGROUND: Constitutional thinness (CT) is a state of low but stable body weight (BMI ≤18 kg/m2). CT subjects have normal-range hormonal profiles and food intake but exhibit resistance to weight gain despite living in the modern world's obesogenic environment. OBJECTIVE: The goal of this study is to identify molecular mechanisms underlying this protective phenotype against weight gain. METHODS: We conducted a clinical overfeeding study on 30 CT subjects and 30 controls (BMI 20-25 kg/m2) matched for age and sex. We performed clinical and integrative molecular and transcriptomic analyses on white adipose and muscle tissues. RESULTS: Our results demonstrate that adipocytes were markedly smaller in CT individuals (mean ± SEM: 2174 ± 142 µm 2) compared with controls (3586 ± 216 µm2) (P < 0.01). The mitochondrial respiratory capacity was higher in CT adipose tissue, particularly at the level of complex II of the electron transport chain (2.2-fold increase; P < 0.01). This higher activity was paralleled by an increase in mitochondrial number (CT compared with control: 784 ± 27 compared with 675 ± 30 mitochondrial DNA molecules per cell; P < 0.05). No evidence for uncoupled respiration or "browning" of the white adipose tissue was found. In accordance with the mitochondrial differences, CT subjects had a distinct adipose transcriptomic profile [62 differentially expressed genes (false discovery rate of 0.1 and log fold change >0.75)], with many differentially expressed genes associating with positive metabolic outcomes. Pathway analyses revealed an increase in fatty acid oxidation ( P = 3 × 10-04) but also triglyceride biosynthesis (P = 3.6 × 10-04). No differential response to the overfeeding was observed in the 2 groups. CONCLUSIONS: The distinct molecular signature of the adipose tissue in CT individuals suggests the presence of augm ented futile lipid cycling, rather than mitochondrial uncoupling, as a way to increase energy expenditure in CT individuals. We propose that increased mitochondrial function in adipose tissue is an important mediator in sustaining the low body weight in CT individuals. This knowledge could ultimately allow more targeted approaches for weight management treatment strategies. This trial was registered at clinicaltrials.gov as NCT02004821.

Genome-wide gene-based analyses of weight loss interventions identify a potential role for NKX6.3 in metabolism.

Hundreds of genetic variants have been associated with Body Mass Index (BMI) through genome-wide association studies (GWAS) using observational cohorts. However, the genetic contribution to efficient weight loss in response to dietary intervention remains unknown. We perform a GWAS in two large low-caloric diet intervention cohorts of obese participants. Two loci close to NKX6.3/MIR486 and RBSG4 are identified in the Canadian discovery cohort (n = 1166) and replicated in the DiOGenes cohort (n = 789). Modulation of HGTX (NKX6.3 ortholog) levels in Drosophila melanogaster leads to significantly altered triglyceride levels. Additional tissue-specific experiments demonstrate an action through the oenocytes, fly hepatocyte-like cells that regulate lipid metabolism. Our results identify genetic variants associated with the efficacy of weight loss in obese subjects and identify a role for NKX6.3 in lipid metabolism, and thereby possibly weight control.

Transcriptome profiling from adipose tissue during a low-calorie diet reveals predictors of weight and glycemic outcomes in obese, nondiabetic subjects.

Background: A low-calorie diet (LCD) reduces fat mass excess, improves insulin sensitivity, and alters adipose tissue (AT) gene expression, yet the relation with clinical outcomes remains unclear.Objective: We evaluated AT transcriptome alterations during an LCD and the association with weight and glycemic outcomes both at LCD termination and 6 mo after the LCD.Design: Using RNA sequencing (RNAseq), we analyzed transcriptome changes in AT from 191 obese, nondiabetic patients within a multicenter, controlled dietary intervention. Expression changes were associated with outcomes after an 8-wk LCD (800-1000 kcal/d) and 6 mo after the LCD. Results were validated by using quantitative reverse transcriptase-polymerase chain reaction in 350 subjects from the same cohort. Statistical models were constructed to classify weight maintainers or glycemic improvers.Results: With RNAseq analyses, we identified 1173 genes that were differentially expressed after the LCD, of which 350 and 33 were associated with changes in body mass index (BMI; in kg/m2) and Matsuda index values, respectively, whereas 29 genes were associated with both endpoints. Pathway analyses highlighted enrichment in lipid and glucose metabolism. Classification models were constructed to identify weight maintainers. A model based on clinical baseline variables could not achieve any classification (validation AUC: 0.50; 95% CI: 0.36, 0.64). However, clinical changes during the LCD yielded better performance of the model (AUC: 0.73; 95% CI: 0.60, 0.87]). Adding baseline expression to this model improved the performance significantly (AUC: 0.87; 95% CI: 0.77, 0.96; Delong's P = 0.012). Similar analyses were performed to classify subjects with good glycemic improvements. Baseline- and LCD-based clinical models yielded similar performance (best AUC: 0.73; 95% CI: 0.60, 0.86). The addition of expression changes during the LCD improved the performance substantially (AUC: 0.80; 95% CI: 0.69, 0.92; P = 0.058).Conclusions: This study investigated AT transcriptome alterations after an LCD in a large cohort of obese, nondiabetic patients. Gene expression combined with clinical variables enabled us to distinguish weight and glycemic responders from nonresponders. These potential biomarkers may help clinicians understand intersubject variability and better predict the success of dietary interventions. This trial was registered at clinicaltrials.gov as NCT00390637.

Role of sulfotransferases in resveratrol metabolism in human adipocytes.

SCOPE: Polyphenols such as resveratrol received interest for their wide-ranging biological benefits, including anti-obesity potential, mimicking effects of caloric restriction with reduced body fat and increased energy expenditure. However, resveratrol is rapidly metabolized, and it is not completely understood which form of resveratrol is responsible for the effects observed within target cells such as adipocytes. Also the role of metabolizing enzymes has not been investigated before. METHODS AND RESULTS: Resveratrol metabolism was evaluated in human adipocytes by UHPLC-MS at low physiological doses. Resveratrol was found to rapidly metabolize into its sulfated form, while resveratrol glucuronides were undetectable. Only resveratrol, but not its sulfated nor glucuronidated forms had an antilipolytic effect on adipocytes. The metabolizing enzyme responsible for sulfation of polyphenols is SULT1A1, and was found to be upregulated upon adipocyte differentiation. Knocking down SULT1A1 in adipocytes led to decreased resveratrol sulfate and increased resveratrol intra- and extracellularly. This lower SULT1A1 activity resulted in an increased antilipolytic effect of resveratrol on adipocytes, as demonstrated by lower glycerol accumulation, which could be attributed to lower activity of the lipolytic protein, perilipin. CONCLUSION: Sulfotransferase activity modulates metabolism of resveratrol in adipocytes with potential consequences on bioavailability and thus metabolic action of this polyphenol.

Structural variation-associated expression changes are paralleled by chromatin architecture modifications.

Copy number variants (CNVs) influence the expression of genes that map not only within the rearrangement, but also to its flanks. To assess the possible mechanism(s) underlying this "neighboring effect", we compared intrachromosomal interactions and histone modifications in cell lines of patients affected by genomic disorders and control individuals. Using chromosome conformation capture (4C-seq), we observed that a set of genes flanking the Williams-Beuren Syndrome critical region (WBSCR) were often looping together. The newly identified interacting genes include AUTS2, mutations of which are associated with autism and intellectual disabilities. Deletion of the WBSCR disrupts the expression of this group of flanking genes, as well as long-range interactions between them and the rearranged interval. We also pinpointed concomitant changes in histone modifications between samples. We conclude that large genomic rearrangements can lead to chromatin conformation changes that extend far away from the structural variant, thereby possibly modulating expression globally and modifying the phenotype. GEO SERIES ACCESSION NUMBER: GSE33784, GSE33867.

Detecting long-range chromatin interactions using the chromosome conformation capture sequencing (4C-seq) method.

Eukaryotic transcription is tightly regulated by transcriptional regulatory elements, even though these elements may be located far away from their target genes. It is now widely recognized that these regulatory elements can be brought in close proximity through the formation of chromatin loops, and that these loops are crucial for transcriptional regulation of their target genes. The chromosome conformation capture (3C) technique presents a snapshot of long-range interactions, by fixing physically interacting elements with formaldehyde, digestion of the DNA, and ligation to obtain a library of unique ligation products. Recently, several large-scale modifications to the 3C technique have been presented. Here, we describe chromosome conformation capture sequencing (4C-seq), a high-throughput version of the 3C technique that combines the 3C-on-chip (4C) protocol with next-generation Illumina sequencing. The method is presented for use in mammalian cell lines, but can be adapted to use in mammalian tissues and any other eukaryotic genome.

Phenotypic consequences of copy number variation: insights from Smith-Magenis and Potocki-Lupski syndrome mouse models.

A large fraction of genome variation between individuals is comprised of submicroscopic copy number variation of genomic DNA segments. We assessed the relative contribution of structural changes and gene dosage alterations on phenotypic outcomes with mouse models of Smith-Magenis and Potocki-Lupski syndromes. We phenotyped mice with 1n (Deletion/+), 2n (+/+), 3n (Duplication/+), and balanced 2n compound heterozygous (Deletion/Duplication) copies of the same region. Parallel to the observations made in humans, such variation in gene copy number was sufficient to generate phenotypic consequences: in a number of cases diametrically opposing phenotypes were associated with gain versus loss of gene content. Surprisingly, some neurobehavioral traits were not rescued by restoration of the normal gene copy number. Transcriptome profiling showed that a highly significant propensity of transcriptional changes map to the engineered interval in the five assessed tissues. A statistically significant overrepresentation of the genes mapping to the entire length of the engineered chromosome was also found in the top-ranked differentially expressed genes in the mice containing rearranged chromosomes, regardless of the nature of the rearrangement, an observation robust across different cell lineages of the central nervous system. Our data indicate that a structural change at a given position of the human genome may affect not only locus and adjacent gene expression but also "genome regulation." Furthermore, structural change can cause the same perturbation in particular pathways regardless of gene dosage. Thus, the presence of a genomic structural change, as well as gene dosage imbalance, contributes to the ultimate phenotype.

Chemical genetic strategy identifies histone deacetylase 1 (HDAC1) and HDAC2 as therapeutic targets in sickle cell disease.

The worldwide burden of sickle cell disease is enormous, with over 200,000 infants born with the disease each year in Africa alone. Induction of fetal hemoglobin is a validated strategy to improve symptoms and complications of this disease. The development of targeted therapies has been limited by the absence of discrete druggable targets. We developed a unique bead-based strategy for the identification of inducers of fetal hemoglobin transcripts in primary human erythroid cells. A small-molecule screen of bioactive compounds identified remarkable class-associated activity among histone deacetylase (HDAC) inhibitors. Using a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics by RNA interference, we have identified HDAC1 and HDAC2 as molecular targets mediating fetal hemoglobin induction. Our findings suggest the potential of isoform-selective inhibitors of HDAC1 and HDAC2 for the treatment of sickle cell disease.

Cell-type-specific long-range looping interactions identify distant regulatory elements of the CFTR gene.

Identification of regulatory elements and their target genes is complicated by the fact that regulatory elements can act over large genomic distances. Identification of long-range acting elements is particularly important in the case of disease genes as mutations in these elements can result in human disease. It is becoming increasingly clear that long-range control of gene expression is facilitated by chromatin looping interactions. These interactions can be detected by chromosome conformation capture (3C). Here, we employed 3C as a discovery tool for identification of long-range regulatory elements that control the cystic fibrosis transmembrane conductance regulator gene, CFTR. We identified four elements in a 460-kb region around the locus that loop specifically to the CFTR promoter exclusively in CFTR expressing cells. The elements are located 20 and 80 kb upstream; and 109 and 203 kb downstream of the CFTR promoter. These elements contain DNase I hypersensitive sites and histone modification patterns characteristic of enhancers. The elements also interact with each other and the latter two activate the CFTR promoter synergistically in reporter assays. Our results reveal novel long-range acting elements that control expression of CFTR and suggest that 3C-based approaches can be used for discovery of novel regulatory elements.

The active FMR1 promoter is associated with a large domain of altered chromatin conformation with embedded local histone modifications.

We have analyzed the effects of gene activation on chromatin conformation throughout an approximately 170-kb region comprising the human fragile X locus, which includes a single expressed gene, FMR1 (fragile X mental retardation 1). We have applied three approaches: (i) chromosome conformation capture, which assesses relative interaction frequencies of chromatin segments; (ii) an extension of this approach that identifies domains whose conformation differs from the average, which we developed and named chromosome conformation profiling; and (iii) ChIP analysis of histone modifications. We find that, in normal cells where FMR1 is active, the FMR1 promoter is at the center of a large ( approximately 50 kb) domain of reduced intersegment interactions. In contrast, in fragile X cells where FMR1 is inactive, chromatin conformation is uniform across the entire region. We also find that histone modifications that are characteristic of active genes occur tightly localized around the FMR1 promoter in normal cells and are absent in fragile X cells. Therefore, the expression-correlated change in conformation affects a significantly larger domain than that marked by histone modifications. Domain-wide changes in interaction probability could reflect increased chromatin expansion and may also be related to an altered spatial disposition that results in increased intermingling with unrelated loci. The described approaches are widely applicable to the study of conformational changes of any locus of interest.

Mapping chromatin interactions by chromosome conformation capture.

Chromosome conformation capture (3C) is one of the only techniques that allows for analysis of an intermediate level of chromosome structure ranging from a few to hundreds of kilobases, a level most relevant for gene regulation. The 3C technique is used to detect physical interactions between sequence elements that are located on the same or on different chromosomes. For instance, physical interactions between distant enhancers and target genes can be measured. The 3C assay uses formaldehyde cross-linking to trap connections between chromatin segments that can, after a number of manipulations, be detected by PCR. This unit describes detailed protocols for performing 3C with yeast Saccharomyces cerevisiae and mammalian cells.

Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1.

Recent evidence suggests that long-range enhancers and gene promoters are in close proximity, which might reflect the formation of chromatin loops. Here, we examined the mechanism for DNA looping at the beta-globin locus. By using chromosome conformation capture (3C), we show that the hematopoietic transcription factor GATA-1 and its cofactor FOG-1 are required for the physical interaction between the beta-globin locus control region (LCR) and the beta-major globin promoter. Kinetic studies reveal that GATA-1-induced loop formation correlates with the onset of beta-globin transcription and occurs independently of new protein synthesis. GATA-1 occupies the beta-major globin promoter normally in fetal liver erythroblasts from mice lacking the LCR, suggesting that GATA-1 binding to the promoter and LCR are independent events that occur prior to loop formation. Together, these data demonstrate that GATA-1 and FOG-1 are essential anchors for a tissue-specific chromatin loop, providing general insights into long-range enhancer function.

Buckwheat honey increases serum antioxidant capacity in humans.

Honey has been known to exert significant in vitro antioxidant activity, in part due to its phenolic content. However, conclusions that the antioxidants in honey are or are not efficacious in the human body cannot be reached if its antioxidant action is not assessed as part of a human study. In the present study, the acute effect of consumption of 500 mL of water, water with buckwheat honey, black tea, black tea with sugar, or black tea with buckwheat honey on serum oxidative reactions was examined in 25 healthy men. Antioxidant capacity of human serum samples was measured using different methods: the oxygen radical absorbance capacity (ORAC) assay, ex vivo susceptibility of serum lipoprotein to Cu(2+)-induced oxidation, and the thiobarbituric acid reactive substances (TBARS) assay. The results showed that the serum antioxidant capacity determined by ORAC increased significantly (p < 0.05) by 7% following consumption of buckwheat honey in water. No significant changes in serum antioxidant capacity could be established after the consumption of any of the other beverages. Ex vivo serum lipoprotein oxidation and TBARS values were not significantly altered after consumption of any of the five beverages. This study provides primary evidence of the in vivo antioxidant activity of buckwheat honey. However, long-term studies and epidemiological data are necessary to investigate whether honey consumption can exert overall antioxidant-related health benefits.

Identification and quantification of antioxidant components of honeys from various floral sources.

Little is known about the individual components of honey that are responsible for its antioxidant activity. The present study was carried out to characterize the phenolics and other antioxidants present in honeys from seven floral sources. Chromatograms of the phenolic nonpolar fraction of the honeys indicated that most honeys have similar but quantitatively different phenolic profiles. Many of the flavonoids and phenolic acids identified have been previously described as potent antioxidants. A linear correlation between phenolic content and ORAC activity was demonstrated (R(2) = 0.963, p < 0.0001). Honeys were separated by solid-phase extraction into four fractions for sugar removal and separation based on solubility to identify the relative contribution of each fraction to the antioxidant activity of honey. Antioxidant analysis of the different honey fractions suggested that the water-soluble fraction contained most of the antioxidant components. Specific water-soluble antioxidant components were quantified, including protein; gluconic acid; ascorbic acid; hydroxymethylfuraldehyde; and the combined activities of the enzymes glucose oxidase, catalase and peroxidase. Of these components, a significant correlation could be established only between protein content and ORAC activity (R(2) = 0.674, p = 0.024). In general, the antioxidant capacity of honey appeared to be a result of the combined activity of a wide range of compounds including phenolics, peptides, organic acids, enzymes, Maillard reaction products, and possibly other minor components. The phenolic compounds contributed significantly to the antioxidant capacity of honey but were not solely responsible for it.

Antioxidant capacity of honeys from various floral sources based on the determination of oxygen radical absorbance capacity and inhibition of in vitro lipoprotein oxidation in human serum samples.

Honeys from seven different floral sources were analyzed for in vitro antioxidant capacity and total phenolic content. Antioxidant capacity was measured by the oxygen radical absorbance capacity (ORAC) assay and by monitoring the formation of conjugated dienes as an index of the inhibition of copper-catalyzed serum lipoprotein oxidation. ORAC values ranged from 3.1 to 16.3 micromol Trolox equivalent/g honey. The darkest colored honeys, such as buckwheat honey, had the highest ORAC values. A linear correlation was observed between phenolic content and ORAC activity of the investigated honeys (p < 0.0001, R (2) = 0.9497). The relationship between the ORAC activity and inhibition of lipoprotein oxidation by the honeys yielded a correlation coefficient of 0.6653 (p = 0.0136). This work shows that honey may be used as a healthy alternative to sugar in many products and thereby serve as a source of dietary antioxidants.