Analysis of polyphenolic metabolites from in vitro gastrointestinal digested soft fruit extracts identify malvidin-3-glucoside as an inhibitor of PTP1B.

Protein-tyrosine phosphatase 1B (PTP1B, EC 3.1.3.48) is an important regulator of insulin signalling. Herein, we employed experimental and computational biology techniques to investigate the inhibitory properties of phenolics, identified from four in vitro gastrointestinal digested (IVGD) soft fruits, on PTP1B. Analysis by LC-MS/MS identified specific phenolics that inhibited PTP1B in vitro. Enzyme kinetics identified the mode of inhibition, while dynamics, stability and binding mechanisms of PTP1B-ligand complex were investigated through molecular modelling, docking, molecular dynamics (MD) simulations, and MM/PBSA binding free energy estimation. IVGD extracts and specific phenolics identified from the four soft fruits inhibited PTP1B (P < 0.0001) activity. Among the phenolics tested, the greatest inhibition was shown by malvidin-3-glucoside (P < 0.0001) and gallic acid (P < 0.0001). Malvidin-3-glucoside (Ki = 3.8 µg/mL) was a competitive inhibitor and gallic acid (Ki = 33.3 µg/mL) a non-competitive inhibitor of PTP1B. Malvidin-3-glucoside exhibited better binding energy than gallic acid and the synthetic inhibitor Dephostatin (-7.38 > -6.37 > -5.62 kcal/mol) respectively. Principal component analysis demonstrated malvidin-3-glucoside PTP1B-complex occupies more conformational space where critical WPD-loop displayed a higher degree of motion. MM/PBSA binding free energy for malvidin-3-glucoside to PTP1B was found to be higher than other complexes mediated by Van der Waals energy rather than electrostatic interaction for the other two inhibitors (-80.32 ± 1.25 > -40.64 ± 1.43 > -21.63 ± 1.73 kcal/mol) respectively. Altogether, we have established novel insights into the specific binding of dietary phenolics and have identified malvidin-3-glucoside as an PTP1B inhibitor, which may be further industrially developed for the treatment of type-2 diabetes.

The anthocyanins in black currants regulate postprandial hyperglycaemia primarily by inhibiting α-glucosidase while other phenolics modulate salivary α-amylase, glucose uptake and sugar transporters.

The hypoglycaemic effects of two Ribes sp. i.e., anthocyanin-rich black currants (BC) were compared to green currants (GC), which are low in anthocyanins to establish which compounds are involved in the regulation of postprandial glycaemia. We determined the effect of the currants on inhibiting carbohydrate digestive enzymes (α-amylase, α-glucosidase), intestinal sugar absorption and transport across CaCo-2 cells. The digestion of these currants was modelled using in vitro gastrointestinal digestion (IVGD) to identify the metabolites present in the digested extracts by LC-MS/MS. Freeze-dried BC and IVDG extracts inhibited yeast α-glucosidase activity (P<.0001) at lower concentrations than acarbose, whereas GC and IVDG GC at the same concentrations showed no inhibition. BC and GC both showed significant inhibitory effects on salivary α-amylase (P<.0001), glucose uptake (P<.0001) and the mRNA expression of sugar transporters (P<.0001). Taken together this suggests that the anthocyanins which are high in BC have their greatest effect on postprandial hyperglycaemia by inhibiting α-glucosidase activity. Phytochemical analysis identified the phenolics in the currants and confirmed that freeze-dried BC contained higher concentrations of anthocyanins compared to GC (39.80 vs. 9.85 g/kg dry weight). Specific phenolics were also shown to inhibit salivary α-amylase, α-glucosidase, and glucose uptake. However, specific anthocyanins identified in BC which were low in GC were shown to inhibit α-glucosidase. In conclusion the anthocyanins in BC appear to regulate postprandial hyperglycaemia primarily but not solely by inhibiting α-glucosidase while other phenolics modulate salivary α-amylase, glucose uptake and sugar transporters which together could lower the associated risk of developing type-2 diabetes.

A randomised, double-blind, cross-over trial to evaluate bread, in which gluten has been pre-digested by prolyl endoprotease treatment, in subjects self-reporting benefits of adopting a gluten-free or low-gluten diet.

The aim of the present study was to determine if the enzyme Aspergillus niger prolyl endoprotease (ANPEP), which degrades the immunogenic proline-rich residues in gluten peptides, can be used in the development of new wheat products, suitable for gluten-sensitive (GS) individuals. We have carried out a double-blind, randomised, cross-over trial with two groups of adults; subjects, self-reporting benefits of adopting a gluten-free or low-gluten diet (GS, n 16) and a control non-GS group (n 12). For the trial, volunteers consumed four wheat breads: normal bread, bread treated with 0·8 or 1 % ANPEP and low-protein bread made from biscuit flour. Compared with controls, GS subjects had a favourable cardiovascular lipid profile - lower LDL (4·0 (sem 0·3) v. 2·8 (sem 0·2) mmol/l; P=0·008) and LDL:HDL ratio (3·2 (sem 0·4) v. 1·8 (sem 0·2); P=0·005) and modified haematological profile. The majority of the GS subjects followed a low-gluten lifestyle, which helps to reduce the gastrointestinal (GI) symptoms severity. The low-gluten lifestyle does not have any effect on the quality of life, fatigue or mental state of this population. Consumption of normal wheat bread increased GI symptoms in GS subjects compared with their habitual diet. ANPEP lowered the immunogenic gluten in the treated bread by approximately 40 %. However, when compared with the control bread for inducing GI symptoms, no treatment effects were apparent. ANPEP can be applied in the production of bread with taste, texture and appearance comparable with standard bread.

A single supplement of a standardised bilberry (Vaccinium myrtillus L.) extract (36 % wet weight anthocyanins) modifies glycaemic response in individuals with type 2 diabetes controlled by diet and lifestyle.

Dietary strategies for alleviating health complications associated with type 2 diabetes (T2D) are being pursued as alternatives to pharmaceutical interventions. Berries such as bilberries (Vaccinium myrtillus L.) that are rich in polyphenols may influence carbohydrate digestion and absorption and thus postprandial glycaemia. In addition, berries have been reported to alter incretins as well as to have antioxidant and anti-inflammatory properties that may also affect postprandial glycaemia. The present study investigated the acute effect of a standardised bilberry extract on glucose metabolism in T2D. Male volunteers with T2D (n 8; BMI 30 (sd 4) kg/m(2)) controlling their diabetes by diet and lifestyle alone were given a single oral capsule of either 0·47 g standardised bilberry extract (36 % (w/w) anthocyanins) which equates to about 50 g of fresh bilberries or placebo followed by a polysaccharide drink (equivalent to 75 g glucose) in a double-blinded cross-over intervention with a 2-week washout period. The ingestion of the bilberry extract resulted in a significant decrease in the incremental AUC for both glucose (P = 0·003) and insulin (P = 0·03) compared with the placebo. There was no change in the gut (glucagon-like peptide-1, gastric inhibitory polypeptide), pancreatic (glucagon, amylin) or anti-inflammatory (monocyte chemotactic protein-1) peptides. In addition there was no change in the antioxidant (Trolox equivalent antioxidant capacity, ferric-reducing ability of plasma) responses measured between the volunteers receiving the bilberry extract and the placebo. In conclusion the present study demonstrates for the first time that the ingestion of a concentrated bilberry extract reduces postprandial glycaemia and insulin in volunteers with T2D. The most likely mechanism for the lower glycaemic response involves reduced rates of carbohydrate digestion and/or absorption.

Using gene expression to predict differences in the secretome of human omental vs. subcutaneous adipose tissue.

The objective of this study was to characterize differences in the secretome of human omental compared with subcutaneous adipose tissue using global gene expression profiling. Gene expression was measured using Affymetrix microarrays (Affymetrix, Santa Clara, CA) in subcutaneous and omental adipose tissue in two independent experiments (n = 5 and n = 3 independent subjects; n = 16 arrays in total, 2 for each subject). Predictive bioinformatic algorithms were employed to identify secreted proteins. Microarray analysis identified 22 gene probe sets whose expression was significantly different with a fold change (FC) greater than 5 in expression in both experiments between omental and subcutaneous adipose tissue. Using bioinformatic predictive programs 11 of these 22 probe sets potentially coded for secreted proteins. Pathway network analysis of the secreted proteins showed that three of the proteins are part of a common pathway network. These proteins gremlin 1 (GREM1), pleiotrophin (PTN), and secretory leukocyte peptidase inhibitor (SLPI) are expressed respectively 43×, 23×, and 5× in omental adipose tissue relative to subcutaneous adipose tissue as determined by real-time PCR. The presence of GREM1, PTN, and SLPI protein in human adipose tissue was confirmed by western blotting. All three proteins are expressed in the human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell line. The expression of GREM1, PTN, and SLPI changed with the differentiation of the preadipocytes into mature adipocytes. Gene expression coupled with predictive bioinformatic algorithms have identified several genes coding for secreted proteins which are expressed differently in omental adipose tissue compared to subcutaneous adipose tissue proving a valid alternative approach to help further define the adipocyte secretome.

Leptin receptor gene expression and number in the brain are regulated by leptin level and nutritional status.

Hormone potency depends on receptor availability, regulated via gene expression and receptor trafficking. To ascertain how central leptin receptors are regulated, the effects of leptin challenge, high-fat diet, fasting and refeeding were measured on leptin receptor number and gene expression. These were measured using quantitative (125)I-labelled leptin in vitro autoradiography and in situ hybridisation, respectively. Ob-R (all forms of leptin receptor) expression in the choroid plexus (CP) was unchanged by high-fat diet or leptin challenge, whereas fasting increased but refeeding failed to decrease expression. (125)I-labelled leptin binding to the CP was increased by fasting and returned to basal levels on refeeding. (125)I-Labelled leptin was reduced by leptin challenge and increased by high-fat feeding. Ob-Rb (signalling form) in the arcuate (ARC) and ventromedial (VMH) nuclei was increased after fasting and decreased by refeeding. Leptin challenge increased Ob-Rb expression in the ARC, but not after high-fat feeding. In general, changes in gene expression in the ARC and VMH appeared to be largely due to changes in area rather than density of labelling, indicating that the number of cells expressing Ob-Rb was the parameter that contributed most to these changes. Leptin stimulation of suppressor of cytokine signalling 3 (SOCS3), a marker of stimulation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway, was unchanged after high-fat diet. Thus, early loss of leptin sensitivity after high-fat feeding is unrelated to down-regulation of leptin receptor expression or number and does not involve the JAK/STAT pathway. The effect of leptin to decrease (125)I-labelled leptin binding and the loss of ability of leptin to up-regulate Ob-Rb expression in the ARC after high-fat feeding offer potential mechanisms for the development of leptin insensitivity in response to both hyperleptinaemia and high-fat diet.

An imbalance in the methionine content of the maternal diet reduces postnatal growth in the rat.

The pregnant rat fed a low-protein diet has become widely used as a model system in the study of the prenatal programming of adult metabolism and disease. When pregnant rats of the hooded Lister strain were fed semisynthetic diets containing 18% or 9% casein supplemented with 0.5% dl-methionine, there was significant postnatal mortality in the group fed the low-protein diet. In a second experiment, dams were fed diets containing 9% casein supplemented with varying concentrations of dl-methionine up to 0.4% (w/w) and compared with a group fed a diet containing 18% casein supplemented with 0.5% dl-methionine. At birth, the pups from dams fed the low-protein diets supplemented with 0.2% dl-methionine or greater were significantly smaller than those of the dams fed the diet containing 18% protein. By 25 weeks of age, the body weight of the offspring of dams fed the low-protein diet supplemented with 0.2% or 0.3% dl-methionine were approximately 10% lower than those in the control group of offspring from dams fed 18% protein supplemented with 0.5% dl-methionine. There were corresponding changes in the weights of the major organs. These data suggest that increasing the dl-methionine supplement in the low-protein diet retards the growth of the fetus and affects the mature adult body weight. In contrast to the findings of other studies that used different formulas of the low-protein diet, the glucose tolerance in the offspring was unaffected by the protein content of the maternal diet at all levels of dl-methionine supplementation. These results suggest that the changes in metabolism of the offspring result from interactions between protein, lipids, and carbohydrates in the maternal diet, rather than a consequence of postnatal growth retardation per se and highlight the importance of considering all components of the maternal diet in the programming mechanism.

Maternal protein intake in the pregnant rat programs the insulin axis and body composition in the offspring.

Evidence to support an association between early nutrition and the development of obesity in the rat is equivocal. In this study we have investigated the postnatal growth, glucose tolerance, and adipocyte function of the offspring from pregnant rats fed with diets containing either 20% or 8% protein during gestation. By 25 weeks of age, the female offspring of dams fed with the diet containing 8% protein had a significantly lower adult body weight due in part to a decrease in body fat. The peak concentration of insulin after oral administration of a glucose dose was significantly lower in both the male and female offspring of the dams fed with the diet containing 8% protein. However, the ability of insulin to stimulate lipogenesis or suppress lipolysis in fat cells isolated from the offspring was not influenced by the prenatal diet. Hepatic phosphoenolpyruvate carboxykinase activity was reduced in female offspring of dams fed with the diet containing 8% protein. These results show that adult body composition is determined during the prenatal period as a result of programming of the insulin axis. This metabolic programming influences hepatic metabolism; however, there is no evidence for a programmed change in adipocyte function.

The expression of growth-arrest genes in the liver and kidney of the protein-restricted rat fetus.

During fetal life, there are periods of rapid cell proliferation, which are uniquely sensitive to nutritional perturbation. Feeding the pregnant rat a protein-restricted diet alters the growth trajectory of major fetal organs such as the kidney. By day 21 of gestation, the ratio of kidney weight to total body weight is reduced in the fetuses of dams fed a protein-deficient diet. In contrast, the ratio of fetal liver weight to total body weight is unchanged. To investigate the mechanisms underlying this disproportionate change in organ growth in the low-protein group, cell proliferation and differentiation have been assessed in the liver and kidney. The steady-state levels of mRNA for the growth-arrest and DNA-damage gene gadd153/CHOP-10, CCAAT enhancer-binding proteins alpha and beta were unaffected by maternal diet in both fetal liver and kidney. The mRNA for alpha-fetoprotein, albumin and hepatic glucokinase were unchanged in the liver, suggesting that maternal protein deficiency does not alter the state of differentiation. The steady-state levels of the mRNA coding for the cyclin-dependent protein kinase inhibitors (p15(INK4a), p19(INK4d), p21(CIP1), p27(KIP1) and p57(KIP2)) were unchanged in the fetal livers but were significantly increased in the kidneys of fetuses from dams fed the low-protein diet. These results show that the asymmetrical growth of the kidney is associated with increases in mRNA for the Cip/Kip cyclin-dependent kinase inhibitors and that these may reflect specific lesions in organ development.