Diana E. Roopchand

Dietary polyphenols promote growth of the gut bacterium Akkermansia muciniphila and attenuate high fat diet-induced metabolic syndrome.

Dietary polyphenols protect against metabolic syndrome, despite limited absorption and digestion, raising questions about their mechanism of action. We hypothesized that one mechanism may involve the gut microbiota. To test this hypothesis, C57BL/6J mice were fed a high-fat diet (HFD) containing 1% Concord grape polyphenols (GP). Relative to vehicle controls, GP attenuated several effects of HFD feeding, including weight gain, adiposity, serum inflammatory markers (tumor necrosis factor [TNF]α, interleukin [IL]-6, and lipopolysaccharide), and glucose intolerance. GP lowered intestinal expression of inflammatory markers (TNFα, IL-6, inducible nitric oxide synthase) and a gene for glucose absorption (Glut2). GP increased intestinal expression of genes involved in barrier function (occludin) and limiting triglyceride storage (fasting-induced adipocyte factor). GP also increased intestinal gene expression of proglucagon, a precursor of proteins that promote insulin production and gut barrier integrity. 16S rRNA gene sequencing and quantitative PCR of cecal and fecal samples demonstrated that GP dramatically increased the growth of Akkermansia muciniphila and decreased the proportion of Firmicutes to Bacteroidetes, consistent with prior reports that similar changes in microbial community structure can protect from diet-induced obesity and metabolic disease. These data suggest that GP act in the intestine to modify gut microbial community structure, resulting in lower intestinal and systemic inflammation and improved metabolic outcomes. The gut microbiota may thus provide the missing link in the mechanism of action of poorly absorbed dietary polyphenols.

The role of adenovirus E4orf4 protein in viral replication and cell killing.

It has only been within the last few years that insights have been gained into the remarkable diversity of functions of the adenovirus early transcription region 4 (E4) products. The polypeptide encoded by E4 open reading frame 4 (E4orf4) has emerged as an enigmatic product. Although it accomplishes certain functions that propel viral replication, it has also been shown to be highly toxic, an effect that could dampen the infectious cycle, but that also might serve to facilitate release of viral progeny. When expressed alone, E4orf4 induces a novel form of p53-independent apoptosis in cancer cells but not in normal human cells, thus making it of potential use in cancer gene therapy. In addition, knowledge of its mechanism of action, especially with regard to its interaction with protein phosphatase 2A (PP2A), could provide insights to develop new small molecule anti-cancer drugs. Thus future studies on E4orf4 should be both informative and potentially valuable therapeutically. In this study we review the current status of knowledge on E4orf4.

Effects of a high fat meal matrix and protein complexation on the bioaccessibility of blueberry anthocyanins using the TNO gastrointestinal model (TIM-1)

The TNO intestinal model (TIM-1) of the human upper gastrointestinal tract was used to compare intestinal absorption/bioaccessibility of blueberry anthocyanins under different digestive conditions. Blueberry polyphenol-rich extract was delivered to TIM-1 in the absence or presence of a high-fat meal. HPLC analysis of seventeen anthocyanins showed that delphinidin-3-glucoside, delphinidin-3-galactoside, delphinidin-3-arabinoside and petunidin-3-arabinoside were twice as bioaccessible in fed state, whilst delphinidin-3-(6″-acetoyl)-glucoside and malvidin-3-arabinoside were twice as bioaccessible under fasted conditions, suggesting lipid-rich matrices selectively effect anthocyanin bioaccessibility. TIM-1 was fed blueberry juice (BBJ) or blueberry polyphenol-enriched defatted soybean flour (BB-DSF) containing equivalent amounts of free or DSF-sorbed anthocyanins, respectively. Anthocyanin bioaccessibility from BB-DSF (36.0±10.4) was numerically, but not significantly, greater than that from BBJ (26.3±10.3). Ileal efflux samples collected after digestion of BB-DSF contained 2.8-fold more anthocyanins than same from BBJ, suggesting that protein-rich DSF protects anthocyanins during transit through upper digestive tract for subsequent colonic delivery/metabolism.

Toxicity of human adenovirus E4orf4 protein in Saccharomyces cerevisiae results from interactions with the Cdc55 regulatory B subunit of PP2A.

The E4orf4 protein of human adenovirus induces p53-independent apoptosis, a process that may promote cell death and viral spread. When expressed alone, E4orf4 kills transformed cells but not normal human cells. The only clear target of E4orf4 in mammalian cells is the Bα (B55) subunit of protein phosphatase 2A (PP2A), a member of one of three classes of regulatory B subunits. Here we report the effects of E4orf4 in Saccharomyces cerevisiae, which encodes two PP2A regulatory B subunits, CDC55 and RTS1, that share homology with mammalian B and B′ subunits, respectively. E4orf4 expression was found to be toxic in yeast, resulting in the accumulation of cells in G2/M phase that failed to grow upon removal of E4orf4. E4orf4-expressing yeast also displayed an elongated cell morphology similar to cdc55 deletion strains. E4orf4 required CDC55 to elicit its effect, whereas RTS1 was dispensable. The recruitment of the PP2A holoenzyme by E4orf4 was entirely dependent on Cdc55. These studies indicate that E4orf4-induced apoptosis in mammalian cells and cell death in yeast require functional interactions with B-type subunits of PP2A. However, some inhibition of growth by E4orf4 was observed in the cdc55 strain and with an E4orf4 mutant that fails to interact with Cdc55, indicating that E4orf4 may possess a second Cdc55-independent function affecting cell growth.

Blueberry polyphenol-enriched soybean flour reduces hyperglycemia, body weight gain and serum cholesterol in mice

Defatted soybean flour (DSF) can sorb and concentrate blueberry anthocyanins and other polyphenols, but not sugars. In this study blueberry polyphenol-enriched DSF (BB-DSF) or DSF were incorporated into very high fat diet (VHFD) formulations and provided ad libitum to obese and hyperglycemic C57BL/6 mice for 13 weeks to investigate anti-diabetic effects. Compared to the VHFD containing DSF, the diet supplemented with BB-DSF reduced weight gain by 5.6%, improved glucose tolerance, and lowered fasting blood glucose levels in mice within 7 weeks of intervention. Serum cholesterol of mice consuming the BB-DSF-supplemented diet was 13.2% lower than mice on the diet containing DSF. Compounds were eluted from DSF and BB-DSF for in vitro assays of glucose production and uptake. Compared to untreated control, doses of BB-DSF eluate containing 0.05-10μg/μL of blueberry anthocyanins significantly reduced glucose production by 24-74% in H4IIE rat hepatocytes, but did not increase glucose uptake in L6 myotubes. The results indicate that delivery of blueberry polyphenols stabilized in a high-protein food matrix may be useful for the dietary management of pre-diabetes and/or diabetes.

Adenovirus protein E4orf4 induces premature APCCdc20 activation in Saccharomyces cerevisiae by a protein phosphatase 2A-dependent mechanism.

ABSTRACT Protein phosphatase 2A (PP2A) has been implicated in cell cycle progression and mitosis; however, the complexity of PP2A regulation via multiple B subunits makes its functional characterization a significant challenge. The human adenovirus protein E4orf4 has been found to induce both high Cdk1 activity and the accumulation of cells in G2/M in both mammalian and yeast cells, effects which are largely dependent on the B55/Cdc55 regulatory subunit of PP2A. Thus, E4orf4 represents a unique means by which the function of a specific form of PP2A can be delineated in vivo. In Saccharomyces cerevisiae, only two PP2A regulatory subunits exist, Cdc55 and Rts1. Here, we show that E4orf4-induced toxicity depends on a functional interaction with Cdc55. E4orf4 expression correlates with the inappropriate reduction of Pds1 and Scc1 in S-phase-arrested cells. The unscheduled loss of these proteins suggests the involvement of PP2ACdc55 in the regulation of the Cdc20 form of the anaphase-promoting complex (APC). Contrastingly, activity of the Hct1 form of the APC is not induced by E4orf4, as demonstrated by the observed stability of its substrates. We propose that E4orf4, being a Cdc55-specific inhibitor of PP2A, demonstrates the role of PP2ACdc55 in regulating APCCdc20 activity.

Stable Binding of Alternative Protein-enriched Food Matrices with Concentrated Cranberry Bioflavonoids for Functional Food Applications

Defatted soy flour (DSF), soy protein isolate (SPI), hemp protein isolate (HPI), medium-roast peanut flour (MPF), and pea protein isolate (PPI) stably bind and concentrate cranberry (CB) polyphenols, creating protein/polyphenol-enriched matrices. Proanthocyanidins (PAC) in the enriched matrices ranged from 20.75 mg/g (CB-HPI) to 10.68 mg/g (CB-SPI). Anthocyanins (ANC) ranged from 3.19 mg/g (CB-DSF) to 1.68 mg/g (CB-SPI), whereas total phenolics (TP) ranged from 37.61 mg/g (CB-HPI) to 21.29 mg/g (CB-SPI). LC-MS indicated that the enriched matrices contained all identifiable ANC, PAC, and flavonols present in CB juice. Complexation with SPI stabilized and preserved the integrity of the CB polyphenolic components for at least 15 weeks at 37 °C. PAC isolated from enriched matrices demonstrated comparable antiadhesion bioactivity to PAC isolated directly from CB juice (MIC 0.4-0.16 mg/mL), indicating their potential utility for maintenance of urinary tract health. Approximately 1.0 g of polyphenol-enriched matrix delivered the same amount of PAC available in 1 cup (300 mL) of commercial CB juice cocktail, which has been shown clinically to be the prophylactic dose for reducing recurring urinary tract infections. CB-SPI inhibited Gram-positive and Gram-negative bacterial growth. Nutritional and sensory analyses indicated that the targeted CB-matrix combinations have high potential for incorporation in functional food formulations.

Food-compatible method for the efficient extraction and stabilization of cranberry pomace polyphenols.

Cranberry pomace is a byproduct of cranberry processing and is comprised of seeds, skins and stems of the cranberry fruit. While cranberry pomace contains beneficial polyphenols, including proanthocyanidins and anthocyanins, it is not a palatable source of these compounds and is typically discarded. In this study, we have developed and optimized a method to extract polyphenols from cranberry pomace using aqueous ethanol, a food grade solvent. Biochemical characterization of the pomace extract showed the presence of a broad range of polyphenols also present in cranberry juice concentrate. By co-drying cranberry pomace extract with a protein-rich food matrix, such as soy protein isolate (SPI), we have developed a method to produce a cranberry polyphenol-SPI complex (CBP-SPI) containing 10% cranberry polyphenols. Unlike dried cranberry pomace extract alone, proanthocyanidins, anthocyanins and total polyphenols were found to be highly stable at 37 °C in the CBP-SPI powder. The extraction and stabilization of cranberry pomace polyphenols using SPI provides an innovative approach for utilizing pomace in the development of novel food ingredients.

Concord Grape Pomace Polyphenols Complexed to Soy Protein Isolate Are Stable and Hypoglycemic in Diabetic Mice

Polyphenols extracted from Concord grape pomace were stabilized by complexation to soy protein isolate (SPI) to produce grape polyphenol-SPI complex (GP-SPI) containing 5% or 10% grape polyphenols. LC-MS and MALDI-TOF analysis showed that a broad range of phytochemicals were present in the grape pomace extract. Anthocyanins and total polyphenols in the GP-SPI complex were stable after a 16-week incubation at 37 °C but were reduced by up 60% in dried grape pomace extract. Compared to vehicle (236 ± 34 mg/dL), a single dose of 300 mg/kg GP-SPI (184 ± 32 mg/dL) or 500 mg/kg GP-SPI (177 ± 28 mg/dL) having 5% grape polyphenols significantly lowered blood glucose in obese and hyperglycemic C57BL/6 mice 6 h after administration. GP-SPI allows the capture of grape pomace polyphenols in a protein-rich food matrix and may be useful as a functional food ingredient for the management of blood glucose levels.

Biochemical Analysis and in Vivo Hypoglycemic Activity of a Grape Polyphenol–Soybean Flour Complex

Defatted soybean flour (DSF) can efficiently sorb, concentrate, and stabilize polyphenols, but not sugars, from Concord grape juice, to yield grape polyphenol-enriched DSF. Sorption of grape polyphenols to DSF particles was dependent on the ratio of DSF and grape juice concentrate used, but not time of mixing or pH. Depending on ratios of starting materials, 1 g of grape polyphenol-enriched DSF contained 1.6-10.4 mg of anthocyanins, 7.5-93.1 mg of proanthocyanidins, and 20.5-144.5 mg of total polyphenols. LC-MS analysis of grape juice samples before and after addition and removal of DSF and eluate from grape polyphenol-enriched DSF confirmed that a broad range of grape compounds were sorbed to the DSF matrix. Finally, grape polyphenol-enriched DSF was able to significantly lower blood glucose levels in hyperglycemic C57BL/6J mice. The data indicate that grape polyphenol-enriched DSF can provide a high-protein, low-sugar ingredient for delivery of concentrated grape polyphenolics.