Synthesis of Fluorescent Proanthocyanidin-Cinnamaldehydes Pyrylium Products for Microscopic Detection of Interactions with Extra-Intestinal Pathogenic Escherichia coli.

Synthesis of proanthocyanidin-cinnamaldehydes pyrylium products (PCPP) was achieved by the condensation reaction of proanthocyanidins (PAC) with cinnamaldehyde and four cinnamaldehyde derivatives. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra of PCPP show masses that correspond to (epi)catechin oligomers attached to single, double, or triple moieties of cinnamaldehydes. Synthesized PCPP exhibited fluorescence at higher excitation and emission wavelengths than PAC. Results indicate that PCPP were more bioactive for agglutinating extra-intestinal pathogenic Escherichia coli (ExPEC) compared to PAC. Scanning electron microscopy indicates that PCPP interact with ExPEC surface structures and suggests that PCPP have a higher affinity with the fimbriae-like structures of ExPEC than PAC. Fluorescent microscopy performed on in vitro and in vivo agglutination assays show that PCPP were entrapping ExPEC in a web-like network, thus demonstrating agglutination of ExPEC. This study demonstrated the potential of PCPP to improve our understanding of the temporal and dynamic interactions of PAC in in vitro and in vivo studies.

Isolation and Characterization of Blueberry Polyphenolic Components and Their Effects on Gut Barrier Dysfunction.

Highbush blueberries contain anthocyanins and proanthocyanidins that have antimicrobial and anti-inflammatory bioactivities. We isolated and characterized three polyphenolic fractions, a total polyphenol fraction (TPF), an anthocyanin-enriched fraction (AEF), and a proanthocyanidin-enriched fraction (PEF), from freeze-dried blueberry powder and evaluated their effects on an in vitro model of gut barrier dysfunction. High-performance liquid chromatography chromatograms illustrate successful fractionation of the blueberry powder into TPF, AEF, and PEF. AEF contained 21 anthocyanins, and PEF contained proanthocyanidin oligomers of (epi)catechin with primarily B-type interflavan bonds. The model uses a strain of Escherichia coli to disrupt a Caco-2 cell monolayer on Transwell inserts. Barrier function was measured by transepithelial electrical resistance (TEER), a marker of membrane permeability. All fractions were able to restore TEER values after an E. coli challenge when compared to the control, while AEF was able to attenuate the E. coli-induced decrease in TEER in a dose-dependent manner.

Aging and caloric restriction impact adipose tissue, adiponectin, and circulating lipids.

Adipose tissue expansion has been associated with system-wide metabolic dysfunction and increased vulnerability to diabetes, cancer, and cardiovascular disease. A reduction in adiposity is a hallmark of caloric restriction (CR), an intervention that extends longevity and delays the onset of these same age-related conditions. Despite these parallels, the role of adipose tissue in coordinating the metabolism of aging is poorly defined. Here, we show that adipose tissue metabolism and secretory profiles change with age and are responsive to CR. We conducted a cross-sectional study of CR in adult, late-middle-aged, and advanced-aged mice. Adiposity and the relationship between adiposity and circulating levels of the adipose-derived peptide hormone adiponectin were age-sensitive. CR impacted adiposity but only levels of the high molecular weight isoform of adiponectin responded to CR. Activators of metabolism including PGC-1a, SIRT1, and NAMPT were differentially expressed with CR in adipose tissues. Although age had a significant impact on NAD metabolism, as detected by biochemical assay and multiphoton imaging, the impact of CR was subtle and related to differences in reliance on oxidative metabolism. The impact of age on circulating lipids was limited to composition of circulating phospholipids. In contrast, the impact of CR was detected in all lipid classes regardless of age, suggesting a profound difference in lipid metabolism. These data demonstrate that aspects of adipose tissue metabolism are life phase specific and that CR is associated with a distinct metabolic state, suggesting that adipose tissue signaling presents a suitable target for interventions to delay aging.

Regional metabolic heterogeneity of the hippocampus is nonuniformly impacted by age and caloric restriction.

The hippocampus is critical for cognition and memory formation and is vulnerable to age-related atrophy and loss of function. These phenotypes are attenuated by caloric restriction (CR), a dietary intervention that delays aging. Here, we show significant regional effects in hippocampal energy metabolism that are responsive to age and CR, implicating metabolic pathways in neuronal protection. In situ mitochondrial cytochrome c oxidase activity was region specific and lower in aged mice, and the impact of age was region specific. Multiphoton laser scanning microscopy revealed region- and age-specific differences in nicotinamide adenine dinucleotide (NAD)-derived metabolic cofactors. Age-related changes in metabolic parameters were temporally separated, with early and late events in the metabolic response to age. There was a significant regional impact of age to lower levels of PGC-1α, a master mitochondrial regulator. Rather than reversing the impact of age, CR induced a distinct metabolic state with decreased cytochrome c oxidase activity and increased levels of NAD(P)H. Levels of hippocampal PGC-1α were lower with CR, as were levels of GSK3ß, a key regulator of PGC-1α turnover and activity. Regional distribution and colocalization of PGC-1α and GSK3ß in mouse hippocampus was similar in monkeys. Furthermore, the impact of CR to lower levels of both PGC-1α and GSK3ß was also conserved. The studies presented here establish the hippocampus as a highly varied metabolic environment, reveal cell-type and regional specificity in the metabolic response to age and delayed aging by CR, and suggest that PGC-1α and GSK3ß play a role in implementing the neuroprotective program induced by CR.

Plasma diacylglycerol composition is a biomarker of metabolic syndrome onset in rhesus monkeys.

Metabolic syndrome is linked with obesity and is often first identified clinically by elevated BMI and elevated levels of fasting blood glucose that are generally secondary to insulin resistance. Using the highly translatable rhesus monkey (Macaca mulatta) model, we asked if metabolic syndrome risk could be identified earlier. The study involved 16 overweight but healthy, euglycemic monkeys, one-half of which spontaneously developed metabolic syndrome over the course of 2 years while the other half remained healthy. We conducted a series of biometric and plasma measures focusing on adiposity, lipid metabolism, and adipose tissue-derived hormones, which led to a diagnosis of metabolic syndrome in the insulin-resistant animals. Plasma fatty acid composition was determined by gas chromatography for cholesteryl ester, FFA, diacylglycerol (DAG), phospholipid, and triacylglycerol lipid classes; plasma lipoprotein profiles were generated by NMR; and circulating levels of adipose-derived signaling peptides were determined by ELISA. We identified biomarker models including a DAG model, two lipoprotein models, and a multiterm model that includes the adipose-derived peptide adiponectin. Correlations among circulating lipids and lipoproteins revealed shifts in lipid metabolism during disease development. We propose that lipid profiling may be valuable for early metabolic syndrome detection in a clinical setting.

A shift in energy metabolism anticipates the onset of sarcopenia in rhesus monkeys.

Age-associated skeletal muscle mass loss curtails quality of life and may contribute to defects in metabolic homeostasis in older persons. The onset of sarcopenia occurs in middle age in rhesus macaques although the trigger has yet to be identified. Here, we show that a shift in metabolism occurs in advance of the onset of sarcopenia in rhesus vastus lateralis. Multiphoton laser-scanning microscopy detects a shift in the kinetics of photon emission from autofluorescent metabolic cofactors NADH and FAD. Lifetime of both fluorophores is shortened at mid-age, and this is observed in both free and bound constituent pools. Levels of FAD and free NADH are increased and the NAD/NADH redox ratio is lower. Concomitant with this, expression of fiber-type myosin isoforms is altered resulting in a shift in fiber-type distribution, activity of cytochrome c oxidase involved in mitochondrial oxidative phosphorylation is significantly lower, and the subcellular organization of mitochondria in oxidative fibers is compromised. A regulatory switch involving the transcriptional coactivator PGC-1α directs metabolic fuel utilization and governs the expression of structural proteins. Age did not significantly impact total levels of PGC-1α; however, its subcellular localization was disrupted, suggesting that PGC-1α activities may be compromised. Consistent with this, intracellular lipid storage is altered and there is shift to larger lipid droplet size that likely reflects a decline in lipid turnover or a loss in efficiency of lipid metabolism. We suggest that changes in energy metabolism contribute directly to skeletal muscle aging in rhesus monkeys.