Publications

Geoffrey Y. Sasaki, Jinhui Li, Morgan Cichon, Rachel E. Kopec, Richard S. Bruno (Molecular Nutrition & Food Research, November 28, 2020)

Catechin‐rich green tea extract (GTE) limits inflammation in nonalcoholic steatohepatitis (NASH) consistent with a Toll‐like receptor 4 (TLR4)‐dependent mechanism. It is hypothesized that GTE supplementation during NASH will shift the hepatic metabolome similar to that attributed to the loss‐of‐TLR4 signaling.

Haley Chatelaine, Priyankar Dey, Xiaokui Mo, Eunice Mah, Richard S. Bruno, Rachel E. Kopec (Molecular Nutrition & Food Research, November 9, 2020)

Persons with metabolic syndrome (MetS) absorb less vitamin E than healthy controls. It is hypothesized that absorption of fat‐soluble vitamins (FSV) A and D₂ would also decrease with MetS status and that trends would be reflected in lipidomic responses between groups.

Kevin Anderson, Nathan Ryan, Arham Siddiqui, Travis Pero, Greta Volpedo, Jessica Cooperstone, Steve Oghumu  (Nutrients, June 4, 2020)

Contact hypersensitivity (CHS) is the most common occupational dermatological disease. Dendritic cells (DCs) mediate the sensitization stage of CHS, while T-cells facilitate the effector mechanisms that drive CHS. Black raspberry (Rubus occidentalis, BRB) and BRB phytochemicals possess immunomodulatory properties, but their dietary effects on CHS are unknown. We examined the effects of diets containing BRB and protocatechuic acid (PCA, a constituent of BRB and an anthocyanin metabolite produced largely by gut microbes), on CHS, using a model induced by 2,4-dinitrofluorobenze (DNFB). Mice were fed control diet or diets supplemented with BRB or PCA. In vitro bone-marrow derived DCs and RAW264.7 macrophages were treated with BRB extract and PCA. Mice fed BRB or PCA supplemented diets displayed decreased DNFB-induced ear swelling, marked by decreased splenic DC accumulation. BRB extract diminished DC maturation associated with reduced Cd80 expression and Interleukin (IL)-12 secretion, and PCA reduced IL-12. Dietary supplementation with BRB and PCA induced differential decreases in IL-12-driven CHS mediators, including Interferon (IFN)-γ and IL-17 production by T-cells. BRB extracts and PCA directly attenuated CHS-promoting macrophage activity mediated by nitric oxide and IL-12. Our results demonstrate that BRB and PCA mitigate CHS pathology, providing a rationale for CHS alleviation via dietary supplementation with BRB or BRB derived anthocyanins.

Anindita Sengupta, Jikang Wu, Justin T. Seffernick, Anice Sabag-Daible, Nicholas Thomsen, Tien-Hao Chen, Angela Di Capua, Charles E. Bell, Brian M.M. Ahmer, Steffen Lindert, Vicki H. Wysocki, Venkat Gopalan ( Journal of Molecular Biology, November 8, 2019)

Salmonella is a foodborne pathogen that causes annually millions of cases of salmonellosis globally, yet Salmonella-specific antibacterials are not available. During inflammation, Salmonella utilizes the Amadori compound fructose–asparagine (F-Asn) as a nutrient through the successive action of three enzymes, including the terminal FraB deglycase. Salmonella mutants lacking FraB are highly attenuated in mouse models of inflammation due to the toxic build-up of the substrate 6-phosphofructose-aspartate (6-P-F-Asp). This toxicity makes Salmonella FraB an appealing drug target, but there is currently little experimental information about its catalytic mechanism. Therefore, we sought to test our postulated mechanism for the FraB-catalyzed deglycation of 6-P-F-Asp (via an enaminol intermediate) to glucose-6-phosphate and aspartate. A FraB homodimer model generated by RosettaCM was used to build substrate-docked structures that, coupled with sequence alignment of FraB homologs, helped map a putative active site. Five candidate active-site residues—including three expected to participate in substrate binding—were mutated individually and characterized. Native mass spectrometry and ion mobility were used to assess collision cross sections and confirm that the quaternary structure of the mutants mirrored the wild type, and that there are two active sites/homodimer. Our biochemical studies revealed that FraB Glu214Ala, Glu214Asp, and His230Ala were inactive in vitro, consistent with deprotonated-Glu214 and protonated-His230 serving as a general base and a general acid, respectively. Glu214Ala or His230Ala introduced into the Salmonella chromosome by CRISPR/Cas9-mediated genome editing abolished growth on F-Asn. Results from our computational and experimental approaches shed light on the catalytic mechanism of Salmonella FraB and of phosphosugar deglycases in general.

Jinhui Li, Geoffrey Sasaki, Priyankar Dey, Chureeporn Chitchumroonchokchai, Allison N. Labyk, Joshua D. McDonald, Joshua B. Kim, Richard S. Bruno (The Journal of Nutritional Biochemistry, March 2018)

Green tea extract (GTE) reduces NFκB-mediated inflammation during nonalcoholic steatohepatitis (NASH). We hypothesized that its anti-inflammatory activities would be mediated in a Toll-like receptor 4 (TLR4)-dependent manner. Wild-type (WT) and loss-of-function TLR4-mutant (TLR4m) mice were fed a high-fat diet containing GTE at 0 or 2% for 8 weeks before assessing NASH, NFκB-mediated inflammation, TLR4 and its adaptor proteins MyD88 and TRIF, circulating endotoxin, and intestinal tight junction protein mRNA expression. TLR4m mice had lower (P < .05) body mass compared with WT mice but similar adiposity, whereas body mass and adiposity were lowered by GTE regardless of genotype. Liver steatosis, serum alanine aminotransferase, and hepatic lipid peroxidation were also lowered by GTE in WT mice, and were similarly lowered in TLR4m mice regardless of GTE. Phosphorylation of the NFκB p65 subunit and pro-inflammatory genes (TNFα, iNOS, MCP-1, MPO) were lowered by GTE in WT mice, and did not differ from the lowered levels in TLR4m mice regardless of GTE. TLR4m mice had lower TLR4 mRNA, which was also lowered by GTE in both genotypes. TRIF expression was unaffected by genotype and GTE, whereas MyD88 was lower in mice fed GTE regardless of genotype. Serum endotoxin was similarly lowered by GTE regardless of genotype. Tight junction protein mRNA levels were unaffected by genotype. However, GTE similarly increased claudin-1 mRNA in the duodenum and jejunum and mRNA levels of occludin and zonula occluden-1 in the jejunum and ileum. Thus, GTE protects against inflammation during NASH, likely by limiting gut-derived endotoxin translocation and TLR4/MyD88/NFκB activation.

Maryam Baniasad, Yongseok Kim, Michael Shaffer, Anice Sabag-Daigle, Ikaia Leleiwi, Rebecca A Daly, Brian M M Ahmer, Kelly C Wrighton, Vicki H Wysocki (Analytical and Bioanalytical Chemistry, February 2022) 

Bottom-up proteomics is a powerful method for the functional characterization of mouse gut microbiota. To date, most of the bottom-up proteomics studies of the mouse gut rely on limited amounts of fecal samples. With mass-limited samples, the performance of such analyses is highly dependent on the protein extraction protocols and contaminant removal strategies. Here, protein extraction protocols (using different lysis buffers) and contaminant removal strategies (using different types of filters and beads) were systematically evaluated to maximize quantitative reproducibility and the number of identified proteins. Overall, our results recommend a protein extraction method using a combination of sodium dodecyl sulfate (SDS) and urea in Tris-HCl to yield the greatest number of protein identifications. These conditions led to an increase in the number of proteins identified from gram-positive bacteria, such as Firmicutes and Actinobacteria, which is a challenging task. Our analysis further confirmed these conditions led to the extraction of non-abundant bacterial phyla such as Proteobacteria. In addition, we found that, when coupled to our optimized extraction method, suspension trap (S-Trap) outperforms other contaminant removal methods by providing the most reproducible method while producing the greatest number of protein identifications. Overall, our optimized sample preparation workflow is straightforward and fast, and requires minimal sample handling. Furthermore, our approach does not require high amounts of fecal samples, a vital consideration in proteomics studies where mice produce smaller amounts of feces due to a particular physiological condition. Our final method provides efficient digestion of mouse fecal material, is reproducible, and leads to high proteomic coverage for both host and microbiome proteins.

Priyankar Dey, Geoffrey Sasaki, Richard Bruno (Handbook of Nutraceuticals and Functional Food, March 2020)

Tea (Camellia sinensis) is the most commonly consumed prepared beverage in the world, and ranks only second to that of water for all beverages consumed. Green tea is rich in polyphenolic catechins, especially epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin. Of these, EGCG has received the most extensive study because it is thought to be primarily responsible for the health benefits of green tea. Although green tea is consumed less frequently than black tea, its popularity has increased due to growing knowledge of its health benefits. For example, epidemiological evidence supports that green tea consumption decreases the risk of cardiometabolic disorders and some forms of cancer. These findings have laid the foundation to define the bioactivities of green tea, with evidence indicating that catechins improve health status through antioxidant, anti-inflammatory, and metabolic benefits.

Douglas R. Moellering, Kelley Smith-Johnston, Christian Kelley, Melissa J. Sammy, Jason Benedict, Guy Brock, Jillian Johnson, Kedryn K. Baskin, Wael N. Jarjour, Martha A. Belury, Peter J. Reiser, Prabhakara R. Nagareddy & Beatriz Y. Hanaoka (Arthritis Research & Therapy, May 2023)

Insulin resistance affects a substantial proportion of patients with rheumatoid arthritis (RA). Skeletal muscle mitochondrial dysfunction results in the accumulation of lipid intermediates that interfere with insulin signaling. We therefore sought to determine if lower oxidative phosphorylation and muscle mitochondrial content are associated with insulin resistance in patients with RA.

Deena Snoke, Connor A. Mahler, Austin Angelotti, Rachel M. Cole, Genevieve C. Sparagna, Kedryn K. Baskin, Martha A. Belury (Biology, December 2022)

Cardiolipin (CL) is a phospholipid unique to the inner mitochondrial membrane that supports respiratory chain structure and function and is demonstrated to be influenced by types of dietary fats. However, the influence of dietary fat on CL species and how this best supports mitochondrial function in brown adipose tissue (BAT), which exhibits an alternative method of energy utilization through the uncoupling of the mitochondrial proton gradient to generate heat, is not well understood. Therefore, the aim of our study was to evaluate metabolic parameters, interscapular BAT CL quantity, species, and mitochondrial function in mice consuming isocaloric moderate-fat diets with either lard (LD; similar fatty acid profile to western dietary patterns) or safflower oil high in linoleic acid (SO), shown to be metabolically favorable in large clinical meta-analyses. Mice fed the SO diet exhibited decreased adiposity, improved insulin sensitivity, and enrichment of LA-containing CL species in BAT CL. Furthermore, mice fed the SO diet exhibit higher levels of OXPHOS complex proteins and increased oxygen consumption in BAT. Our findings demonstrate that dietary consumption of LA-rich oil improves metabolic parameters, increases LA-containing CL species, and improves BAT function when compared to the consumption of lard in mice during diet-induced weight gain.

Connor A Mahler, Deena B Snoke, Rachel M Cole, Austin Angelotti, Genevieve C Sparagna, Kedryn K Baskin, Ai Ni, Martha A Belury (Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions, December 2023)

Hepatic mitochondrial dysfunction is a major cause of fat accumulation in the liver and individuals with fatty liver conditions have hepatic mitochondrial structural abnormalities and a switch in the side chain composition of the mitochondrial phospholipid, cardiolipin, from poly- to monounsaturated fatty acids. Linoleic acid (LA), an essential dietary fatty acid, has been shown to remodel nascent cardiolipin (CL) to its tetralinoleoyl cardiolipin (L₄CL, CL with 4 LA side chains) form, which is integral for mitochondrial membrane structure and function to promote fatty acid oxidation. It is unknown, however, whether increasing LA in the diet can increase hepatic L₄CL concentrations, and improve mitochondrial respiration compared with a diet rich in monounsaturated and saturated fatty acids.

A. Alsudayri, S. Perelman, M. Brewer, A. Chura, M. DcDevitt, C. Drerup, L. Ye (April 2024)

Enteroendocrine cells (EECs) are crucial for sensing ingested nutrients and regulating feeding behavior. How gut microbiota regulate the nutrient-sensing EEC activity is unclear. Our transcriptomic analysis demonstrates that commensal microbiota colonization significantly increases the expression of many genes associated with mitochondrial function. Using new methods to image EEC cytoplasmic and mitochondrial Ca²⁺ activity in live zebrafish, our data revealed that it is dynamically regulated during the EEC development process. Mature EECs display an increased mitochondrial-to-cytoplasmic Ca²⁺ ratio. Mitochondria are evenly distributed in the cytoplasm of immature EECs. As EECs mature, their mitochondria are highly localized at the basal membrane where EEC vesicle secretion occurs. Conventionalized (CV) EECs, but not germ-free (GF) EECs, exhibit spontaneous low-amplitude Ca²⁺ fluctuation. The mitochondrial-to-cytoplasmic Ca²⁺ ratio is significantly higher in CV EECs. Nutrient stimulants, such as fatty acid, increase cytoplasmic Ca²⁺ in a subset of EECs and promote a sustained mitochondrial Ca²⁺ and ATP increase. However, the nutrient-induced EEC mitochondrial activation is nearly abolished in GF zebrafish. Together, our study reveals that commensal microbiota are crucial in supporting EEC mitochondrial function and maturation.