bluuebunny/crossref_metadata_2025 huggingface dataset:
Antidepressant treatment increases cAMP signaling by translocating Gas from lipid rafts and increasing association with type 6 adenylyl cyclase (AC6) independent of action at monoamine transporters.
Authors: Schappi Jeffrey M , Rasenick Mark
Published: November 2023

Antidepressants of different chemical classes promote sustained increases in cAMP, due, at least in part to the redistribution of Gαs from lipid rafts into non‐raft membrane fractions resulting in increased Gαs functional coupling with adenylyl cyclase (AC). This has been demonstrated in both rats and cultured neural and glial cells by a number of techniques, including cell fractionation, functional assays, and imaging studies such as Fluorescence Recovery After Photobleaching (FRAP). Unlike animal models, C6 glioma lack monoamine reuptake transporters, suggesting that mechanisms of antidepressant response entail more than inhibition of neurotransmitter reuptake inhibition. This is noteworthy, since inhibition of 5HT or NE uptake is quite rapid even though clinical effects of antidepressants require several weeks (and several days in the cellular models). Furthermore, while these neural and glial cells showed an “antidepressant response”, Gαs localization and cAMP production in kidney epithelial cells like COS7 and HEK293 were unchanged by antidepressant treatment. Similarly, membranes from liver and kidney of rats treated chronically with antidepressant did not show the same response as brain from those same animals. In this study we sought to determine whether cellular antidepressant response, with respect to increased cAMP signaling and Gαs localization, is dependent on the type of AC isoform expression. Cell lines “insensitive” to antidepressant treatment, such as HEK293, become responsive after transfection with AC6, showing translocation of Gαs from lipid rafts, both by cell fractionation and by FRAP. Likewise, knockdown of AC6 but not other adenylyl cyclase isoforms abolishes the antidepressant response in responsive cell lines such as C6 glioma. Thus, it is suggested that AC6 performs a anchoring function for Gαs outside of rafts, affixing the translocated Gαs into the non‐raft domain and facilitating an antidepressant‐induced increase in adenylyl cyclase activity.
The Golgi Arf‐GEFs Gea1 and Gea2 Integrate Signals to Coordinate Vesicle Formation
Authors: Gustafson Margaret Ann , Fromme J. Christopher
Published: November 2023

A major challenge for eukaryotic cells is regulating the transport of protein cargoes among compartments and to and from the plasma membrane. How does a cell guarantee the efficient trafficking of appropriate cargoes to the only the right place at the right time? We and others have shown that at the Golgi complex, the cell's sorting house, decisions about packaging cargo rely on coordination of multiple signals.

In Saccharomyces cerevisiae (and similarly in other eukaryotes including humans), this coordination relies heavily on small GTPases. The GTPase Arf1 acts as a central molecular switch: when switched on (GTP‐bound), it recruits cargoes, adapters, and coat proteins needed to form a vesicle at the Golgi. Three homologous proteins, called guanine nucleotide exchange factors (Arf‐GEFs), are responsible for exchanging GTP for GDP on Arf1 at the Golgi to activate the molecular switch. Sec7 (BIG1/2 in humans) turns on Arf1 to package secretory cargoes at the trans‐Golgi network (TGN), while Gea1 and Gea2 (GBF1 in humans) help Arf1 package retrograde cargoes into COPI vesicles for intra‐Golgi and Golgi‐to‐ER traffic. We have previously shown that Sec7 utilizes autoinhibition, positive feedback, and interactions with multiple small GTPases to regulate its activity and localization. Identifying the corresponding information used by Gea1/2 to make the decision about when and where to activate Arf1 is crucial to understanding how trafficking is regulated between the three Golgi Arf‐GEFs.

We have identified three players in the decision‐making process for Gea1/2. First, all C‐terminal domains of each protein are required for regulating recruitment of the GEFs to membranes in vitro and some are required for viability of cells or cellular localization, highlighting the complex nature of coordinated signals. Second, the composition of the membrane surface is important: unlike Sec7, Gea1/2 prefer neutral to negatively‐charged membranes in vitro, paralleling the more neutral early Golgi compartments and negatively‐charged, phosphatidylserine‐rich TGN in vivo. Lastly, the Rab GTPase Ypt1 (Rab1 in humans) aids in recruiting Gea1/2 to membranes in vitro, in agreement with prior findings that overexpression of Ypt1 can suppress temperature sensitive mutations in Gea1/2.

Together, these results begin to reveal the careful orchestration of retrograde Golgi membrane trafficking through coincidence detection. By sensing multiple signals for activation and recruitment, the Golgi Arf‐GEFs are able to carefully calibrate their activity and localization to achieve specificity in cargo packing and timing of vesiculation. Our ongoing work focuses on identifying the signals that fine‐tune compartment specificity within the Golgi and understanding the molecular mechanisms of these complementary interactions.

Support or Funding Information

This work was funded by the NIH (T32 GM07273 and R01 GM098621) and by the Harry and Samuel Mann Outstanding Graduate Student Award.
Variants in the Toll‐interacting protein gene (rs5743899) and susceptibility to Plasmodium falciparum malaria infection in West Africa
Authors: Farid Iman , Funwei Roland I , Agyingi Smith , Snyder Tara , Falade Catherine , Ojurongbe Olusola , Thomas Bolaji N
Published: November 2023

Malaria remains a very significant disease, with high rates of morbidity and mortality, contributing to economic inequities in endemic countries. The parasite is widely distributed with extensive genetic polymorphisms, increasing rates of drug resistance and lack of an effective vaccine. Dysregulated immune response, especially focused on signaling or pathogen recognition factors in immune system pathway is critical to understanding disease severity or pathophysiology. Toll‐interacting protein (TOLLIP) regulates human toll‐like receptor signaling pathways, and its polymorphisms have been associated with infectious disease. To elucidate whether TOLLIP polymorphisms are associated with Plasmodium falciparum infections in West Africa, we examined the genetic frequency of TOLLIP gene polymorphisms (rs5743899 and rs3750920) in 110 patients, with symptomatic, clinical malaria (diagnosis confirmed by microscopy and polymrease chain reaction) and 371 controls (PCR‐negative), recruited from south‐west Nigeria. Genomic DNA samples between groups were analyzed with a polymerase chain reaction and restriction fragment length polymorphism assay. Our results show that the Tollip rs5743899 mutant variant had the lowest frequency compared to the wild type variant, in addition to an extensive disparity between malaria‐infected patients and uninfected controls. There was a significant difference in the genotypic frequency of the rs5743899 mutant variant between malaria‐infected and control groups respectively in West Africa (13.7% versus 20.8%; p=0.09), but not in the rs3750920 polymorphism (57.3% versus 49.3%; p=0.16). This present observation reveals that Tollip gene rs5743899 mutant variant is potentially associated with Plasmodium falciparum malaria infection in West Africa. Interestingly, the most common haplotype among both groups is H2 (40.4% versus 44.1% for malaria‐infected and uninfected controls respectively), combining the mutant variant for both Tollip polymorphisms. Conclusively, this report shows that susceptibility to malaria infection in West Africa and potential contribution to disease severity or preponderance is significantly associated with host genetic variation, with toll‐interacting protein gene a significant member.

Support or Funding Information

Funding provided by College of Health Sciences and Technology Faculty Development and Laboratory Support Fund, Rochester Institute of Technology
Construction of a System for the Study of Protein‐Induced Membrane Tubules
Authors: Rivera Alba Katiria González , Forest Katrina T.
Published: November 2023

Communication between cells and the environment is crucial to life, and correspondingly cells have evolved complex signaling pathways. For example, bacteria have a repertoire of cellular structures that mediate adhesion, motility, virulence, etc. Some of the well‐studied systems are pili, flagella, and multiple secretion systems. Recently, more attention has been paid to membranous structures such as secreted vesicles and membrane appendages. These structures have been shown to play roles in adhesion, cell‐to‐cell communication, and pathogenesis. Membrane appendages appear to be very diverse structures with possible distinct assembly mechanisms. For instance, membrane appendages can appear as tubular structures with a size range of 20–70 ηm in width that extend from the bacterial membrane for very long distances. These membrane appendages are sometime referred as nanotubes and in some instances they have been shown to play a role in metabolic exchange. One study showed that Delftia sp. can form nanopods as the result of the release of outer membrane vesicles that fused together1. Other membrane tubules have been shown to contain protein filaments, such as sheathed flagella and unnatural pili.

In our laboratory we attempted to construct a heterologous system for bacterial pilus assembly. However, we found that under special conditions we could trick E. coli to elaborate structures that resembled nanotubes. In our initial studies we expressed different proteins from the Pseudomonas aeruginosa Type‐4 pilus system in E. coli Bl21‐AI, E. coli BL21 (DE3), or E. coli NovaBlue (DE3) and induced protein expression with IPTG and/or arabinose when necessary. Using negative staining and electron microscopy, we observed tubular structures (about 20–40 ηm wide, and longer than 1 μm). Remarkably although formation of these extensions was dependent on the major component of the pilus, PilA, it was independent of pre‐pilin cleavage by pre‐pilin peptidase; we found long membrane tubules in the presence of a non‐cleavable pilin. This system promises a means to investigate in detail how bacteria can build nanotubes and how this process is affected by the presence of transmembrane proteins.

Support or Funding Information

NIH 5 T32 GM008349
Effects of Curcumin on Vesicular Stomatitis Virus (VSV) Infection and Dicer‐1 Expression
Authors: Ahmed Jemil , Tan Yuxiao , Ambegaokar Surendra
Published: November 2023

Vesicular stomatitis virus (VSV), part of Rhabdoviridae, can cause severe illness in many important livestock, such as horses, cattle, and sheep. Curcumin (CUR) is a polyphenolic extract from the spice turmeric, and has a variety of pharmacological benefits including anti‐inflammatory, antioxidant and anti‐viral activities. However, there is limited work on its effects on VSV infection, or a mechanistic description of its anti‐viral properties. The initial goal of this study was to determine if CUR could restrict VSV infection in Vero cells, and if so, to determine the minimal concentration required for restriction. We observed that 10μM of CUR provided robust inhibition of recombinant VSV‐EGFP infection of Vero cells, as measured via plaque assay and fluorescence, with approximately 33% reduced infection at MOI 0.0002 and nearly 90% reduction at MOI 0.00002 after 24 hours. We hypothesized RNAi mechanisms may be effective at restricting VSV infection, and measured Dicer‐1 mRNA via qPCR. We found cells infected with VSV‐EGFP and treated with CUR had higher levels of Dicer‐1 expression than VSV‐EGFP infected cells treated with vehicle (DMSO); Actin was used as reference gene. Preliminary results indicate CUR alone has limited effect on Dicer‐1 expression in the absence of VSV infection. This may indicate that VSV infection itself alters Dicer‐1 expression; CUR restriction of VSV infection may rescue the effect on Dicer‐1 expression by inhibiting VSV replication through an as yet unidentified mechanism. It is know that VSV infection induces oxidative stress to promote apoptosis, and thus the antioxidant properties of curcumin may be the primary effect on VSV restriction, which future studies will investigate.

Support or Funding Information

Dept. of Botany & Microbiology, Neuroscience Program, and Summer Science Research Program at Ohio Wesleyan Univ.; Ohio Wesleyan Univ. Theory‐to‐Practice Grant.
Metabolic Stress Drives Keratinocyte Defenses Against Staphylococcus aureus Infection
Authors: Wickersham Matthew Adam , Wachtel Sarah , Lung Tania Wong Fok , Jacquet Rudy , Soong Grace , Richardson Anthony , Parker Dane , Prince Alice
Published: November 2023

OBJECTIVE

Human skin is commonly colonized and infected by Staphylococcus aureus. Under normal conditions skin is relatively hypoxic. Thus, we postulated that the burden of staphylococcal infection would result in competition for glucose and stimulation of HIF1a signaling to initiate keratinocyte metabolic and immune responses to infection.

METHODS

The metabolic activity of primary keratinocytes in response S. aureus metabolic mutants was monitored using a SeaHorse analyzer to measure oxygen consumption and extracellular acidification. A transcriptomic analysis of keratinocyte responses to S. aureus USA300 infected keratinocytes was performed and key results were confirmed by qRT‐PCR and immunoblot. The immunological consequences of Hif1a signaling were demonstrated in vitro, as the metabolic and inflammatory activities of primary keratinocytes were studied through qRT arrays, western blots, ELISA assays and confocal imaging. The in vivo consequences of the glycolytic response to infection were confirmed in vivo in a murine model of skin infection.

Using a combination of metabolic and transcriptomic methodologies, we found that S. aureus infection is sensed as a metabolic stress by keratinocytes. This is sufficient to induce HIF1a signaling, which promotes IL‐1b production and generates components of the aerobic glycolysis pathway to meet the metabolic requirements of infection. Staphylococci capable of glycolysis, including WT and agr mutants readily induced HIF1a. In contrast, pyk glycolytic mutants failed to compete with keratinocytes for their metabolic needs. Although S. aureus activate the Akt/mTOR cascade, inhibition of this pathway did not inhibit the keratinocyte cytokine response to infection. However, 2‐DG mediated suppression of glycolysis blocked keratinocyte production of IL‐1b in vitro and significantly exacerbated the S. aureus cutaneous infection in a murine model.

CONCLUSIONS

S. aureus is perceived as a metabolic stress by infected keratinocytes. These keratinocytes respond through HIF1a signaling, increasing glycolytic activity and generating IL‐1b. S. aureus glycolytic mutants, independent of toxin production, fail to activate keratinocytes.

Support or Funding Information

FUNDING: The results presented in this abstract were supported by the National Institutes of Health under award number RO1AI103854.
Identification of NADPH‐dependent Glutamate Dehydrogenase in Yarrowia lipolytica
Authors: Nelson Kjersten , Juco Karen , Tamayo Lizeth , Nicaud Jean‐Marc , Trotter Pamela J.
Published: November 2023

The recent mapping of the genome of the nonconventional oleaginous yeast species Yarrowia lipolytica has sparked interest in its usefulness as a model for fat metabolism and as an alternative to Saccharomyces cerevisiae. However, for Yarrowia lipolytica to be more effective as a model organism, more information must be obtained about its metabolism. This study focused on the gene that encodes NADPH‐dependent glutamate dehydrogenase (GDH) in Y. lipolytica. The aims of this study were to test gene complementation in S. cerevisiae and to analyze the phenotype of gene deletion in Y. lipolytica. The NADPH‐GDH gene in Y. lipolytica (ylGDH1/3) was identified by homology to NADPH‐dependent GDHs in S. cerevisiae (scGDH1 and scGDH3). For the complementation test, the ylGDH1/3 was amplified by PCR, cloned into the S. cerevisiae pBEVY‐L vector under control of the constitutive GPD/ADH1 promoter and verified by restriction digests and sequencing. The pBEVY‐L‐ylGDH1/3 was introduced into a S. cerevisiae null mutant strain that lacks any GDH activity. The subsequent gain of GDH activity to a level greater than that in wild type demonstrated the NADPH‐dependent GDH activity of the protein encoded by ylGDH1/3. For gene disruption in Y. lipolytica, overlap extension PCR was used to create a ylGDH1/3 disruption cassette containing the selection marker LEU2 (gdh1/3::LEU2) from Y. lipolytica. The cassette was transformed into Y. lipolytica forming a mutant strain via homologous recombination. PCR analysis confirmed the disruption and enzyme activity measurements indicate a significant decrease in NADPH‐dependent GDH activity in the knockout strain, which further suggests that ylGDH1/3 in Y. lipolytica encodes NADPH dependent GDH. Future investigations include examining how the NADH dependent GDH activity is affected in the mutant strain. This valuable information about the yeast's metabolism may further aid researchers in understanding the metabolic pathways, as well as using Y. lipolytica as a model to learn more about metabolic diseases such as diabetes and obesity.
Different Lipids in Synaptic Vesicle and Synaptosome Membrane
Authors: Lewis Kenneth T , Maddipati Krishna R. , Naik Akshata R. , Jena Bhanu P.
Published: November 2023

Supramolecular 15 nm cup‐shaped lipoprotein structures called porosomes present at the presynaptic membrane, mediate neurotransmitter release via the transient fusion of synaptic vesicles at its base via SNAREs. Major lipids associated with the isolated neuronal porosome complex have been determined to be unique in composition. Since lipids are known to greatly influence various membrane protein association and functions including membrane fusion, the composition of lipids in synaptic vesicle and the synaptosome membrane, was hypothesized to be unique and tightly regulated. To test this hypothesis, the lipidome of isolated synaptosome, synaptosome membrane, and synaptic vesicles, was determined using mass spectrometry. Results from the study demonstrate a unique lipid composition of the synaptic vesicle compared to the synaptosome membrane, reflecting on the tight regulation of nerve cells in maintaining the lipid composition of both organelles.

Support or Funding Information

Work presented in this article was supported in part by the National Science Foundation grants EB00303, CBET1066661 (BPJ); the WSU Interdisciplinary Biomedical Systems Fellowship and the Thomas C. Rumble University Graduate Fellowship (KTL); and the WSU Interdisciplinary Biomedical Systems Fellowship (ARN).
Investigating The Spatiotemporal Distribution Patterns of PCID2 between The Nucleus And The Centrosome
Authors: Flores Regino K , Resendes Karen K
Published: November 2023

PCID2 is a subunit of the TREX‐2 mRNA export complex that is also involved in the process of CRM1‐mediated nuclear protein export. Nuclear export is driven by RanGTP, which triggers a structural modification of CRM1 resulting in increased affinity for the leucine‐rich NES. Subsequent GTP hydrolysis following translocation across the NPC causes the CRM1‐cargo complex to dissociate. Karyopherins can also serve functional roles at non‐nuclear locations in the cell. For example, both CRM1 and Ran are found at the centrosome in addition to the nucleus. Specific factors facilitate mitosis by restricting the centrosome to duplicate once during the cell cycle through alternating nuclear and centrosomal localization. Specifically, CRM1 has an integral role in localizing nucleophosmin to the centrosomes to facilitate this “licensing” process. Leptomycin B inhibition of CRM1 causes dissociation of nucleophosmin from the centrosomes. In doing so it leads to centrosome reduplication within the same cell cycle. In addition to its nuclear function, PCID2 is also localized to the centrosome in a subset of cells. The function of PCID2 at the centrosome is not yet elucidated, but we demonstrate here that PCID2 is in fact present in CRM1 complexes. This evidence gives us reason to believe that PCID2 may serve a role at the centrosome that is supplementary to that of CRM1 and Ran. We hypothesize that PCID2 is localized to the centrosome as a part of the CRM1‐RanGTP complex and may translocate from the nucleus to the centrosome. To investigate the relationship between PCID2 in the nucleus and at the centrosome, we performed immunofluorescence of PCID2 in HeLa cells treated with Leptomycin B, a CRM1‐dependent nuclear export inhibitor. Preliminary results shows that LMB treatment causes a decrease in nuclear intensity of CRM1 and a potential decrease in PCID2, while causing an increase in the incidence of cells exhibiting punctate staining of PCID2. Because we know that LMB treatment leads to unregulated duplication of centrosomes, these findings suggest a correlation between increased PCID2 punctate staining and multiple centrosome duplications. This is consistent with our previous work showing that PCID2 is most commonly localized to the centrosome in G2 when duplication is occurring. These findings could also imply that PCID2 has a role in the localization of centrosomal components or mitotic proteins to the centrosome from the nucleus. Future experiments will investigate whether increased PCID2 punctate staining is indicative of a role in excess centrosome duplication or whether LMB is instead altering the CRM1/PCID2 localization pattern, causing centrosomal accumulation of PCID2.
Functional Insights on the Human Adenine Nucleotide Translocator Interactome
Authors: Acoba Michelle Grace , Lu Ya‐Wen , Kandasamy Selvaraju , Huang Tai‐Chung , Nirujogi Raja , Pandey Akhilesh , Claypool Steven M.
Published: November 2023

The adenine nucleotide translocator (ANT), by mediating the exchange of ADP for ATP across the inner mitochondrial membrane, lies at the core of oxidative phosphorylation (OXPHOS). Discrepancies in ANT expression and/or activity resulting to reduced energy metabolism are linked to a wide spectrum of pathologies, including lactic acidosis, progressive external opthalmoplegia, muscular dystrophy and cardiomyopathy. In yeast, the major isoform was modeled for a long time to exist in isolation, but was demonstrated to physically interact with respiratory supercomplexes (RSCs) exclusively in the context of the phospholipid cardiolipin (CL). OXPHOS in CL‐null yeast is remarkably decreased in optimal situations and completely eliminated under stressful conditions, pointing to the physiological significance of these protein‐protein associations. In this study, we determined that human ANTs also have the capacity to assemble into high molecular weight complexes. Additionally, we defined the interactome of two human ANT isoforms using an unbiased proteomics approach. Similar to the yeast orthologue, we saw RSCs and other solute carriers as binding partners, which was surprising given that the organization of yeast and mammalian ANTs is different, with Complex I missing in yeast. To probe the physiological relevance of the ANT‐RSC interaction, we deleted the major ANT isoform in HEK293, which unexpectedly only had a moderate effect on OXPHOS, indicating that the minimal levels of the other isoforms can still support respiratory capacity. As a reciprocal inquiry, we ablated OXPHOS function and found that ADP/ATP transport by ANTs is impaired. These results provide insights regarding the physiological relevance of evolutionarily conserved interactions of proteins important for mitochondrial function.

Support or Funding Information

This work was supported by the National Institutes of Health (R01HL108882) to S.M.C and pre‐doctoral fellowships from the American Heart Association (16PRE31140006 to M.G.A. and 12PRE11910004 to Y‐W.L.).
Impact of Acute Dietary Vitamin A Intake on the Measured Variable (Plasma Retinol Specific Activity) Used in Prediction of Total Body Vitamin A Stores by Retinol Isotope Dilution
Authors: Ford Jennifer Lynn , Green Joanne Balmer , Green Michael H
Published: November 2023

Retinol isotope dilution (RID) equations are used to estimate vitamin A (VA) status (total body stores; TBS) and the efficacy of VA intervention programs after ingestion of labeled VA. Researchers have been concerned that dietary VA intake between dose administration and blood sampling (i.e., during isotope equilibration) might significantly affect TBS predictions. We recently published equations (Green et al. J Nutr 2016;146:2137–42 and 2407–11) developed from the “Olson equation” (Furr et al. Am J Clin Nutr 1989;49:713–6) that predict TBS from 1/specific activity (SAp; tracer: tracee) of plasma retinol 4 d after dosing plus several correction factors related to dose absorption and retention, equilibration of tracer between plasma and stores, and VA catabolism during equilibration; we showed that the measured variable, SAp, is the factor in the equation that has the greatest impact on TBS. To address the concern about VA intake after dosing, we used the powerful tool of model‐based compartmental analysis to simulate the effect of restricted VA intake (0 μg/d) vs. recommended intakes (400 μg/d for children and 800 μg/d for adults) on SAp at 4 d, assuming VA fractional catabolic rates of 2.2%/d (children) or 0.5%/d (adults). Intakes of 400 or 800 μg/d, compared with 0, resulted in dilution of 4 d SAp by <5%. Our results indicate that the effect of intake is small because dietary VA input represents <10% of total daily retinol input into plasma. We also further examined the contribution of 1/SAp to TBS predictions derived by model‐based compartmental analysis (WinSAAM) for data from three adult populations (Cifelli et al. J Nutr 2008;138:971–7 and Green et al. J Nutr 2016;146:2129–36). These analyses indicate that, across a wide range of TBS (35 – 2500 μmol), 1/SAp at 4 d and TBS were significantly correlated and the measured variable accounts for the vast majority of the variance in model‐determined TBS. Our results suggest that newly ingested dietary VA is a minor contributor to total daily plasma retinol input compared with VA in readily exchangeable storage pool(s); in other words, VA in exchangeable pools has a greater impact on SAp at 4 d after tracer dosing than VA intake during this period. In conclusion, these data suggest that, at VA intakes near the RDA for children and adults, dietary VA does not dilute plasma retinol specific activity at 4 d after dosing to any substantial degree and thus should have a minimal effect on this variable in the RID equation.

Support or Funding Information

International Atomic Energy Agency, Bill and Melinda Gates Foundation (149953), and College of Health and Human Development, Penn State University
Hepatic Uptake of Vitamin A is Independent of Retinoic Acid Pretreatment in Neonatal Rats
Authors: Li Yaqi , Green Michael H. , Tan Libo , Ross A. Catharine
Published: November 2023

Previous studies have shown that total retinoid concentration in the liver of neonatal rats would increase in response to the VARA supplementation, which is a combination of vitamin A (VA) and retinoic acid (RA). Later studies have further demonstrated that a single dose of VA may stimulate the hepatic uptake, which in turn, significantly elevated the liver total retinol concentration. In current study, we gave the neonatal rats the RA treatment prior to the VA supplementation, and examined the distinct effect of RA on VA metabolism and kinetics in the liver tissue of these young animals. Sprague‐Dawley male and female rats were mated, and the dams were fed a VA‐marginal diet (0.35 mg retinol equivalents/kg diet) and were allowed to consume food and water ad libitum. On postnatal day (P) 2 and 3, pups from experimental group received an oral dose of RA (0.625μg/g body weight), while other pups received canola oil as the control treatment. Then, an oral dose of VA (6 μg retinyl palmitate/g body weight), containing 2 μCi of [3H]‐retinol as a tracer, was administered to all pups on P4. Pups (n = 4–6/treatment/time point) were euthanized from 25 min to 30 h, a total of 12 time points, after dose administration and the liver were collected. Tissue total retinoid mass was determined by ultra‐performance liquid chromatography (UPLC), and the tissue tracer level was measured by liquid scintillation counting. Results showed that in both oil‐pretreated and RA‐pretreated groups, the fraction of retinyl ester and retinol entering the liver increased continuously and peaked at 10 h. After that, the fraction of retinol slightly declined and remained at the same level until the end of the study. However, the fraction of retinyl ester decreased gradually until the 20hr time point and showed a trend of increase afterward. At the same time, liver vitamin A content of both groups increased gradually and peaked firstly at 10 h, after a slight decrease it reached a second peak at 20 h and declined in the following period of time. In conclusion, our findings indicate that liver uptake of dietary vitamin A may be tightly controlled and would not be affected by RA supplementation.

Support or Funding Information

NIH grant HD006982
Spirulina maxima Extract Protects against TMT‐induced cognitive disorder in SH‐SY5Y cells
Authors: Chei Sungwoo , Seo Young‐Jin , Koh Eun‐Jeong , Choi Jia , Hwang Ji‐Hyun , Lee Yeon‐Joo , Song Ji‐Hyeon , Choi Seo‐Yun , Park Min‐Ki , Lee Boo‐Yong
Published: November 2023

Spirulina maxima, which is used as a food additive, is a microalgae rich in protein and other essential nutrients. In the resent study, it has been shown that spirulina maxima has antioxidant, antiobesity and anticancer effects. We performed with Spirulina maxima extraction using ultrasonication. Treatment of trimethyltin(TMT) induces neurotoxicity in central nervous system and is used in model of cognitive disorders. TMT is a neurotoxic compound which causes a dramatic pattern of neuronal cell death. In this results, treatment with TMT decreased cell viability. We observed that Spirulina maxima ameliorates decreasing cell viability induced by TMT through using MTT assay in SH‐SY5Y cells. Spirulina maxima extract increased protein levels of p‐CREB and BDNF. Additionally, spirulina maxima extract suppresses expression of oxidative‐associated factors. In this results, spirulina maxima extract improves the neuroprotective related proteins against TMT‐induced cognitive disorders in SH‐SY5Y cells.
Characterization of a Hepcidin Knockout Rat, a Novel Model of Iron Overload
Authors: Flores Shireen RL , Wang Xiaoyu , Ha Jungheun , Doguer Caglar , Wang Tao , Collins James F
Published: November 2023

Genetically‐modified mouse models of altered iron homeostasis have made invaluable contributions to our understanding of the regulation of iron metabolism in humans. Emerging evidence, however, suggests that mice may not be the best model for all aspects of human physiology/pathophysiology. For example, mice display a resistance to liver cirrhosis and diabetes, which is important when investigating iron‐overload disorders, where dysfunction of the endocrine pancreas and liver damage are common consequences of excess iron deposition. Fortunately, recent advances in technology have now made genetic manipulation feasible in rats, making it possible to produce genetically‐modified rats that may better resemble some (but not all) human diseases, including those that arise from iron dysregulation.

Objective

In order to make comparative observations between rats and mice in regards to regulation of iron metabolism, we have developed a hepcidin knockout (Hamp−/−) Sprague‐Dawley (SD) rat. To our knowledge, this is the first attempt at direct genetic manipulation of the Hamp gene in rats. Unlike, many mouse strains used for iron biology research, SD rats are outbred, better reflecting genetic diversity in the human population.

Rationale

The rationale for this experimental approach was based upon the numerous pathophysiologic differences noted between mouse models of hereditary hemochromatosis (HH) (a group of genetic iron‐overload disorders) and humans with HH.

Hypothesis

We hypothesized that the Hamp−/− rats would exhibit iron loading, in the liver, heart and pancreas, similar to what is seen in humans with juvenile HH (which may arise from mutations in the Hamp gene). Moreover, since rats have a longer life span than mice, we postulated that they would be more likely to develop tissue damage associated with iron loading.

Results

Our initial characterization has confirmed that the Hamp−/− rats develop early and severe multivisceral iron overload, with sparing of splenic macrophages. Interestingly, the Hamp−/− rats displayed a periportal pattern of hepatic iron deposition, similar to what is seen in humans, but distinct from the centrolobular iron‐loading pattern seen in Hamp−/− mice.

Conclusions

These data demonstrate that rats lacking hepcidin do indeed load iron, and further that key differences may exist between mice and rats in terms of iron loading patterns and regulation of iron metabolism. It is thus possible that rats may better model disturbances of iron metabolism as compared to mice, although additional studies are required to definitively make this determination.

Support or Funding Information

Supported by NIH grant 1R01 DK074867 (to J.F.C.).
Association Between Weight‐related Health Behaviors and Weight Perception in Adolescents with Overweight or Obesity
Authors: Borton Kelley A. , Hahn Samantha L. , Sonneville Kendrin R.
Published: November 2023

Weight misperception is the belief that one is of a normal or healthy body weight despite being overweight or obese. Weight misperception has been established as protective against further weight gain in adolescents, in comparison to those who accurately perceive their elevated weight status. However, less is known about the individual factors that may contribute to this relationship. Therefore, our objective was to identify health and weight related behaviors that may contribute to weight misperceivers gaining less weight over time. Using data from the Youth Risk Behavior Survey, a nationally representative sample of adolescents enrolled in grades 9–12, we specifically examined those that met the criteria with overweight and obesity (n=4,536). Participants were classified as either accurate perceivers of their overweight or obese weight status (n=1368), or misperceivers (n=3103). Logistic regression analyses were run using SPSS, using weight misperception as the exposure variable, and examining various weight related health behaviors as the outcomes, controlling for sex, race/ethnicity, and grade. Adolescents who misperceived their weight were less likely to be trying to lose weight (OR: 0.169, 95% CI: 0.144–0.199, p<0.001). Weight misperceivers were also more likely to eat vegetables at least two times per day (OR: 1.235, 95% CI: 1.067–1.430, p=0.005), and to eat fruit or drink 100% fruit juice at least two times per day (OR: 1.169, 95% CI: 1.015–1.346, p=0.030). Weight misperceivers were less likely to spend three or more hours per day playing computer games (OR: 0.169, 95% CI: 0.144–0.199, p<0.001). Adolescents who misperceived their weight were more likely to regularly eat breakfast (OR: 1.125, 95% CI: 0.969–1.305), and less likely to spend three hours per day watching television (OR: 0.995, 95% CI: 0.859–1.153), but the odds ratios were not statistically significant. Weight misperceivers were also more likely to drink at least one soda per day (OR:1.158, 95% CI: 0.991–1.354), but this was not significant. Adolescents with overweight or obesity who misperceive their weight as normal are more likely to engage in a number of healthy weight‐related behaviors, which may be a potential explanation for why those that misperceive their weight, gain less weight over time. Identifying both positive and negative health behaviors that differ based on perception status that may lead to weight change, can help researchers determine possible intervention targets that focus on health behaviors for adolescents who are overweight or obese.
Effects of a high‐fat diet on spontaneous locomotor activity and blood metabolic biomarkers in Sprague Dawley rats
Authors: Chia‐Ying Wu , Yang Yan‐Ting , Chen Ching‐Han , Chang Jui‐Chou , Tsai Shiow‐Chwen , Chen Chung‐Yu , Chen Chiao‐Nan , Bernard Jeffrey R. , Liao Yi‐Hung
Published: November 2023

Purpose

Obesity commonly results from the combination of excessive caloric intake and lack of physical activity. However, it is not well known if a chronic high‐fat diet leads to negative changes in resting physical activity level, which would be expected to accelerate the development of obesity. Therefore, the purpose of this study was to investigate whether a 10‐week high‐fat diet would suppress spontaneous locomotor activity in Sprague Dawley rats.

Methods

Sixteen male Sprague Dawley rats were randomly assigned into one of two treatment groups: 1) standard diet (SD: 10% fat, 20% protein and 70% carbohydrate) or 2) high‐fat diet (HFD: 45% fat, 20% protein and 35% carbohydrate). After one‐week acclimation, rats were fed either the SD or HFD for 10‐weeks. Throughout the dietary intervention period, body weight, water and food intake were recorded on a weekly basis. In addition, body composition (via DEXA) and spontaneous locomotor activity (via image analysis) were measured at the 9‐week time point. Immediately following the completion of the dietary intervention fasting blood glucose, plasma insulin, HbA1c and the hormones leptin and adiponectin were assess. Thereafter, an intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed at two‐day intervals in order to evaluate whole‐body glucose metabolism. All results were analyzed using a t‐Test. Significance was set at p≤0.05.

Results

Spontaneous locomotor activity (inactive time, standing frequency, movement distance, and movement velocity) were not different between SD and HFD treatment groups. Consumption of the high‐fat diet significantly increased weight gain and increased fat accumulation (whole body and visceral part), while significantly decreasing muscle mass compared to the control diet (p<0.05). In addition, rats fed the high‐fat diet showed a significant increase (by ~310%) in circulating leptin levels compared to rats fed the standard diet (p<0.05), whereas the adiponectin levels were not different between treatment groups. However, blood glucose, HbA1c and the homeostatic model assessment (HOMA) were all significantly elevated for HFD compared to SD (p<0.05). In addition, the area under the curve (AUC) for the ITT and IPGTT tests were also significantly greater for the HFD treatment group (p<0.05).

Conclusion

This investigation demonstrates that despite negative changes in body composition, impaired whole‐body glycemic control, and spontaneous locomotor activity were not affected by a high‐fat diet in Sprague Dawley rats. Thus, these results suggest that excessive caloric intake may play a more critical role in the development of obesity rather than the lack of spontaneous locomotor activity.
Anti‐Obesity Effects of Solar Salt Prepared from Concentrated New Sea Water on 3T3‐L1 Adipocytes
Authors: Park Kun‐Young , Park Eui‐Seong , Ju Jaehyun , Choi Shin‐Il
Published: November 2023

The anti‐obesity effect of Korean solar salt manufactured from concentrated new sea water on 3T3‐L1 adipocytes was studied. Our experimental groups consisted of S1 (conventionally manufactured solar salt by mixture of concentrated old‐ and new sea water), S2 (charcoal‐ and magnetic filtered during S1 preparation), S3 (solar salt made by concentrated new sea water), and S4 (charcoal‐ and magnetic filtered during S3 preparation). S3 suppressed lipid droplet formation in the differentiated adipocytes (oil‐red O staining), reduced secretion of leptin. Also, S3 significantly suppressed gene expression of lipogenesis‐ and adipogenesis‐related factors (PPARγ, SREBP‐1c, C/EBPα, LXR, LPL, FAS, DGAT and MTP; p<0.05), while promoting β‐oxidation‐related factors (PPARα and CPT‐1). Taken together, S3 was found to have anti‐obesity efficacy by suppression of lipogenesis and adipogenesis and promotion of β‐oxidation‐based lipolysis, probably owing to different processing methods to eventually lead to changes in mineral composition of the salt. Thus, a processing method of solar salt affects the anti‐obesity effect of the product.

Support or Funding Information

This work was supported by a grant 20130290 to Solar Salt Research Center of Mokpo National University from Ministry of Oceans and Fisheries of Korea.
Epigenetic Regulation of Tumor Necrosis Factor (Tnf) in Skeletal Muscle by Chronic Caloric Restriction
Authors: Hernandez‐Saavedra Diego , Goldberg Zachary Jacob , Wang Alex Po‐Hsiang , Chen Hong , Pan Yuan‐Xiang
Published: November 2023

Caloric restriction (CR), without malnutrition, is considered an effective dietary intervention to extend lifespan and quality of life. Such effects include those related to musculoskeletal health where chronic CR restores the phenotype of aged individuals to that of the young. We demonstrated that CR reverted the effect of a high fat diet and effectively reducing body mass. In skeletal muscle of the CR group, nutrient sensing and degradation pathways were activated. This could impact the canonical inflammatory marker TNF and related pathways, which are involved in the metabolism and physiology of the muscle. However, the epigenetic mechanisms elicited by CR on muscle inflammation remain elusive. In our study, 4‐week old male Sprague‐Dawley rats were fed a high fat diet (HF, 45% Kcal from fat) ad libitum for 4 months. Animals were then divided into HF ad libitum, maintenance group fed a control diet (ML, with 100% energy requirement at baseline adjusted with body weight), and the caloric restriction group (CR, 25% energy reduction). A control group (AL) was fed a control diet ad libitum. The dietary intervention continued for six months before animals were sacrificed and skeletal muscle samples were collected for gene expression and epigenetic analyses. In the CR group, gene expression was increased for the p50 subunit of the inflammatory marker Nfκb, but a marked decrease in gene expression occurred for Ikbkɛ, Tnf, Ifnγ, Il‐1b and Il‐6 compared to both the HF and ML groups. Gene expression of DNA methyltransferase Dnmt1 was reduced by CR to the level similar to AL, whilst Dnmt3a was increased in ML and CR groups which were both previously exposed to HF. Moreover, gene expression of histone deacetylases (Hdac1, Hdac3), and H3K4 methyltransferase (Kmt2b) were upregulated in ML and CR groups, eliciting a lasting HF effect to part of the histone modifying machinery. On the other hand, histone methylases Suv39h1 and Ezh2 were downregulated by CR. CpG density analysis for the Tnf rat gene revealed two CpG‐rich regions within the gene body in intron 1–2 (182 bp) and exon 4 (146 bp), as well as two highly dense CpG islands at the 3′‐downstream region (100 bp and 480 bp). Further analysis was performed to confirm the methylation and histone modification status within these regions. The findings strongly indicate that chronic CR regulates myogenesis and wasting through transcriptional targeting of Tnf by recruiting DNA methyltranferases and histone modifiers at different regions with the TNF locus.
Effects of Sulforaphane in the Tumor Microenvironment of Triple‐Negative Breast Cancer
Authors: Slattery Ed , Cline Troy , Housley Lauren
Published: November 2023

Triple negative breast cancer (TNBC), an aggressive breast cancer phenotype that has a high rate of recurrence, is diagnosed in approximately 35,000 new breast cancer cases each year in the United States. Treatment options are limited due to the lack of common therapy targets in this subtype (estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2). Since cancer progression is linked to the supportive role of healthy human cells found in the tumor microenvironment (TME), including tumor associated macrophages (TAMs), new cancer chemoprevention therapies are being sought that disrupt the recruitment and supportive activity of these tumor associated cells. There is evidence that sulforaphane (SFN), a well‐tolerated isothiocyanate derived from cruciferous vegetables, may prevent the progression of breast cancer. SFN has been shown to directly decrease cell viability in TNBC cell lines, reduce mammary tumor growth in animal models, and modify cell proliferation markers in human breast tissue. Yet, effects in human tissue have been less pronounced than in isolated cancer cells, possibly due to cell interactions in the TME. Thus, there is a critical need to determine mechanisms of SFN action in the TME of TNBC to design more effective chemoprevention and treatment strategies. TAMs, which are recruited to the TME by chemokines, are known to aid in the growth and survival of breast cancer cells. One group reported a decrease in chemokine expression in human epithelial cells following SFN treatment. The objective of this study was to establish the effect of SFN treatment on cell proliferation and chemokine secretion of human TNBC cells (MDA‐MB‐231) grown under the influence of TAMs. A conditioned‐media approach was used to model non‐contact interactions found in the TME using THP‐1 human monocytes and MDA‐MB‐231 human TNBC cells. THP‐1 cells were differentiated in 30% (v/v) cancer‐conditioned media to create TAMs. MDA‐MB‐231 cells were then grown in 30% (v/v) TAM‐conditioned media for the remaining experiments. TAM‐educated MDA‐MB‐231 were treated with SFN (10 μM) or vehicle‐control. Alterations in cell proliferation and chemokine secretion were observed in the MDA‐MB‐231 cell line after SFN treatment. This research provides evidence of the potential use of SFN in chemoprevention strategies for particularly aggressive forms of breast cancer.

Support or Funding Information

California State University: 1) Student Award for Research & Creativity, 2) Research, Scholarly and Creative Activities Program