bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutational analysis of the Arabidopsis thaliana lysoglycerophospholipid acyltransferase AT1g78690
Authors: Nag Saparja , Garrett Teresa A.
Published: November 2023

Headgroup‐acylated glycerophospholipids (HAGPLs) are a class of poorly understood lipids responsible for supporting cell division and maintenance of the outer cell membrane in Escherichia coli. Overexpression of AT1g78690 in E. coli results in the accumulation of the HAGPL acylphosphatidylglycerol (acyl‐PG). However, this peripheral membrane protein, native to Arabidopsis thaliana, does not directly acylate PG but in vitro is a lysophospholipid acyltransferase that catalyzes the formation of di‐acylated glycerophospholipids, PE or PG, from mono‐acylated glycerophospholipid substrates, 1‐acyl lyso PE or 1‐acyl lyso PG respectively. AT1g78690 bears 39% and 41% sequence identity to human and Drosophila tafazzin, respectively, a monolysocardiolipin remodeling enzyme implicated in Barth Syndrome. Mutations to tafazzin are implicated in altered cardiolipin metabolism found in Barth Syndrome patients. To understand the catalytic and kinetic properties of AT1g78690, several amino acids that are 100% conserved among members of the lysophospholipid acyltransferase (LPLAT) superfamily, threonine‐63, asparagine‐66, histidine‐67, aspartate‐72, and arginine‐92 were targeted for mutation to alanine. Histidine‐67 and arginine‐92 are particularly interesting because mutation of analogous amino acids in tafazzin are associated with Barth Syndrome. Following cloning into pET28a for overexpression in E. coli, membranes will be isolated and used as the enzyme source for a thin layer chromatography based in vitro enzyme assay that follows the transfer of radiolabeled palmitate from palmitoyl Co‐A to lyso PE to form PE. Kinetic parameters, including the Michaelis‐Menten constant (Km) and catalytic efficiency (kcat) as well as substrate specificity will be examined to better understand the catalytic mechanism of AT1g78690 and its homologs.

Support or Funding Information

National Science Foundation Research at Undergraduate Institutions Grant #1516805 and #1152463
Saccharomyces cerevisiae Coq10, A Putative START Domain Protein Binds Coenzyme Q And Late‐Stage Q‐Biosynthetic Intermediates
Authors: Tsui Hui , Clarke Catherine
Published: November 2023

Coenzyme Q (ubiquinone or Q) functions as an essential redox‐active lipid in respiratory electron and proton transport in cellular energy metabolism, and is an important lipid‐soluble antioxidant in cellular membranes. In Saccharomyces cerevisiae, at least 13 nuclear‐encoded genes COQ1‐COQ11, YAH1, and ARH1 have been identified as necessary for Q biosynthesis and its function in respiration and as an antioxidant. Many of the Coq polypeptides responsible for Q biosynthesis assemble into a high molecular weight, multi‐subunit complex localized to the matrix side of the mitochondrial inner membrane. This study focuses on characterizing the Coq10 polypeptide, a putative steroidogenic acute regulatory (StAR)‐related lipid transfer (StART) domain protein in S. cerevisiae. Unlike Q‐less coq1‐coq9 deletion mutants, yeast coq10 null mutants contain near wild‐type steady‐state levels of Q6, but are nonetheless respiratory deficient. Yeast coq10 null mutants show decreased de novo Q biosynthesis and accumulate high levels of the early Q‐intermediates, especially during early log phase of growth. The structure of Caulobacter crescentus CC1736, an ortholog of yeast Coq10, contains a hydrophobic tunnel characteristic of StART domain proteins. Purified CC1736 is capable of binding Q and a farnesylated analog of a late‐stage Q biosynthetic intermediate. Overexpression of CC1736 in the coq10 null mutant is able to rescue the respiratory‐deficient phenotype and partially restore efficient Q biosynthesis, suggesting a strong functional conservation. These observations suggest the Coq10 StART domain protein is required for efficient Q biosynthesis, crucial for Q function in respiratory electron transport, and for Q to function as an antioxidant. Coq10 is speculated to be a Q‐binding protein interacting with the multi‐subunit Coq polypeptide complex and/or respiratory complexes, facilitating final delivery of Q from its site of synthesis to sites of function. In order to better understand the biological function of yeast Coq10 we describe the isolation and characterization of the high molecular weight Coq10 oligomer and are characterizing lipid ligand interactions with mature Coq10. The results will provide important clues about the physiological role of Coq10 as well as the molecular interactions required for its function. This research was supported by NSF MCB‐1330803 and Ruth L. Kirschstein National Research Service Award GM007185.

Support or Funding Information

This research was supported by NSF MCB‐1330803 and Ruth L. Kirschstein National Research Service Award GM007185.
Elevated Uptake of Glycerophosphoinositol Through the Git1 Permease Causes Cell Growth Inhibition in Saccharomyces cerevisiae
Authors: Ziegler Phillip , Nikiforov Alexiy , Robinson Benjamin , Bakalinsky Alan , O'Donnell Allyson , Patton‐Vogt Jana
Published: November 2023

The Git1 permease is responsible for the transport of the phospholipid metabolites glycerophosphoinositol (GPI) and glycerophosphocholine (GPC) in Saccharomyces cerevisiae. GPI is produced via the deacylation of phosphatidylinositol (PI), primarily by the phospholipase Plb3. Extracellular GPI is then transported into the cell via Git1 under phosphate or inositol limiting conditions, where it is catabolized to form inositol and glycerol‐3‐phosphate. Inside the cell, the inositol can then be utilized to produce PI. Unpublished data suggests that the α‐arrestins Aly1 and Aly2 regulate the endocytosis of the Git1 permease, as Git1 is retained at the cell surface in an aly1Δaly2Δ mutant. Importantly, provision of the Git1 substrate GPI to aly1Δaly2Δ cells constitutively expressing GIT1 results in growth inhibition. This inhibition is not observed when the other Git1 substrates, GPC and glycerol‐3‐phosphate (G3P), are provided. We are identifying proteins that are involved in this growth inhibition by GPI. To this end, tools have been developed to employ the Yeast Genomic Tiling Collection (a previously constructed library containing >95% of the functional yeast genome in 1,588 plasmids) to screen for genes that restore cell growth upon overexpression. S. cerevisiae cells constitutively expressing GIT1 and containing the tiling library will be screened for growth in the presence of an inhibitory concentration of GPI. In total, our results suggest a novel role for GPI in growth regulation.

Support or Funding Information

National Institute of Health Grant NIH R15 GM104876
IDENTIFICATION OF INHIBITORS OF ACSVL3, A THERAPEUTIC TARGET IN GLIOMA
Authors: Clay Emily , Shi Xiaohai , Liu Yanqiu , DiRusso Concetta C. , Black Paul N. , Watkins Paul A.
Published: November 2023

The objective of this study was to identify inhibitors of the enzyme Very Long Chain Acyl‐CoA Synthetase 3 (ACSVL3; SLC27A3). ACSVL3 is present in human glioma cells but not healthy glial cells, and knocking out ACSVL3 in the U87 human glioblastoma cell line has previously been shown to result in slower growth rate, fewer and smaller tumors formed in xenografts, and disrupted Akt signaling. Therefore, an inhibitor of its activity is desired for both understanding its mechanism of action in tumorigenesis and for potential chemotherapeutic usage. Several drugs and compounds that were previously shown to inhibit the homologous enzyme ACSVL1 (SLC27A2) were studied to see if they had similar inhibitory effects on ACSVL3. COS‐1 cells stably expressing ACSVL1 or ACSVL3 were harvested and cell pellets assayed for activation of [1‐14C]stearic acid (C18:0, a preferred ACSVL3 substrate) to its CoA derivative in the absence and presence of inhibitors. We also developed a fluorescence‐based assay using COS‐1 cells stably expressing a chimeric ACSVL1/3 protein containing the regulatory domain of ACSVL1 and the catalytic domain of ACSVL3. The regulatory domain of ACSVL1 conferred the ability to uptake the fluorescent fatty acid analog, C1‐BODIPY‐C12, in a process requiring functional ACSVL3 enzyme activity. Most ACSVL1 inhibitors tested also inhibited ACSVL3 using in vitro acyl‐CoA synthetase assays. These compounds inhibited BODIPY fatty acid uptake in COS‐1 cells expressing chimeric ACSVL1/3 in a dose‐dependent manner. One compound, Grassofermata/CB5, was a significantly more potent inhibitor of stearic acid activation by ACSVL3 than by ACSVL1 and was tested further. CB5 was the most potent inhibitor of stearic acid activation in U87 glioma cells. To assess effects on cell growth, CB5 was added to U87 cells in culture. At a concentration of 3 μM, CB5 drastically slowed cell growth rate but did not lead to increased cell mortality. The effects of CB5 on cell growth were reversible; when CB5 was removed from cells after several days of treatment, they reverted to a faster growth rate. A dose response curve of cell growth to CB5 concentration revealed a narrow therapeutic window. In conclusion, many known inhibitors of ACSVL1 are also potent inhibitors of ACSVL3, and the small molecule CB5 shows promise as a potential chemotherapeutic drug and tool to understand the mechanism(s) underlying the role of ACSVL3 in glioma.

Support or Funding Information

Kennedy Krieger institutional funds.
Dual Site Mechanism Governs Sphingosine Kinase 1 Membrane Binding and Biological Function
Authors: Pulkoski‐Gross Michael , Truman Jean‐Philip , Salama Mohamed , Clarke Christopher , Hannun Yusuf A. , Obeid Lina
Published: November 2023

Sphingosine kinase 1 (SK1) is an enzyme at a critical junction in sphingolipid metabolism as it can regulate the balance between the pro‐apoptotic lipids ceramide and sphingosine and the pro‐survival lipid sphingosine 1‐phosphate. Here we identify a novel membrane binding motif in SK1 which is responsible for interaction with anionic phospholipids in the membrane, such as phosphatidic acid (PA). We believe that this newly identified site works in conjunction with a previously identified hydrophobic site in order to regulate the biological function of SK1. In vitro biochemical analysis shows that the anionic binding site is responsible for binding to PA. However, both sites are needed to effectively bind to liposomes containing PA. This provides evidence for SK1's binding to membranes being dictated by both sites. In cells, these sites are important for membrane binding as shown in both fractionation experiments as well as membrane localization by confocal imaging. However, this effect isn't limited to biochemical observations as these sites also affect the biology of SK1.

It has been shown that SK1 is involved in regulating cell motility in cancer cells including invasion and involvement in the phosphorylation of Ezrin‐Radixin‐Moesin (ERM) proteins. We show that the mutation of either of these sites, in cells that overexpress these mutant SK1s and in which endogenous SK1 is knocked down with CRISPR technology, is sufficient to abrogate invasion of colon cancer HCT116 cells to invade through the Matrigel. Furthermore, when these mutants are combined this dual site mutant is no longer able to result in the phosphorylation of ERM proteins. Finally, membrane disruption with methyl β‐cyclodextrin, induces pit formation and co‐localizes SK1 and Endophilin‐2 as previously shown. However, when either site is mutated this co‐localization no longer happens even in the presence of Endophilin‐2 associated pits showing these sites are important for SK1s function in endocytosis. Altogether, our data suggest that SK1 uses a dual site mechanism to ensure that it can function at membranes as well as find the correct context in which it should be active.

Support or Funding Information

This work was supported by NIH grants P01CA097132 to LMO and YAH as well as a National Research Service Award F31 CA196315 to MPG.
Acyl‐CoA Thioesterase 1 regulates hepatic lipid metabolism and PPAR‐α signaling
Authors: Franklin Mallory P , Sathyanarayan Aishwarya , Mashek Douglas
Published: November 2023

Peroxisome proliferator‐activated receptor alpha (PPAR‐α) is a major transcription factor that governs the expression of fatty acid oxidation (FAO) and anti‐inflammatory genes during fasting. Acyl‐CoA thioesterase 1 (ACOT1) hydrolyzes acyl‐CoAs to long‐chain free fatty acids and Coenzyme A during fasting. ACOT1 activity seemingly removes acyl‐CoA substrate for mitochondrial FAO and produces long‐chain free fatty acids that could serve as ligands to activate PPAR‐α. The objective of this research was to identify the role of ACOT1 in hepatic fasting lipid metabolism. To test our objectives, we injected C57Bl/6 mice with an adenovirus harboring shRNA targeted to ACOT1 or a scrambled control. Week‐long liver specific knockdown of ACOT1 increased FAO in vivo measured by serum β‐hydroxybutyrate and reduced liver triglyceride. These data correlated to results from in vitro fatty acid tracer studies in which ACOT1 knockdown increased FAO and triglyceride turnover. Despite increased FAO, ACOT1 knockdown reduced the expression of PPAR‐α target genes involved in FAO. In addition, ACOT1 knockdown led to increased expression of inflammatory and oxidative stress genes, due to reduced PPAR‐α dependent anti‐inflammatory signaling. Moreover, in vitro experiments exhibited increased PPAR‐α reporter activity in COS7 cells overexpressing ACOT1. The ability of ACOT1 to increase PPAR‐α activity was dependent on its catalytic activity, suggesting that ACOT1 produces fatty acids that serve as ligands for PPAR‐α. To confirm this theory, mice were supplemented with a synthetic PPAR‐α ligand, Wy‐14643, which rescued the expression of PPAR‐α target genes and mitigated the inflammation and oxidative stress observed in response to ACOT1 knockdown. Importantly, ACOT1 exhibits nuclear localization during prolonged fasting or under β‐adrenergic stimulation, suggesting a local pool of free fatty acids activate PPAR‐α.Together these studies suggest that ACOT1 coordinates the rate of fatty acid oxidation while simultaneously producing fatty acids that activate PPAR‐α, potentially in the nucleus, to protect the liver from inflammation and oxidative stress.

Support or Funding Information

NIH/NIDDK T32 Training grant, Minnesota Obesity Training Program Predoctoral Fellowship (DK083250), awarded to Mallory Franklin.
Macrophage catabolism of aggregated lipoproteins using a novel extracellular compartment regulates lipid accumulation during atherosclerosis
Authors: Singh Rajesh K , Haka Abigail S , Barbosa‐Lorenzi Valeria C , Asmal Arky , Lund Frederik , Xiong Yuquan , Chin Harvey F , Grosheva Inna , Hla Timothy , Maxfield Frederick R
Published: November 2023

Despite impressive advances in research, prevention, and treatment, atherosclerotic vascular disease remains the leading cause of death in the developed world. Mechanisms of cholesterol accumulation in the arteries have been studied intensively, but the in vivo contributions of different pathways leading to lipid accumulation and foam cell formation are not understood. In the arteries, low‐density lipoprotein (LDL) is aggregated and bound to the extracellular matrix. When such aggregated LDL is presented to macrophages, they form a novel acidic, hydrolytic compartment that is topologically extracellular, to which lysosomal enzymes are secreted. Using state‐of‐the‐art quantitative and high resolution microscopy techniques, characterization of compartment morphology reveals how macrophages use local actin polymerization to drive plasma membrane remodeling at the interface with aggregated LDL. This leads to sequestration of aggregated LDL into topologically convoluted structures that allow acidification, catabolism and internalization of LDL. Such compartments are observed in vivo in murine atherosclerotic plaque macrophages interacting with cholesterol rich deposits. We find that a TLR4/MyD88/Syk/PI3 kinase/Akt dependent signaling pathway in macrophages regulates the formation of such catabolic compartments. Consistent with this, deficiency of TLR4 in vivo can protect macrophages from lipid accumulation in murine atherosclerotic plaques. Herein, we provide compelling evidence for a novel form of catabolism that macrophages use to degrade aggregated LDL in vivo during atherosclerosis and this process leads to foam cell formation, cell death and promotes disease progression.

Support or Funding Information

The project described was supported by National Institutes of Health grants R37‐DK27083 and R01‐HL092234. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. R.K.S is an American Heart Association Stanley Stahl Postdoctoral Fellow (ID: 15POST22990022). V.C.B.L is supported by a fellowship from CNPq “Ciência sem Fronteiras” Brazilian program. H.F.C is a Merck Life Sciences Research Foundation Fellow. The authors declare no competing financial interests.
Novel function of tetraspanin‐interacting protein IGSF3 in the regulation of glycosphingolipid metabolism
Authors: Schweitzer Kelly , Justice Matthew J. , Bronova Irina , Leach Sonia M. , Berdyshev Evgeny V. , Petrache Irina
Published: November 2023

Rationale

We have previously identified in a patient with severe emphysema with stable chromosomal translocation a disruption in the gene encoding for Immunoglobulin Superfamily, Member 3 (Igsf3) accompanied by low levels of this membrane‐anchored protein. We set out to identify the function of Igsf3, which is predicted to interact with other membrane proteins, tetraspanins. cDNA microarray analyses identified that loss of IGSF3 significantly altered gene expression in the glycosphingolipid (GSL) pathway. Since GLS interaction with tetraspanins at the plasma membrane (dubbed as glycosynapse microdomain) inhibits integrin‐mediated cell functions, we hypothesized that Igsf3 is required for the glycosynapse control of cell motility.

Methods

Tetraspanin‐enriched microdomains (TEMs) were isolated using density gradient‐ultracentrifugation from human bronchial airway epithelial cells (Beas2b) and from primary immortalized human lung lymphocytes isolated from the consenting subject. Beas2b were transduced or transfected with adenoviral‐ and mammalian‐ IGSF3 plasmid vectors; or with siRNA and lentiviral‐shRNA. GSL were measured by LC‐MS/MS and cell migration and proliferation were assessed by standard techniques.

Results

Co‐immunoprecipitation studies identified that within TEMs, Igsf3 colocalized with the tetraspanins CD9, CD81, CD82, and CD151, as well as with integrinb1 (Itgb1). As expected, when compared to other cellular fractions, TEMs were enriched in cholesterol, ceramides, and glycosylceramides (GC). IGSF3 knockdown significantly increased C16‐GC and C22‐GC (by 5.2‐ and 6.2‐ fold, respectively; p<0.0001), whereas IGSF3 overexpression tended to decrease glycosylceramide levels. Loss of Igsf3 by either siRNA or shRNA significantly delayed wound repair in a cell monolayer scratch assay (p<0.0001) and inhibited cell migration (p<0.03), but did not affect cell proliferation.

Conclusions

Igsf3 controls glucosylceramide abundance in TEMs and is required for cell migration. While the mechanism by which Igsf3 regulates the GSL metabolism remains to be investigated, our results suggest that Igsf3 may be an important component of the glycosynapse with a functional role in cellular motility and injury repair.

Support or Funding Information

Wollowick Chair in Respiratory Research; Medresearch AEF_Schweitzer
Rottlerin diminished the lipid accumulation in 3T3‐L1 cell line and enhanced the uncoupling of oxidative phosphorylation in D16 cell line via LRP6 mediated pathway
Authors: Ryu Yungsun , Jeong Jeongho , Go Gwang‐woong
Published: November 2023

Rottlerin is a polyphenol compound abundant in Mallotus philippensis. Rottlerin is well known as a PKCδ and mTOR inhibitor, followed by for its anti‐carcinogenic properties. It was recently demonstrated that rottlerin down‐regulates the level of LRP6, which is a novel nutraceutical target for hyperlipidemia and obesity. However, the precise mechanism remains largely unknown. Thus, we hypothesized that rottlerin would reduce adiposity in different adipocytes. We used 3T3‐L1 cell line as a white adipocyte model and D16 cell line as a brown adipocyte model for in vitro analysis. Cell cytotoxicity assay (MTT), lipid accumulation assay (oil red O staining), and immunoblotting were performed in the differentiated adipocytes. Rottlerin did not affect cell cytotoxicity in both differentiated 3T3‐L1 and D16 cell lines (up to 20 μM and 5 μM, respectively). In 3T3‐L1, rottlerin significantly reduced lipid accumulation in a dose‐dependent manner (5, 10, and 20 μM, P < 0.05); lipogenic enzymes including SCD1 and DGAT1 also correspondingly decreased (P < 0.05). More interestingly, SREBP1, a principal lipogenic transcription factor, also remarkably diminished in a dose‐dependent manner (P < 0.01). In differentiated D16, rottlerin enhanced the levels of UCP1 and PGC‐1α, indicating increased non‐shivering thermogenesis; likewise, the level of PRDM16, a co‐regulator of brown adipose development, was enhanced (P < 0.01). LRP6/β‐catenin was reduced in both 3T3‐L1 and D16 cell lines (all P < 0.001). Further analysis of the LRP6‐related pathway is required. In conclusion, rottlerin reduced lipid accumulation in 3T3‐L1 and improved uncoupling of oxidative phosphorylation in D16 via the LRP6 mediated pathway.
Detection of a Perilipin‐5 Splice Variant
Authors: Ranzau Brodie , Dubreuil Daniel M. , Hubbell Theresa , Tansey John T.
Published: November 2023

Alternative splicing occurs throughout the human genome, leading to multiple proteins from a single gene. The resulting proteins can be nearly identical or vastly different in how they function. The perilipins are a family of lipid droplet associated proteins that among other functions regulate interaction of lipases with their substrates. Alternative splicing within the perilipin family has been observed in perilipins 1 and 3, giving rise to proteins with varying functions. In the course of our studies on perilipin 5, an immunoblot signal was observed that corresponded to an approximately 35 kDa protein. Based on this data we hypothesized that perilipin 5 also undergoes alternative splicing. Western blot analysis of this shortened form of perilipin 5 has revealed its expression in C2C12 cultured myoblasts and in heart and liver mouse tissue. Further analysis of perilipin 5 cDNA through reverse transcriptase PCR provided evidence that this protein is a result of alternative splicing. Based on the current naming convention for the perilipin proteins, we have termed this protein perilipin 5b. The objective of the current study is to develop DNA based probes for use in northern blot analysis of perilipin 5. The genomic DNA sequence of perilipin 5 was analyzed to identify areas where splicing events could occur, and probes were designed accordingly to detect the different variants of perilipin 5. In the analysis of different mouse tissue samples, these probes have been used to detect which of the perilipin 5 splice variants are being expressed under different feeding conditions. Ongoing studies are seeking to determine the function of perilipin 5b.
Methionine restriction decreases fat mass in C57BL/6 mice via increasing endogenous hydrogen sulfide production
Authors: Wang Yanan , Zhang Jiahong , Guo Haitao , Yan Biao , Shi Yonghui , Le Guowei
Published: November 2023

Dietary methionine restriction (MR) increases lifespan, reduces adiposity in diverse organisms. However, the mechanisms underlying the healthy benefits of adiposity reduction remain largely unknown. This study aimed to investigate the effects of MR on the activity of the transsulfuration pathway (TSP) enzyme cystathionine γ‐lyase (CGL), hydrogen sulfide (H2S) production, and lipid metabolism in high‐fat diet mice. C57BL/6 mice were fed a high fat diet (HFD) containing either 0.86% methionine (CF) or 0.17% methionine (MR). In the 23th week, lipid profiles, the activity of CGL and the level of H2S in liver were determined. The mRNA level of CGL and the relactive genes (FAS, ACC‐1, SREBP1c, CYP7A1, CPT1 and PPARα) expression of lipid metabolism in mice liver were assayed by real‐time PCR. The protein level of CGL was measured by western blot. We found that compared with CF group, MR significantly decreased the level of triglyceride and cholesterol in mice plasma. The fat synthesis genes (FAS, ACC‐1 and SREBP1c) of MR group were down‐regulated and the fatty acid oxidation genes (CYP7A1, CPT1 and PPARα) were increased. And we found MR significantly increased mRNA and protein expression of CGL, and increased H2S production in mice liver. We concluded that MR can attenuate high‐fat diet‐induced adiposity possibly through increasing endogenous H2S production.

Support or Funding Information

This research was supported by program of “Collaborative innovation center of food safety and quality control in Jiangsu Province”, the National Natural Science Foundation of China (No. 31571841), State Key Laboratory of Food Science and Technology of Jiangnan University in China (SKLF‐ZZB‐201609), and China Postdoctoral Science Foundation (2015M571669).
Angiopoietin‐like 4 Directs Uptake of Dietary Fat Away From Adipose During Fasting
Authors: Cushing Emily Malcolm , Davies Brandon
Published: November 2023

Misregulation of lipid metabolism has been implicated in numerous disease states including metabolic syndrome, diabetes mellitus, and atherosclerosis. Angiopoietin‐like 4 (ANGPTL4) is a fasting‐induced inhibitor of lipoprotein lipase (LPL) and regulator of plasma triglyceride metabolism. ANGPTL4 is expressed most highly in adipose tissue and liver, but is also expressed in several other tissues including heart and skeletal muscle, and is also present in the plasma. We used gene expression assays in combination with triglyceride clearance and lipase activity assays to examine the kinetics of Angptl4 induction in various tissues during a fasting time course and the effects of global ANGPTL4 deficiency on triglyceride uptake in mice. Angptl4 was strongly induced early in fasting (within 2 to 4 hours), and this induction was not attributable to circadian rhythms. Consistent with previous reports we found that plasma triglyceride levels in Angptl4–/− mice were lower than wild‐type littermate controls, but interestingly, this was only the case in the fasted state. Using chylomicrons containing 3H‐labeled triglycerides we found that Angptl4–/− mice deliver more triglycerides to adipose tissue than wild‐type mice. Again, this difference was only apparent in the fasted state as Angptl4–/− mice showed no significant differences in uptake compared to wild‐type mice in the refed state. Our data support a model where Angptl4 is induced early in fasting and acts locally to divert uptake of fatty acids and triglycerides away from adipose tissues and toward triglyceride‐fueled tissues such as heart and skeletal muscle.

Support or Funding Information

1R01HL130146‐01A1, NIH/NHLBI (Davies) 06/01/16–03/31/21
A new anti‐atherogenic strategy
Authors: Ma Chuanrui , Zhang Wenwen , Duan Yajun , Chen Yuanli , Han Jihong
Published: November 2023

The progress on the selective liver X receptor β (LXRβ) modulators, which can reduce atherosclerosis without activation of lipogenesis, is limited. Metformin, a medicine for diabetes treatment, activates AMPKα to enhance energy metabolism. In this study, we investigate if metformin can enhance T0901317 (LXR ligand)‐inhibited atherosclerosis while eliminating T0901317‐activated hypertriglyceridemia. We randomly divided the proatherogenic apoE deficient (apoE−/−) mice into 4 groups (15 mice/group), and fed them a high‐fat diet (HFD), HFD containing T0901317 [1 mg/kg bodayweight/day (mpk)], metformin (100 mpk), or combined T0901317 and metformin, respectively for 16 weeks. After treatment, we collected mouse aorta and liver samples, and determined atherosclerotic lesions and liver steatosis, respectively. We found that T0901317 alone inhibited en face and aortic root sinus lesions, and the inhibition was enhanced by co‐treatment with metformin. The co‐treatment of T0901317 and metformin also enhanced lesion stability by increasing collagen content in lesion caps while reducing necrotic cores and calcification within lesions. Macrophage/foam cell formation and accumulation were inhibited by the co‐treatment which can be attributed by the LXR‐activated ABCA1/ABCG1 expression. T0901317 induced a severe fatty liver and hyperglyceridemia which was substantially blocked by metformin. Mechanistically, metformin had little effect on T0901317‐induced hepatic LXRα/β expression. However, it selectively reduced T0901317‐mediated LXRα (the main transcription factor controlling lipogenesis) but not LXRβ, nuclear translocation. Thereby, metformin blocked expression of T0901317‐induced lipogenic genes, SREBP1c, FASN and ACC, while increased ACC phosphorylation. Moreover, metformin favored energy dissipation process by inducing ATGL, HSL, PPARα and CAT and inhibiting DGAT1 expression. Furthermore, we used AMPKα knockout HepG2 cells to determine the effect of metformin relied on AMPKα expression. Taken together, our study demonstrates that addition of metformin to T0901317 not only enhances T0901317‐inhibited atherosclerosis, but also blocks T0901317‐induced severe hepatic lipid accumulation, fatty liver and hypertriglyceridemia, which suggests that the co‐treatment of metformin and T0901317 may function as a safe and novel therapy for atherosclerosis treatment.

Support or Funding Information

The National Science Foundation of China Grants 81473204 to J Han and 81573427 to Y Duan
Multifaceted Role of Glycan interactions on Clathrin‐Independent Endocytosis of MHC1 and CD59
Authors: Mathew Mohit Philip , Donaldson Julie G.
Published: November 2023

Glycosylation is an often overlooked post‐translational modification that is unique in its complexity and variety. In fact, altered glycosylation is a hallmark of cancer. Although these changes have been viewed as passive by‐products of cancer metabolism (with efforts primarily directed at their use as biomarkers for detection), more recent results have shown that these glycans can serve important functional roles. The role that glycan interactions can play in clathrin‐independent endocytosis (CIE) is an area of increasing interest in particular because CIE is a form of endocytosis which, unlike clathrin mediated endocytosis (CME), does not have well defined canonical machinery. As a result, studying the role glycans can play in this setting could help elucidate some of the regulatory and molecular mechanisms that govern these trafficking pathways.

In this study we show that the perturbation of global cellular glycosylation patterns by metabolic flux modulation affects clathrin independent endocytosis. Interestingly, these changes in glycosylation appear to have certain cargo‐specific effects. For example, treatment of cells with N‐Acetyl Glucosamine (that corresponds to an increase in production of branched glycans) leads to an increase in major histocompatibility complex Class I (MHCI) internalization while at the same time mediating a decrease in endocytosis of CD59, a GPI‐anchored protein.

In order to better understand the mechanism behind these glycosylation mediated effects we focused on the role of Galectin 3. Galectin3 is a galactose binding pentameric lectin that is produced in the cytoplasm of cells, is secreted and binds to glycans on the surface of the cell. The multivalent binding potential of galectin 3 makes it unique from other galectins and essential in forming the galectin lattice which can be a major determinant of membrane characteristics. We found that knocking‐down the expression of Galectin 3 led to a decrease in MHCI uptake and an increase in CD59 uptake. Whereas, the inhibition of all glycan‐galectin interactions by lactose treatment inhibits the uptake of both CIE cargo. Our results suggest that glycans play a role in CIE that is quite nuanced, with each cargo affected in a specific manner and different galectins having possibly competing effects. These treatments did not affect the uptake of transferrin, a CME cargo protein, further suggesting a CIE specific role for glycosylation.

These findings are interesting for a couple of reasons: 1) The elucidation of the role glycans play in CIE could lead to a more complete understanding of how these still mysterious pathways are regulated and 2) This could open a window into how altered glycosylation can alter the progress of cancer via the CIE pathway.
Suboptimal Thiamin Status Is Common Among Women Of Reproductive Age And Children ≤ 5 y In Cambodia: Findings From The National Micronutrient Survey
Authors: Whitfield Kyly C , Smith Geoffry , Chamnan Chhoun , Karakochuk Crystal D , Kuong Khov , Dijkhuizen Marjoleine A , Hong Rathavuth , Berger Jacques , Green Tim J , Wieringa Frank T
Published: November 2023

Thiamin deficiency is thought to be an issue in Cambodia because case reports of beriberi are not uncommon, and several studies across Southeast Asia have reported low thiamin concentrations among women of reproductive age (WRA), children <5 y, and breastfed infants. However, due to a lack of population‐representative data, the magnitude of this public health issue is currently unknown. The objective of this study was to assess biochemical thiamin status using erythrocyte thiamin diphosphate (eTDP) concentrations among a nationally representative sample of WRA (15–49 y) and children ≤ 5 y, and to classify thiamin status using various cut‐offs from the literature. Non‐fasting venous blood samples were collected as part of a National Micronutrient Survey linked to the 2014 Cambodian Demographic and Health Survey, and eTDP was measured among 719 women and 761 children using HPLC‐FLD. The median (SD) age of women was 30 (6) y, and children (46% girls) were 42 (17) mo. Women had lower mean (95% CI) eTDP of 150 nmol/L (146–153) compared to children, 174 nmol/L (171–179; P < 0.001). Thiamin status was assessed using 10 different cut‐offs from the literature, none of which were developed in relation to clinical presentation of beriberi. Thiamin status varied dramatically depending on the cut‐off employed (70 nmol/L and 180 nmol/L): from 2% to 78% among women, and <1% to 58% among children. Deficiency was most prevalent among infants aged 6–12 mo (n=50); even using the most conservative cut‐offs (from the Institute of Medicine), 4% were deficient (<70 nmol/L) and 14% had marginal status (70–90 nmol/L). More research is required to develop more useful, clinically meaningful thiamin status cut‐offs to define thiamin status and to attain global consensus on their use. Immediate action is required, however, to develop interventions to increase thiamin intake, and thus improve thiamin status, among women and children in Cambodia and throughout Southeast Asia.
Effect of DM extracts on the Atopy Dermatitis in human keratinocytes
Authors: Kim Hyesook , Lee Minhee , Kwon Han Ol , Park Soo‐jeung , Lee Dasom , Kim Yong Jae , Kim Eun , Lee Jeongmin
Published: November 2023

Atopic dermatitis (AD), a pruritic chronical inflammatory skin disease is associated with a personal or family history of atopy such as asthma of respiratory diseases and or allergic rhinitis. The prevalence rate is increasing recently. This study was to evaluate the inhibitory effects performance by respectively of DM extracts (DME) from the tumor necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ)‐induced inflammatory response in human keratinocytes. (HaCaT cells). HaCaT cells were each treated with 250 μg/mL respectively of DME water extract, 20% ethanol extract, and 50% ethanol extract. Also, investigated the effects of DME on the production of histamine in A23187 and PMA‐stimulated Mast cells. (MC/9 cells). We found that treatments with DME decreased the production of pro‐inflammatory cytokines in TNF‐α, IFN‐γ‐stimulated HaCaT cells, and the decrease in histamine release in the A23187 and PMA‐simulated Mast cells.
MODE‐K Cells as a Model to Study the Gut Epithelial Response: An Outside‐in and Inside‐out Approach
Authors: Crockett Erica K. , Washburn Karley F. , Graef Jennifer L. , Lucas Edralin A. , Smith Brenda J.
Published: November 2023

The gut epithelium serves as a barrier between the intestinal lumen and immune cells residing in gut‐associated lymphoid tissues. These epithelial cells are required to respond to stimuli from the outside‐in (e.g., microbiome and dietary components), as well as from the inside‐out (e.g., cytokines and growth mediators). This study evaluated the utility of a gut epithelial cell model and its response to inside‐out (tumor necrosis factor; TNF‐α) or outside‐in (E. coli lipopolysaccharide; LPS) immune challenges. Experiments were designed to evaluate alterations in cell viability, proliferation, and gene expression using the MODE‐K cell line which was derived from the small intestine of C3H/HeJ mice. For all experiments, MODE‐K cells were grown at 37°C with 5% CO2/95% humidified air in RPMI GlutMAX media supplemented with 10% FBS, 1% HEPES, 1% NEAA, 1% sodium pyruvate, and 0.1% β‐mercaptoethanol. Cell viability and proliferation were assessed using trypan blue exclusion and MTT assays at 12, 24, and 48 hours. Doses of TNF‐α and LPS were selected based on MTT results and cell viability (≥90%). MODE‐K cells were cultured in 12‐well plates and treated with TNF‐α (0, 0.1 or 1.0 ng/mL) or LPS (0, 0.1, 1.0, or 10 ug/mL). Cells were harvested after 2 or 6 hours and total RNA was extracted, followed by qRT‐PCR using Cyclo as the invariant control. Both doses TNF‐α up‐regulated (p<0.05) the relative abundance of interleukin‐6 (Il6), a pro‐inflammatory cytokine, at 2 hrs, whereas only the higher dose of TNF‐α up‐regulated Il6 at 6 hrs. The relative abundance of Tgfb, a cytokine involved in T‐cell differentiation, was elevated with the 1.0 TNF‐α dose at 2 hrs, but not by 6 hrs. The gene expression of the anti‐inflammatory cytokines, interleukin‐10 (Il10) and ‐4 (Il4), was not detectable at either time point. Expression of toll‐like receptor 4 (Tlr4), a pattern recognition receptor, was suppressed (p<0.05) at 2 hrs with all doses of TNF‐α and LPS. This response was normalized by 6 hrs, with the exception of the 1.0 TNF‐α treatment. All doses of LPS, representing the outside‐in stimulus, up‐regulated Il6 at both time points. Likewise, all doses of LPS up‐regulated Tnfa (p<0.05) at 2 hrs, while only the two highest doses increased (p<0.05) Tgfb. No alterations in these responses were observed after 6 hrs. These findings demonstrate that the MODE‐K cells respond to the pro‐inflammatory cytokine, TNF‐α, from the gut mucosa, as well as endotoxins present in the intestinal lumen (i.e., LPS), demonstrating the importance of both the mucosal immune system and the microbiome in initiating epithelial cell signaling. This cell culture model can potentially serve as a system to study the gut epithelial cells' in‐side out and outside‐in responses and to understand the modulating effects of dietary bioactive components.

Support or Funding Information

Oklahoma Center for the Advancement of Science and Technology (HR14‐126)
Prenatal food and micronutrient interventions in rural Bangladesh remain cost‐effective when assessing both favorable and unfavorable outcomes: Cost‐effectiveness analysis of the MINIMat trial on under five‐mortality and stunting.
Authors: Svefors Pernilla , Selling Katarina Ekholm , Shaheen Rubina Ekholm , Persson Lars Åke Ekholm , Lindholm Lars
Published: November 2023

Background

Few studies analyze the cost‐effectiveness of nutrition interventions in low‐income countries, possibly deterring large‐scale implementation. Nutrition interventions may have favorable as well as unfavorable effects. The Maternal and Infant Nutrition Interventions in Matlab (MINIMat) with early prenatal food and micronutrient supplementation reduced infant mortality and were reported to be very cost‐effective. However, the multiple micronutrients (MMS) resulted in an increased risk of stunted growth in infancy and early childhood. This unfavorable outcome was not included in the previous cost‐effectiveness analysis. The aim of this study is to evaluate whether the MINIMat interventions remain cost‐effective in view of both favorable (decreased under‐five year mortality) and unfavorable (increased stunting) outcomes.

Method

Pregnant women in rural Bangladesh, where food insecurity still is prevalent, were randomized to early (E) or usual (U) invitation to food supplementation and daily doses of 30 mg, or 60 mg iron with 400 μgm of folic acid, or MMS with 15 micronutrients including 30 mg iron and 400 μgm of folic acid. E reduced stunting at 4.5 years compared with U, MMS compared with Fe60 increased stunting at 4.5 years while the combination EMMS reduced infant mortality compared with UFe60. The outcome measure used was disability adjusted life years (DALY), a measure of overall disease burden that combines years of life lost due to premature mortality (under five‐year mortality) and years lived with disability (stunting). Incremental cost effectiveness ratios were calculated using cost data from already published studies.

Results

By incrementing UFe60F to EMMS, one DALY could be averted at a cost of US$26.

Conclusion

When both favorable and unfavorable outcomes were included in the analysis early prenatal food and multiple micronutrient interventions remained highly cost effective and seem to be meaningful from a public health perspective.
FEASIBILITY OF INTEGRATING CALCIUM AND IRON‐FOLIC ACID SUPPLEMENTATION TO PREVENT PREECLAMPSIA AND ANEMIA IN PREGNANCY IN PRIMARY HEALTHCARE FACILITIES IN KENYA
Authors: Omotayo Moshood Olanrewaju , Dickin Katherine , Pelletier David , Martin Stephanie , Kung'u Jacqueline , Stoltzfus Rebecca
Published: November 2023

Background

Calcium (Ca) supplementation to prevent preeclampsia can save maternal and newborn lives, but there are no program models for integration into existing antenatal care (ANC) platforms. Many countries have existing antenatal iron supplementation programs, however feasibility of integrating Ca supplementation has not been examined.

Methods

We used a program impact pathway model to guide design of integrated Ca and iron‐folic acid (IFA) supplementation in Kenya. We provided healthcare providers with job aids (posters and counseling cards), trained them on counseling techniques and supplementation guidelines, and developed behavior change materials for pregnant women (calendar and poster). We allocated health facilities to prescribe either 1.0 or 1.5 g/d Ca, with standard IFA. We collected implementation data from 16 facilities and 990 women through record review, spot‐checks and exit interviews and carried out descriptive analyses. We also modeled effects of supplementation on percentage of the population meeting recommended daily allowance (RDA).

Results

Supplements and job aids were available during 90% of facility spot‐check episodes; calendar availability was lower (78%). Over 98% of clients received Ca and IFA supplements, but only 76% received enough Ca supplements to last between ANC visits. Among clients that still had pills by return date, adherence was 77% and 83% for the IFA and Ca regimen, respectively. In the most conservative modeling scenario, over 75% of participants were estimated to meet the RDA, when 1.5 g/d of Ca supplements was prescribed. Only 54% were estimated to meet the RDA when 1.0 g/d was prescribed.

Conclusion

This study illustrates a systematic approach for integrating Ca supplementation into primary health care and demonstrates that such integration is feasible when contextual bottlenecks are addressed. Policy makers and program planners should pay attention to supply chain, healthcare worker dispensing behaviour, and appropriateness of regimen for their settings.

Support or Funding Information

Funding for this research was provided by the Micronutrient Initiative
A new tool for Researchers: The USDA Branded Food Products Database
Authors: Haytowitz David B. , Pehrsson Pamela R. , McKillop Kyle A. , Moore Gary , Finley John , Fukagawa Naomi K.
Published: November 2023

The USDA Branded Food Products Database is the result of a Public‐Private Partnership (PPP), whose goal is to enhance public health and the sharing of open data by complementing the USDA National Nutrient Database for Standard Reference (SR) with nutrient composition of branded foods and private label data provided by the food industry. Members of the current PPP include: 1) Agricultural Research Service (ARS), USDA; 2) International Life Sciences Institute (ILSI) North America; 3) GS1 US; 4) 1WorldSync; and 5) Label Insight. These data are publicly available on NDL's Web site and are updated continuously. Users include, but are not limited to, federal agencies, the research community, international databases, proprietary databases and end users, the food industry, the media and consumers. Data from Label Insight and ILSI NA partner companies using GS1 US and 1Worldsync have been received and processed through a cooperative agreement with the University of Maryland's Joint Institute for Food Safety and Applied Nutrition; USDA's Nutrient Data Laboratory developed and performed quality control checks on the data and the companies supplying the data have been informed of any problems, which they will have an opportunity to resolve. The data is housed on a server hosted by USDA's National Agricultural Library and currently contains approximately 68,000 food items and is expected to double in size by spring 2017. The USDA Branded Food Products Database is clearly identified as distinctive but connected to the USDA National Nutrient Database for SR. It is accessed through the same search program (ndb.nal.usda.gov). Examples of data and search strategies will be presented.