bluuebunny/crossref_metadata_2025 huggingface dataset:
GLP1 targets lncRNA Gas5 in diabetic adipocytes
Authors: Lui Ashley , Kraja Gearta , Rupani Rea , Bader Deena , Patel Niketa
Published: June 2021

Diabetes type 2 affects 10% of the US population whereas 80% of patients affected are insulin resistant. We previously showed that lncRNA Gas5 was downregulated in the serum of T2DM patients. Here we sought to investigate the underlying molecular mechanisms of Gas5. Our data shows that Gas5 is down regulated in type II diabetic adipocytes and their secretome. Depletion of Gas5 in non‐diabetic adipocytes resulted in decreased glucose uptake. Using an unbiased diabetes transcriptomics screen, we demonstrated that this reduction of Gas5 resulted in a decrease of both insulin receptors A and B. Glucagon‐Like Peptide‐1 mimetics are new therapeutics available for diabetic patients and have recently been seen as the leading treatment for patients who are no longer responsive to metformin. Our data has shown treatment with GLP‐1 in adipocytes results in an increase in Gas5 resulting in an increase in glucose uptake. Gas5 has been recognized as a biomarker for T2DM, but this is the first time it has been studied mechanistically in adipocytes and researched as a therapeutic target.

Support or Funding Information

VAMR

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Novel Components in Fibroblast Growth Factor Signal Transduction
Authors: Webb Jason , Gaudenzi Carolina , Eichelberger Eric , Stefinko Mariya , Alonso Yanely , Ahmed Fardin , Voisine Cindy , Stern Michael , Lo Te‐Wen
Published: June 2021

Fibroblast Growth Factor Receptors (FGFRs) are a type of receptor tyrosine kinase (RTK) that control many developmental processes. These include cell proliferation, cell migration, and cell survival. Mutations to these FGFRs and their pathways in humans result in developmental disorders such as achondroplasia, craniosynostosis, and thanatophoric dysplasia. To better understand FGFR signaling specificity we use the nematode C. elegans as a model. C. elegans contain a single FGFR, EGL‐15 which is imperative for sex myoblast migration and fluid homeostasis. Defects in the processes mediated by the EGL‐15 FGFR result in striking phenotypes that can be used to discover additional signaling components of the EGL‐15 pathway. For example, hyperactivation of EGL‐15 signaling causes the worm to accumulate excessive amounts of a clear fluid in its body which is known as the Clr phenotype. The isolation of suppressors of Clr (soc) mutants has helped to identify many of the core components of EGL‐15 signaling. A previous soc screen identified the adaptor protein SEM‐5 which links RTK activation to the activation of the RAS/MAPK pathway.

SEM‐5 is known to bind to EGL‐15 at two tyrosine residues (Y1009 and Y1087). However, previous work suggests that removal of these binding sites does not confer a Soc phenotype. To verify this, we utilized three different CRISPR/Cas9 mutants. One mutant contains the Y1009F mutation, the second mutant contains the Y1087F mutation, and the third mutant contains both the Y1009F and Y1087F mutations. Each of these mutations was tested for their ability to suppress the Clr phenotype associated with the hyperactivation of the EGL‐15 signaling pathway. Neither the Y1009F or Y1087F mutation alone or together, results in a Soc (suppressor of Clr) phenotype. This would suggest that a key component that links EGL‐15 to SEM‐5 has yet to been identified. We performed an additional modified suppressor screen to isolate these potential “links” between EGL‐15 and SEM‐5 and are in the process of mapping and identifying these components.

Support or Funding Information

Ithaca College H&S Summer Scholars, Ithaca College CSTEP and FERP Funding, NIH R15 (RGM122001A), MNEIU Student Center For Science Engagement

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of UCP2 protein expression in normal RIN‐5F and UCP2 inhibited cells
Authors: Willis Jacquelyn , Clemons Tameka
Published: June 2021

Uncoupling protein 2 (UCP2) is a mitochondria integral membrane protein that transports protons across the intermembrane space. Traditionally, the function of UCP2 is to impact oxidative phosphorylation by uncoupling ATP synthesis. However, a cell signaling role for UCP2 has recently been investigated. UCP2 expression has been shown to have expression in pancreatic beta cells and the presence of UCP2 has been suggested to lead to a decrease in the release of insulin. Although amylin is co‐secreted with insulin, studies on the relationship between amylin and UCP2 have had little investigation. A known function of amylin is to block glucagon secretion, which leads to a reduction in the rate of glucose production by decreasing the rate of gastric emptying. This study analyzed the protein expression level of amylin in normal RIN‐5F cells and RIN‐5F cells that have been transfected with a UCP2 siRNA construct. The study included stimulating the cells with either 11mM or 30 mM of glucose and comparing the protein expression level in the two cell lines. Western blot analysis was conducted and the results suggest a difference in UCP2 and amylin levels in normal RIN‐5F cells in comparison to the transfected RIN‐5F cells.

Support or Funding Information

National Science Foundation Research Initiation Award #24430

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Cushing Syndrome Mutation of Protein Kinase A C‐subunit Disrupts the Internal Allosteric Network Affecting Regulation and Substrate Specificity
Authors: Walker Caitlin , Wang Yingjie , Olivieri Cristina , Karamafrooz Adak , Casby Jordan , Bathon Kerstin , Taylor Susan S. , Calebiro Davide , Bernlohr David A. , Veglia Gianluigi
Published: June 2021

Genetic alterations of the PRKACA gene coding for the catalytic subunit of the CAMP‐dependent protein kinase A (PKA‐C) are linked to cortisol‐secreting adrenocortical adenomas resulting in the onset of Cushing's syndrome. Among those, a single mutation (L205R) located in the P+1 loop of the kinase's active site has been found in 30–60% of patients. It has been proposed that L205R hinders the interactions of PKA‐C with its regulatory (R) subunit generating a constitutively active kinase causing hyperphosphorylation of downstream substrates. However, the x‐ray structures of PKA‐C wild‐type and L205R are essentially identical and the aberrant mechanism of function of the mutant remains unknown. To gain mechanistic insights into the multifarious effects of this single mutation, we carried out solution NMR spectroscopy in concert with binding thermodynamics as well as molecular dynamics simulations. We found that this single mutation, in addition to changing the protein‐protein interface, disrupts the internal allosteric network of the kinase and the network of interactions between the small and large lobes. The ternary complex of L205R with nucleotide and pseudo‐substrate displays significantly altered allosteric interactions, leading to the disruption of the interactions with R‐subunit, the binding with endogenous protein kinase inhibitor (PKI), and modifying substrate specificity. Our results show that this single mutation has global effects on the structure of the kinase that leads to the dysregulation and aberrant phosphorylation profiles.

Support or Funding Information

This work was supported by the National Institutes of Health, GM100310 (G.V.) and S10 OD021536 (G.V.)., and the CRC/Trasregio 166 (Project C1 to DC) as well as the IZKF Würzburg (grant B‐281 to DC). KB was partially supported by a fellowship through a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of Würzburg.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TRPM8 channel role in mesenchymal stem cells osteogenic differentiation
Authors: del Pilar Torres Yolima , Henao Juan Carlos , Barreto Alfonso , Rodríguez Viviana , Mejía Camila
Published: June 2021

Mesenchymal stem cells (MSCs) have properties that make them a good model to use in regenerative medicine. MSCs proliferation and differentiation are complex processes involving the activation of several molecules that induces changes in intracellular calcium concentration. Different ion channels are responsible for calcium influx including TRP channels. The present study evaluates the expression and functional activity of TRPM8 channels in human mesenchymal stem cells. Cells were characterized by the expression of the specific surface markers CD90+, CD105+ and CD34− and by the ability to differentiate into adipogenic and osteogenic linage. Western blot, immunocytochemistry and flow cytometry were performed to determine TRPM8 protein expression. Patch clamp recordings and fluorescence imaging with Fluo‐4 AM were used to analyze channel activity. We found that the agonist menthol induces an increase in ionic currents which were incompletely abolished by treatment of the cells with the antagonist BCTC. Changes in intracellular calcium concentration were detected after addition of menthol, suggesting an effect induced by activation of TRPM8 channels. Alizarin red staining showed that the TRPM8 agonist Icilin increased osteogenic differentiation. On the contrary, the TRPM8 antagonist BCTC decreased cell differentiation. These results were confirmed by real time PCR with the marker Alkaline Phosphatase (ALP). Results from this study suggest that the activity of TRPM8 channels in mesenchymal stem cells is related with regulation of osteogenic differentiation.

Support or Funding Information

This work was supported by Departamento Administrativo de Ciencia, Tecnología e Innovación, COLCIENCIAS, Project: Evaluación del papel de canales TRPM8 en la diferenciación de células madre mesenquimales derivadas de tejido adiposo; Grant ID 120365843092.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interactions between doripenem and NDM metallo‐β‐lactamases as inspiration for future generation antibiotics
Authors: Orischak Matthew , Shurina Ben , VanPelt Jamie , Page Richard
Published: June 2021

Antibiotic resistant bacterial infections are a growing threat to patients globally. Bacteria are evolving rapidly and developing new modes of antibiotic resistance. One of these resistance modes is the expression of a family of proteins that chemically inactivate antibiotics. β‐lactamases act on antibiotics that contain a β‐lactam ring such as penicillin. New Delhi metallo‐β‐lactamase (NDM) was selected as the primary focus for this project as it confers multi‐drug resistance, is an increasing threat to public health, and there are currently no FDA approved inhibitors for NDM. Doripenem is an antibiotic notable for its relative stability as a carbapenem, a subclass of β‐lactam antibiotics, and is often used as a last resort antibiotic. Unfortunately, NDM is capable of hydrolyzing doripenem. Despite this, doripenem may still serve as a promising scaffold from which new derivatives could be designed for use as next generation antibiotics that are resistant to β‐lactamase mediated hydrolysis. Alternatively, doripenem derivatives might be designed to act as inhibitors for β‐lactamases such as NDM. In order to better engineer doripenem derivatives for either of these purposes, structural information detailing the interactions between NDM and doripenem is required. While there are existing structures of NDM in complex with other substrates such as meropenem and imipenem, there was no structure of doripenem in complex with NDM. Our lab has successfully solved an X‐ray crystal structure of hydrolyzed doripenem bound to the active site of NDM‐4 at 2 Å resolution. Our crystal structure of NDM‐4 in complex with doripenem reveals novel interactions between the sulfonamide functional group at the R2 position of doripenem and the surface of NDM‐4. Previously published crystal structures of NDM complexed with carbapenem substrates do not show strong interactions with the functional groups at the R2 position. Future work will explore doripenem derivatives and investigate their resistance to hydrolysis or potential use as β‐lactamase inhibitors.

Support or Funding Information

NIH R35 GM128595

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Isoleucine Serves as a Precursor for the Synthesis of β‐Alanine in Plants
Authors: Rouhier Kerry A , Perrett Morgan E , Goldfarb Margo H , Reinhart Peter E , Wilczewski‐Shirai Kai C , Raymond‐Schmidt Lisa , Gothard Megan E , Piras Crystal A
Published: June 2021

The non‐protein amino acid β‐alanine serves as an athletic performance enhancer and as a key precursor in the production of food additives and pharmaceuticals. In plants, β‐alanine is important for the synthesis of pantothenate (vitamin B5) and subsequently coenzyme A through a condensation reaction with pantoate. It is well established that the source of pantoate is valine, however the synthesis of β‐alanine in plants is less‐well established. There is some evidence that plants synthesize β‐alanine from spermine (or other poly‐amines), uracil, and propionate, but evidence has only been recorded in very specific plant systems, such as stress‐tolerant Limonium latifolium. In this project we sought to confirm the synthesis pathway of β‐alanine in Arabidopsis and the more economically relevant grain, wheat. We hypothesized that another branched‐chain amino acid, isoleucine, could serve as a source of β‐alanine in plants. Isoleucine degradation concludes with the production of propionyl‐CoA, which is further metabolized to acetyl‐CoA in a similar manner to β‐oxidation. Therefore, in order for isoleucine to serve as a precursor, the intermediate malonate semialdehyde must react via an unknown aminotransferase to produce β‐alanine. Using transgenic Arabidopsis plants and Hard Red Winter wheat, seedlings were treated with isotopically‐labeled precursors and extracts were analyzed by both NMR and GC‐MS. Results show that β‐alanine can be synthesized from both propionate and isoleucine via the proposed propionyl‐CoA metabolic pathway. Given these new findings and the prior evidence that valine serves as the precursor for pantoate, branched‐chain amino acids look to play an important role in the synthesis of pantothenate and coenzyme A in plants.

Support or Funding Information

Clare Boothe Luce Undergraduate Scholars program funded by the Henry Luce Foundation (Grant #9601), Kenyon Summer Science Scholars Program funded by Kenyon College, Kenyon Summer Science Cascade Program funded by the Sherman Fairchild Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Increase of Efficiency of RuBisCO
Authors: Link Tina , Stanovski Lisa , Fisher Joseph , Kona Abhishek , Lu Allison , Nguyen Lan , Rajput Aashita , Saberi Zara
Published: June 2021

Carbon dioxide (CO2) is the primary source of carbon for life on Earth. As such, the efficient incorporation of CO2 into organic matter is of significant importance. Ribulose‐1,5 bisphosphate carboxylase/oxygenase (RuBisCO), sequenced from the cyanobacteria Synechococcus PCC6301, is a 64.8 k‐Da photosynthetic enzyme involved in the oxidation‐reduction process of carbon fixation, assimilating inorganic carbon into bioavailable organic carbon. RuBisCO is composed of two subunits: a large L chain and a smaller S chain, with a length of 467 amino acids. Each chain consists of eight sub‐chains and weighs 51.87 k‐Da and 12.93 k‐Da respectively. RuBisCO, the most abundant protein in the world, is slow‐reacting, fixing about 3–10 CO2 molecules each second per molecule of enzyme. During the catalysis of RuBisCO, the formation of an active site intermediate is able to react with either CO2 or Oxygen (O2). Approximately 25% of the time, RuBisCO will bind with O2 instead of CO2. This misbinding has a significant impact on the enzyme's effectiveness within the Calvin cycle as well as other photosynthetic processes. The functionality error of RuBisCO means that the enzyme exhausts large quantities of ATP in order to process the O2 and then attempt to bind to CO2. Minimizing RuBisCO's intermediate active site's availability can reduce the occurrence of photorespiration. While variations to RuBisCO exist between different photosynthetic species, all species experience inefficiencies due to oxygenation to some extent. Efforts to discover more efficient carboxylation through random mutagenesis of RuBisCO lead to the discovery of a mutant that catalyzes the carbon fixation reaction with greater speed, accuracy, and precision. The resultant mutated RuBisCO was chosen through selection in E. coli. Mutations in the C‐terminal loop 6 region and a substitution mutation at 331 of valine to alanine, both within the large subunit, decreased the specificity factor by almost 40%. RuBisCO's enlarged specificity factor could potentially benefit the development and growth of many different species of plants. The potential increase in crop productivity could have a substantial impact on global food distribution. Walton High School SMART Team has designed a 3D model of RuBisCO using JMOL to investigate the relationship between structure and function.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Avocado Oil on Metabolic Profile and Development of Diabetic Nephropathy in Goto‐Kakizaki Rats
Authors: Ortiz‐Avila Omar , Saavedra‐Molina Alfredo , Cortés‐Rojo Christian
Published: June 2021

Uncontrolled glucose and lipid metabolism during diabetes, caused by a decrease in the synthesis of regulatory substances such as insulin and adiponectin, promotes the development of diabetic complications. One of the most frequent and devastating complications is diabetic nephropathy, which is characterized by high levels of proteinuria due to inadequate glomerular filtration. The aim of this study was to evaluate the effect of avocado oil on metabolic profile and the development of diabetic nephropathy in a model of type 2 diabetes.

Goto‐Kakizaki rats were used as a model of type 2 diabetes, and avocado oil was administered orally for 3, 6 and 12 months. The results showed that treatment with avocado oil maintained for a longer time adiponectin levels, however had no effect on insulin levels. The treatment improved the lipid profile in rats by lowering cholesterol, triglyceride and LDL levels and maintaining HDL concentrations. Another important aspect was that avocado oil did not result in weight gain in diabetic rats. When evaluated renal function, the oil partially counteracted the increase in proteinuria observed in the diabetic group, while in the histopathological evaluation diabetic rats showed signs of diabetic nephropathy such as accumulation of extracellular matrix in the glomerulus and thickening of the basement membrane starting from 3 months of treatment. Treatment with avocado oil delayed the development of these alterations until 6 months.

These results show that avocado oil improved several parameters of the metabolic profile and delayed the development of diabetic nephropathy, probably due to the components on avocado oil, since some of them have antioxidants functions, while others, such as oleic acid, have a beneficial effect on the metabolism of lipids and carbohydrates.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Effects of De Novo Lipogenesis Inhibitors on plasma lipids and liver PNPLA3
Authors: pocai alessandro
Published: June 2021

Obesity and genetic polymorphisms such as the patatin‐like phospholipase domain‐containing 3 (PNPLA3‐I148M; 49% allele frequency in Hispanics) predispose to non‐alcoholic steatohepatitis (NASH). NASH patients are at increased risk of death by cardiovascular and liver‐related complications. Hence, there is an urgent need to develop new strategies for preventing and treating NASH. Increased de novo lipogenesis (DNL) and liver lipid deposition are processes associated with the early stage of the disease.

Inhibition of liver acetyl‐CoA carboxylase (ACCi) in NASH patients reduces DNL and liver triglycerides (TG) and improves noninvasive markers of fibrosis. Unexpectedly, ACCi results in a significant rise in plasma TG, a major concern given the already high risk of CV disease in NASH patients. Hence, alternative strategies to reduce DNL devoid of the lipid liability might be beneficial for liver steatosis in NASH. Previous studies have demonstrated that diacylglycerol acyltransferase 2 inhibition (DGAT2i) lowers DNL and improves inflammation and fibrosis in rodents. Here, we conducted a 2‐week study in diet‐induced obese (DIO) mice comparing the effects of ACCi versus DGAT2i on lipid metabolism.

Thirty‐six DIO mice were treated with vehicle, ACCi (30 mg/kg PO) or DGAT2i (10 mg/kg and 30 mg/kg PO). ACCi and DGAT2i resulted in significant reduction in liver fat mass (−43% for ACCi vs. −23 and −29% for DGAT2i), however, ACCi significantly increased plasma TG (+49%, ACCi treatment vs. vehicle; p < 0.001). ACCi increased mRNA expression of liver sterol regulatory element‐binding protein 1c (SREBP‐1c, +40%) and of the lipogenic genes stearoyl‐CoA desaturase‐1 (SCD1; +650%), ELOVL fatty acid elongase 6 (ELOVL6; +373%), and Glycerol‐3‐phosphate acyltransferase 1 (GPAT1; +96.4 %). In the same study, DGAT2i resulted in no change of plasma TG while reducing expression of SCD1 (−92% and −95%), ELOVL6 (−16% and −10%) and GPAT1 (−32% and −39%) mRNA. DGAT2i decreased PNPLA3 expression (−71% and −79%; DGAT2i 10 and 30 mg/kg vs. vehicle; p<0.05) while ACCi increased PNPLA3 (+2400%, ACCi vs. vehicle; p<0.001). PCSK9 plasma levels were significantly reduced (−67%; p < 0.001) in DGAT2i treated mice. In contrast, ACCi treated mice exhibited a significant increase of plasma PCSK9 levels (226%; p < 0.001).

Inhibition of de novo lipogenesis with DGAT2 and ACC inhibitors are potential promising strategies to lower liver lipids in NASH patients. Our results also demonstrated that DGAT2i and ACCi inhibition have opposite effects on liver PNPLA3 and plasma PCSK9

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional decline in brown adipose progenitors during BAT remodeling
Authors: Ruan Hai‐Bin , Huang Zan , Zhang Zengdi
Published: June 2021

In humans, there is a striking decrease in the prevalence and activity of brown adipose tissue (BAT) with age and obesity; however, the cellular and molecular mechanisms of BAT remodeling are almost completely unknown. Rodent models have been widely used to study BAT but they possess constitutively active BAT throughout life even at thermoneutrality; therefore, complementary animal models that possess characteristics of human BAT remodeling are needed. Here, we found that BAT progressively whitened in rabbits in a similar manner as in humans. As rabbits mature, preadipocytes in BAT displayed reduced capacity to be differentiated into brown adipocytes, when transplanted into immune‐deficient mice. RNA‐sequencing of brown preadipocytes identified that, during BAT remodeling, genes enriched for muscle contraction and mitochondrial function were down‐regulated, while genes involved in lipid and amino acid metabolism were up‐regulated. Knocking down genes regulating muscle and mitochondrial function in rabbit brown preadipocytes blunted their ability to become mature brown adipocytes in vitro and in vivo. We also identified follistatin like 1 (FSTL1) as a novel “batokine” that is specifically expressed by brown preadipocytes but not mature brown adipocytes. FSTL1 gradually reduced its expression when BAT whitens in rabbits, but was stably expressed in mouse BAT during life. Deleting FSTL1 specifically in the Myf5 lineage in mice reduced BAT size, UCP1 expression, and body temperature in newborns, suggesting the constitutive expression of FSTL1 prevents BAT whitening in mice. Taken together, we identify potential molecular mechanisms underlying functional decline in the browning ability of preadipocytes that contribute to BAT remodeling in rabbits and possibly humans.

Support or Funding Information

American Diabetes Association (18‐IBS‐167)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fluoxetine Ameliorates the Depressive‐Like Behaviors and Metabolic Abnormalities Induced by Chronic High‐Fat Diet
Authors: Chiu Yen‐Ju , Chen Yun‐Wen
Published: June 2021

Major depressive disorder (MDD) is one of the most common psychiatric disorders, and frequently co‐occurs with multiple comorbidities, especially Type 2 diabetes mellitus. Selective serotonin reuptake inhibitors (SSRIs) are the most widely used class of antidepressants. This study aimed to investigate the effects of fluoxetine (Prozac®), a SSRIs, on metabolic abnormalities induced by chronic high‐fat diet (HDF). Male HDF‐induced insulin resistance mice were administrated with fluoxetine (10 mg/kg and 20 mg/kg) by intraperitoneal injection for four weeks. The behavioral tests showed that fluoxetine significantly reversed the depressive‐like behaviors induced by chronic HDF, including the reduced sucrose preference and increased immobility time in the forced swimming test and tail suspension test. Moreover, fluoxetine also reduced serum level of glucose and improved blood lipid profiles in HFD‐treated mice. Furthermore, fluoxetine improved impaired glucose tolerance and insulin sensitivity. Our results indicated that fluoxetine could ameliorate depressive‐like behaviors and metabolic abnormalities induced by chronic HFD, which suggested that the multiple effects of fluoxetine could be beneficial for patients with depression and/or metabolic syndrome.

Support or Funding Information

This work is supported by Ministry of Science and Technology, Taiwan (MOST 106‐2320‐B‐006‐058‐MY3)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Animal Model to Investigate the Pathophysiology of Atherosclerosis Through Regulation of Cholesterol Homeostasis and the Highly Atherogenic LDL‐L5
Authors: Chang Christopher , Yang Tzu‐Ching , Lu Shao‐Chun , Chang Po‐Yuan
Published: June 2021

The electronegative low‐density lipoprotein (LDL) L5 is elevated in hypercholesterolemic plasma and can induce apoptosis in coronary endothelial cells. The atherogenic potential of L5 renders it an important therapeutic target for cardiovascular disease but the exact mechanisms how plasma L5 concentrations can be regulated by cholesterol intake remains unclear. In this study, we developed an animal model to investigate the inducibility of plasma L5 by cholesterol and test the LDL‐lowering effect of ezetimibe, an inhibitor of intestinal cholesterol absorption, in male Golden Syrian hamsters. The hamsters were fed diets containing high fat and 1% cholesterol (HFC diet) for 2 weeks and LDLs were purified from hamster plasma by ultracentrifugation followed by ion exchange liquid chromatography. The eluting patterns of LDL in hamsters revealed five subfractions, named L1 to L5, which were similar to those obtained from hypercholesterolemic human plasma. HFC‐fed hamsters had highly elevated plasma L5, expressed as L5/LDL ratio, of 48.8±13.4% (n=6) compared with the hamsters fed with normal chow diet (18.9±2.7%; n=4). When the cholesterol absorption inhibitor ezetimibe was added to the HFC diets at the dose of 1mg/day/kg body weight, the plasma L5 levels were significantly reduced compared to non‐ezetimibe group (32.1±11.3% vs. 48.8±13.4%; n=6). Hamsters fed with normal chow diet also demonstrated a modest reduction of L5 with ezetimibe (14.1±11.3% vs. 18.9±2.7%; n=4). The time interval required to reduce plasma L5 by ezetimibe was only two to four weeks, consistent with the cholesterol‐lowering effect of ezetimibe in human. Therefore, we have established a novel hamster‐L5 model to investigate the pathophysiology of atherosclerosis in vivo. This animal model can also serve as a screening tool to identify LDL‐ and L5‐lowering therapy for cardiovascular disease.

Support or Funding Information

This work was supported by grants NSC101‐2320‐B‐002‐026 from the National Science Council and MOST‐106‐2314‐B‐002 ‐156 ‐MY2 (Dr. Po‐Yuan Chang) from the Ministry of Science and Technology, Taipei, Taiwan.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RNA seq Analysis of Livers from Mice Lacking Fatty Acid Transport Protein 2 (FATP2) Demonstrate Metabolic Linkages in Genes Involved in PPARa‐responsive Lipid Metabolic Pathways
Authors: Perez Vincent , Behrens Mark , DiRusso Concetta C. , Black Paul N.
Published: June 2021

Fatty acid transport protein 2 (FATP2) is highly expressed in liver, small intestine and kidney where it functions in both the uptake of exogenous long chain fatty acids and in the activation of very long chain fatty acids. Previous studies from this lab have found that when the fatty acid uptake is attenuated using the FATP2‐specific inhibitor Lipofermata, cells that express this protein are protected from lipotoxicity. These observations support the notion that FATP2 functions as a gate in regulating the uptake of exogenous fatty acids, which are linked to downstream lipid metabolic events. In the present study, we completed a comprehensive RNA seq analysis to address the impact of deleting FATP2 in the liver on the transcriptome. Wild type (C57BL/6NJ) and FATP null (FATP2−/−) mice (5 weeks old) were maintained on standard chow diet for 6 weeks. Over this period of time, the FATP2−/− mice had reduced weight gain and lowered blood triglyceride levels compared with control mice. A number of lipid metabolic genes in the liver were increased in the FATP2−/− mice that mapped to the gene ontology categories beta‐oxidation, peroxisome biogenesis, fatty acid trafficking and arachidonic fatty acid/eicosanoid metabolism. These metabolic systems are linked through activity of the transcription factor PPARa suggesting endogenous expression was elevated possibly through increases in a proximal ligand when FATP2 is absent. This correlated with increased expression of phospholipase A2. To address these changes further, we employed untargeted (NMR) and targeted (LC‐MS/MS) metabolomics experiments, which showed there were no major differences in the abundance of substrates in central carbon metabolism between the wild type and FATP2−/− livers, but there was a significant increase in the eicosanoids LXA4 and PGJ2 in the FATP2−/− livers. Taken together, the expression of FATP2 appears to have a broad impact on the expression of key lipid metabolic genes that may be linked through increased levels of bioactive lipid mediators.

Support or Funding Information

This work has been supported by the Agricultural Research Division of the University of Nebraska.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cross Regulation of Phospholipid and Unsaturated Fatty Acid Biosynthesis
Authors: Dible Sarah , McDonough Virginia
Published: June 2021

Considering the requirement for unsaturated fatty acids (UFAs) to produce phospholipids, we investigated the potential cross regulation of phospholipid and UFA biosynthesis. The Δ‐9 desaturase, encoded by OLE1, is the sole source of endogenous UFAs in Saccharomyces cerevisiae. Inspection of the OLE1 promoter revealed several sequences that are close matches to the regulatory sequence, UASINO, found in many phospholipid biosynthetic genes, and is the target of regulation by Ino2p, Ino4p and Opi1p. We then examined if these proteins also play a role in regulation of OLE1 expression. Reporter gene analysis showed that when uracil prototrophy was placed under control of the OLE1 promoter, poor expression was observed in an opi1 mutant. When lacZ was placed under control of the OLE1 promoter, reporter assays confirmed that expression was poor in the opi1 mutant. Fatty acid analysis revealed that normal regulation of OLE1 occurred in response to unsaturated fatty acid (UFA) supplement in the opi1 mutant, indicating that post‐transcriptional regulation is still functional. Finally, the 2 hybrid assay was used to examine potential interactions between Opi1p and Ole1p, Ino2p, Ino4p and known OLE1 regulators Mga2p and Spt23p. While no unexpected Opi1p interactions were detected, we observed that Ino4p interacts with Mga2p in this assay. This preliminary work indicates that phospholipid regulators also play a role in regulating OLE1 expression.

Support or Funding Information

We acknowledge the support of the Hope College Biology Department, and the Hope College Dean of Natural and Applied Sciences.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acyl‐CoA Synthetase 6 Mediates Brain Docosahexaenoic Acid (DHA) Enrichment and Neuroprotection
Authors: Fernandez Regina F , Kim Sora Q , Ellis Jessica M
Published: June 2021

The omega‐3 fatty acid, docosahexaenoic acid (DHA), is enriched in the central nervous system and thought to protect against neurological dysfunction. Yet, studying the role of brain DHA metabolism in the development of neurological dysfunction has remained limited because the biochemical mechanisms regulating DHA enrichment in the brain remain unclear. To define a critical regulatory node in brain DHA biochemistry we targeted long chain acyl‐CoA synthetase 6 (Acsl6) because this enzyme initiates cellular fatty acid metabolism, is enriched in the brain and prefers DHA as its substrate. Genetically deleting Acsl6 in mice resulted in large and specific reductions (35–72%) in DHA‐containing phospholipids across tissues that highly express Acsl6 (i.e. the central nervous system). Acsl6−/− mice develop age‐related deleterious neurological pathology characterized by increased astrogliosis and microglia. Surprisingly, this pathology develops in the absence of alterations in pro‐ and anti‐inflammatory lipid mediators, suggesting alternative mechanisms mediating neuroinflammation in Acsl6‐induced DHA deficiency. Together our findings demonstrate a novel central nervous system DHA‐deficient model susceptible to aging‐induced neuroinflammatory pathology.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CysLT1R regulates cys‐LT‐mediated calcium influx and oxidized LDL‐uptake in macrophages
Authors: Pokhrel Sabita , Duah Ernest , Paruchuri Sailaja , Thodeti Charles
Published: June 2021

Cysteinyl leukotrienes (cys‐LTs), LTC4, LTD4, LTE4 are potent inflammatory lipid mediators that act through two distinct G‐protein‐coupled receptors, CysLT1R and CysLT2R. Although cys‐LTs are shown to induce vascular leakage and atherosclerosis, the molecular mechanism by which cys‐LTs modulate endothelial and macrophage functions are not well understood. Atherosclerosis is the chronic inflammatory disease that accounts for the global epidemic of cardiovascular diseases (CVD), mainly driven by endothelial dysfunction. We have recently shown that cys‐LTs regulate endothelial cell (EC) dysfunction via CysLT2R, enhancing EC contraction and permeability. Further, cys‐LTs enhance TNFα‐mediated up‐regulation of VCAM‐1 and attachment of THP‐1 cells to endothelium. Therefore, we further focused on cys‐LT‐mediated effects in Raw and THP‐1‐derived macrophages. We observed that macrophages express mainly CysLT1R and flux calcium via CysLT1R in response to LTD4. Further, LTD4 significantly promoted oxidized low density lipoprotein (ox‐LDL) uptake by macrophages. Importantly, LTD4 treatment enhanced the expression of a scavenger receptor CD36, Oxidized low‐density lipoprotein receptor 1 (OLR1), Monocyte chemoattractant protein 1 (MCP1) and Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC1α) transcripts suggesting that these receptors could mediate LTD4‐induced ox‐LDL uptake by macrophages contributing to atherosclerosis.

Support or Funding Information

NIH R15 grant (1R15HL133918), and James Foght Professorship support.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Cysteinyl Leukotriene 2 Receptor in Tumor Angiogenesis, Permeability and Metastasis
Authors: Teegala Lakshminarayan Reddy , Duah Ernest , Kondeti Vinay , Adapala Ravi K , Keshamouni Venkateshwar G , Kanaoka Yoshihide , Austen K Frank , Thodeti Charles K , Paruchuri Sailaja
Published: June 2021

Cysteinyl leukotrienes (cys‐LTs) are pro‐inflammatory mediators that enhance vascular permeability through distinct receptors (CysLTRs). We found that CysLT2R regulates angiogenesis in isolated mouse endothelial cells (EC) and in Matrigel implants in WT mice, and enhances EC contraction and permeability via Rho‐dependent myosin light chain 2 and VE‐cadherin axis. Since solid tumors utilize aberrant angiogenesis for their growth and metastasis and their vessels exhibit vascular hyper‐permeability, we hypothesized that CysLT2R via its actions on endothelium might regulate tumor growth. Both tumor growth and metastases of adoptively transferred syngeneic Lewis lung carcinoma (LLC) cells are significantly reduced in CysLT2R null mice (Cysltr2−/−) compared to WT and CysLT1R null mice (Cysltr1−/−). In WT recipients of LLC cells, CysLT2R expression is significantly increased in the tumor vasculature, compared to CysLT1R. Further, tumor vasculature in Cysltr2−/− recipients exhibited significantly improved integrity as revealed by increased pericyte coverage and decreased leakage of intravenously administered Texas Red‐labeled dextran. Administration of a selective CysLT2R antagonist significantly reduced LLC tumor volume, vessel density, dextran leakage and metastases in WT mice, highlighting CysLT2R as a VEGF‐independent novel regulator of vasculature, promoting risk of metastasis. Thus, both genetic and pharmacologic findings establish CysLT2R as a gateway for angiogenesis and endothelial cell dysregulation in vitro and ex vivo and in vivo model with a mouse tumor. Finally, analysis of primary human lung adenocarcinoma data set revealed a strong correlation between poor survival and high CysLT2R expression, suggesting CysLT2R as a possible target for intervention.

Support or Funding Information

James Foght Professorship Support, AHA GIA 15GRNT 25670004

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the Perilipin 5 11‐mer Region
Authors: Tucker Trent M. , Wei Derek T , Tansey John T
Published: June 2021

Lipid storage‐related diseases have become prevalent throughout the nation and are responsible for afflictions such as obesity and type II diabetes. Proteins such as the perilipin family serve a key role in the modulation of lipolysis. Although this protein family plays such a major role in biochemical processes, little information has been discovered pertaining to the structure of the Perilipins. Structure is central to understanding the function of a protein, and how that protein will interact will other molecules. There has been data showing the structure of the C‐terminal domain of perilipin 3; however, structural information for the other family members is yet, unknown. The current study is aimed to determine the structure of the 11‐mer region in Perilipin 5. The 11‐mer region is implicated in the interactions between Perilipin 5 and lipids. Bioinformatics data were obtained on the 11‐mer sequence. The secondary structure of the 11‐mer region was characterized using Phyre2, and RaptorX and viewed using molecular visualization software including PyMol and NGL. These models predicted the 11‐mer region to have an amphipathic alpha helix which allowed Perilipin 5 to bind to lipid droplets. Additional studies are being performed using protease protection assays and characterization of a synthetic peptide that mimics the 11‐mer region. Characterization on the 11‐mer region of Perilipin 5 will further help elucidate how this region of perilipin 5 associates with lipid droplets.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Correlating Indigenous Peruvian Women's Distance to Cusco and Their Access to Healthcare, and Investigating the Effect of Cat's Claw Extracts on HeLa Cells
Authors: Lamba Arleen , Oakes Alissa , Roberts Lia , Deprele Sylvine , Nogaj Luiza
Published: June 2021

Uncaria tomentosa, or Cat's Claw, is a large woody vine plant that is indigenous to the Amazonian rain forest. The roots and bark of cat's claw have been used for 2000 years as an herbal remedy for ailments ranging from chronic fatigue to gastrointestinal inflammation, and more recently as an anticancer remedy. To explore the use of Cat's Claw among indigenous women of Peru, the Global Women in STEM Program at Mount Saint Mary's University designed and administered in person a survey that explores socioeconomic factors, access to healthcare, and the use of natural remedies. Survey results showed that Peruvian women often utilize natural remedies to alleviate a wide range of ailments, including cancer. Cat's claw was found to be a common remedy in our survey.

Previous chemical analysis of Cat's Claw showed an abundance of polyphenolic compounds such as vanillic acid, gallic acid, and caffeic acid, which are reducing agents. These compounds have been shown to have protective properties against oxidative damage. From the chemical analysis, we hypothesized that both a simple decoction and polyphenolic extract from cat's claw will increase cell viability and decrease cytotoxicity in HeLa cells. In order to test this, we isolated the phenolic compounds of Cat's Claw by soxhlet extraction and compared its effects against previously synthesized vanillic, gallic, and caffeic acids using MTT and LDH Cytotoxicity assays. Our results showed that the purified Cat's Claw extract increased cell viability and decreased cytotoxicity, in comparison to the decocted crude extract. The crude extract exhibited a clear concentration‐dependent trend with a steady decrease in viability and increase in cytotoxicity as the volume of crude extract increased. Similar to the purified extract: the synthesized vanillic, caffeic, and gallic acids showed high cell viability and low cytotoxicity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐Fat Diet Causes Mitochondrial Dysfunction and Renal Fibrosis in Minipigs
Authors: Chien Miao‐Ju , Li Sin‐Jin , Chen Ching‐Yi
Published: June 2021

Obesity is a common and complex health problem which has been dramatically increasing in the world. It is also considered as an independent risk factor for chronic kidney disease. Kidney is one of the most energy‐demanding organs in the human body, which has the second highest mitochondrial content and oxygen consumption only next to the heart. The purpose of this study was to investigate the nexus between mitochondrial function and kidney injury by using dietary‐induced obese minipig model.

Materials and methods

Six‐month‐old Lee‐Sung minipigs were assigned randomly into 2 groups and fed either a control diet (C) or a high‐fat diet (HFD) for six months, the kidney was collected for the analysis.

Results

Compared with C group, HFD group had heavier body weight, thicker back fat, higher plasma levels of glucose, triacylglycerol and total cholesterol. Furthermore, the biomarkers of kidney injury, including blood creatinine, urine creatinine, urine urea, and urine protein were higher in the HFD group than the C ones. A heavier kidney was observed in the HFD pigs; whereas a smaller ratio of kidney weight to body weight was found in the HFD group than C group, implying a less effective loading in the kidney function of HFD pig. In histologic section of kidney, disarranged structure and more glomerular fibrosis were observed in the HFD pigs. Moreover, HFD caused a decreased antioxidant capacity in the kidney, indicating an oxidative stress. Whether HFD caused the renal injury through mitochondrial mechanisms, we analyzed the ATP generation and found that HFD kidney displayed a lower ATP production than C group did. The mitochondrial‐related genes in kidney were analyzed as well. The mRNA expression of SIRT1, which has a role in variety of mitochondrial processes, including redox homeostasis and mitochondrial biogenesis was downregulated by HFD. PGC1‐1a, a nutritional sensor in the kidney, was inhibited by HFD, suggesting a decrease in mitochondrial biogenesis. UCP2, activated by mitochondrial ROS, was upregulated in HFD group compare with C group. The mRNA expressions of Opa1 and Fis1, markers of mitochondrial dynamics, were downregulated by HFD, suggesting a mitochondrial dysfunction.

In conclusion, long term HFD feeding in Lee‐Sung minipigs induced obesity, dyslipidemia and renal injury. Mitochondrial dysfunction might partially contribute to the progression of obesity‐related renal damage.

Support or Funding Information

This study was sponsored by the Ministry of Science and Technology, Taiwan (MOST107‐2313‐B‐002‐049).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Enhanced Conformational Sampling Methods for GPCRs
Authors: Serrano Erik , Khleif Rafeed
Published: June 2021

G protein‐coupled receptors (GPCRs) are known to possess multiple active conformational states in nature, however, studying these conformations is extremely challenging as majority of the functionally important conformations have high energy compared to the lowest energy conformation. We are developing a method called Enhanced Conformational Markov‐state Sampling in Membrane BiLayer Environment (EnCoMSeMBLE) that would enhance our search of the conformational landscape of GPCRs and that can be applied to a‐helical transmembrane proteins in general. It enables a level of conformational sampling not achievable by classical or accelerated molecular dynamics (MD) simulations or Markov‐State Models (MSM). This method combines brute force conformational sampling of helix‐helix interactions in the membrane with MD simulations and Markov‐State modeling to identify functionally important conformations. This method is being tested by application to the Glucagon Like peptide‐1 receptor (GLP1‐R), a class B GPCR, and the muscarinic acetylcholine M2 receptor, a class A GPCR, both of which have been crystallized in inactive as well as active conformations. The comparison of the activation landscapes of class A and class B GPCRs is beginning to provide key similarities and differences in activation across these very distinct GPCRs. This detailed understanding of the GPCR activation, enabled by our method, complements major structural biology efforts underway targeting GPCRs, where it is very challenging to map out the structural landscape and activation pathways of GPCRs.

Support or Funding Information

BUILD PODER is supported through a grant from the National Institutes of General Medical Sciences (NIGMS), grants # # 8TL4GM11897

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.