bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Status of Perilymphatic Mast Cells is Critical for Lymphatic and Immune Functions
Authors: Gashev Anatoliy A. , Dusio Giuseppina F. , Gasheva Olga Yu. , Skoog Hunter , Tobin Richard , Peddaboina Chander , Meininger Cynthia , Newell‐Rogers M. Karen , Zawieja David C. , Nizamutdinova Irina Tsoy
Published: November 2023

Mesenteric lymphatic vessels surrounded by mast cells which inhabit in high density in close proximity to lymphatic wall. In the events of acute inflammation mast cells, upon activation, release numerous bio‐active and, in particular, vasoactive mediators that continuously modulate the mesenteric lymphatic vessels and surrounding tissues. In current study we attempted to establish mechanistic links between the functional status of mast cells and the responses of mesenteric lymphatic vessels and perilymphatic mesenteric tissues to acute inflammatory insult in adulthood and elderly. We used an in vivo model of acute peritoneal inflammation induced by lipopolysaccharide (LPS) treatment (24 hours after single IP injection of LPS in dose of 10 mg/kg body weight into adult (9‐mo) and aged (24‐mo) F‐344 rats). We analyzed contractility of isolated mesenteric lymphatic vessels, mast cell activation, activation of nuclear factor‐κB (NF‐κB) without and with stabilization of mast cells by cromolyn or blockade of all types of histamine receptors and production of 27 major pro‐inflammatory cytokines in adult and aged perilymphatic mesenteric tissues and blood. We found that the reactivity of aged contracting mesenteric lymphatic vessels to LPS‐induced acute inflammation was abolished and that activated mast cells trigger NF‐κB signaling in the mesentery through release of histamine. The aging‐associated activation of mast cells maintains chronic histamine‐dependent activation of NF‐κB signaling in aged perilymphatic mesenteric tissues and limits NF‐κB activation in aged mesentery in response to acute inflammation. These findings correlate with our previously published findings which link together chronic activation of aged mast cells with diminished recruitment/activation of MHC class II+ cells and eosinophils towards/near aged mesenteric lymphatic vessels during acute inflammation (Chatterjee V, Gashev A.A. Amer. J. Physiol., 2012, Lymphatic Research and Biology, 2014). Cumulatively our studies indicate that healthy functional status of mast cells is critical for proper performance of the lymphatic and immune functions. In healthy adults the mast cells‐derived histamine is necessary trigger of the NF‐κB‐mediated reactions of mesentery to acute inflammation. The decreased ability of the aged immune system to respond to acute inflammatory challenge in peritoneal compartment of the body appears to be generated by chronically activated mast cells.

Support or Funding Information

NIH grants AG030578, DK099161, DK076169 and HL123420, research funds from Texas A&M University Health Science Center College of Medicine and Department of Medical Physiology, and from the Raleigh R. White IV endowment fund from Baylor Scott & White Foundation.
Mapping the apoptotic process with super resolution microscopy in kidney cells challenged with high glucose
Authors: Bernhem Kristoffer , Zhang Liang , Fontana Jacopo , Nilsson Linnéa , Scott Lena , Brismar Hjalmar , Aperia Anita
Published: November 2023

The role of the Bcl‐family proteins in the mitochondrial apoptotic process is well described with biochemical and molecular methods as well as with studies of isolated mitochondria and transfected cell lines. There is yet no proof of principle that this description of apoptosis conforms with the in vivo process in primary mammalian cells. Here we have used Stimulated Emission Depletion (STED) microscopy to study the apoptotic process in immune‐stained rat renal epithelia cells exposed to 20 mM glucose (HG) and to study its rescue by ouabain. To assess distance between Bcl proteins, we used the nearest‐neighbor algorithm. The anti‐apoptotic protein Bcl‐xl was predominantly expressed on mitochondria in control cells, and remained so throughout the process, although its abundance decreased. After 2h HG the apoptosis‐inducing protein BAD had translocated from the cytoplasm to the mitochondria where it clustered with Bcl‐xL. This occurred before an increase in reactive oxygen species and was dependent on activation of the PI3K –AKT pathway. According to current concepts, Bcl‐xl interacts with the apoptotic protein Bax on the mitochondria under control conditions to translocate Bax back to the cytosol. We found that Bax started to accumulate on the mitochondria after 4h HG and, surprisingly, that the interaction between Bcl‐xL and Bax became more pronounced during the course of the apoptotic process. After 6h HG Bax also interacted with the non‐specific ion transporter VDAC; an interaction described to lead to penetration of the inner mitochondrial membrane and mark the point of no return. Our group has previously identified an anti‐apoptotic signaling pathway that is triggered by ouabain binding to Na,K‐ATPase and activates PI3K and AKT. Here we show that 10 nM ouabain rescues from apoptosis by counteracting the AKT dependent inhibition of BAD activation. This study reveals new information about interaction between Bcl family of proteins during the apoptotic process and about mechanism and rescue from kidney cell death in poorly controlled diabetes.
Culture Medium Fatty Acid Withdrawal Prompts Insulin‐Producing Cell Death
Authors: Rosim Mariana Papaléo , Nunes Viviane Abreu , Lensen Sigurd , Curi Rui , Azevedo‐Martins Anna Karenina
Published: November 2023

Fatty acids (FAs) are important components of cell membranes and precursors of many bioactive molecules. The deficiency of FA in the diet has been associated with the development of human diseases (e.g. neuronal diseases) and tissue damage in experimental animal models. FAs regulate several cell functions including insulin secretion. We investigated herein the effects of FA withdrawal in the culture medium on insulin producing cell (RINm5F) metabolism, survival, and response to death cell stimuli. Cells were cultivated in a lipid‐reduced fetal bovine serum (LRS) medium for 24, 48 or 72 h. Cell proliferation was measured by BrdU incorporation into cells and glucose metabolism was analyzed by measurement of 14CO2 production from [U‐14C]‐glucose. Production of reactive oxygen species (ROS) was determined using dichlorofluorescein diacetate assay. After the culture period, cells were exposed to death inducers (either UV radiation or staurosporine). In order to evaluate the effect of FA deprivation on RINm5F cell survival and susceptibility to death induction, and the mechanism of cell death, we analyzed, by flow cytometry, loss of plasma membrane integrity, DNA fragmentation, phosphatidylserine externalization and activation of caspases 3, 8 and 9. The removal of FAs from the culture medium increased the number of dead cells as assessed by loss of plasma membrane integrity and occurrence of DNA fragmentation. Cells cultured in LRS exhibited higher cell death rates as compared to those cultivated with conventional serum. Cultivation in LRS for 48 hours increased numbers of cells with externalized phosphatidylserine, activation of caspases, raised production of ROS and glucose oxidation. In conclusion, FA deprivation induced RINm5F cell death through apoptosis and exacerbated the cell death response to staurosporine and UV radiation treatments. Cell death was associated with changes in generation of ROS and glucose oxidation.

Support or Funding Information

The authors thank the financial support of FAPESP, CAPES and CNPq
Sirtuin 1 regulation of mitophagy and function in hindlimb muscles of aging mice
Authors: Myers Matthew J. , Mohamed Junaith S. , Alway Stephen E.
Published: November 2023

Age‐related muscle wasting, or sarcopenia, results in a functional decline in muscle strength, mobility, and overall independence of aging populations. Muscles of old animals and humans have reduced levels of repair, which contributes to sarcopenia. Aging also reduces muscle mitophagy, which results in an accumulation of dysfunctional mitochondria, but sirtuin 1 (SIRT1) appears to improve mitophagy.

Purpose

The objective of this project was to determine the long‐term effects of SIRT1 on mitophagy, repair, and function of muscles in aging mice.

Methods

Cardiotoxin (naja mossambica) or the vector control was injected into the tibialis anterior or gastrocnemius of one leg of wild type, SIRT1 overexpresser, adult skeletal muscle‐specific knockout, and satellite cell‐specific knockout mice that were 20wk or 80wk of age. The muscles were analyzed 21 days post‐injection.

Results

The data show that SIRT1 increased in vivo fatigue‐resistance, the percentage of type I fibers, muscle force, mitochondrial function and mitophagy in muscles of aged mice after injury.

Conclusion

Our results suggest that targeting activation of SIRT1 is a potential therapeutic strategy for offsetting muscle sarcopenia in aging.
Palmitic Acid Methyl Ester Regulates Proliferation of Human Mesenchymal Stem Cells
Authors: Lin Jian‐Hong , Yang Kun‐Ta
Published: November 2023

BACKGROUND

Human mesenchymal stem cell (hMSCs) have unique multilineage differentiation and self‐renewal capabilities that can be used for cell therapy and regenerative medicine. Although MSCs can be easily isolated from many tissues such as bone marrow, adipose tissue, and umbilical cord etc., it is still challenging to obtain sufficient numbers of cells for clinical use. In vitro environments, chemical cues such as fatty acid affect stem cell proliferation. Palmitic acid methyl ester (PAME) is released from adipose tissue and synthesized through transesterification of palmitic acid(PA) with methanol. PAME was shown to possess potent anti‐inflammatory and anti‐fibrotic. However, the effect of PAME on MSCs proliferation is unknown.

PURPOSE

In this study, we investigated effects and mechanisms of PAME on proliferation of the cultured hMSCs.

METHODS

hMSCs were cultured in αMEM for 1 day, and then treated with PAME for 2 days. We performed MTT and BrdU assays to determine the proliferate capacity of these cells, and evaluated cell cycle and mitochondrial reactive oxygen species (ROS) using flow cytometry. The action of PAME on signaling pathway was analyzed by using Western Blots and Quantitative real time‐PCR.

RESULTS

Our results show that PAME inhibited the proliferation of hMSCs in a dose‐dependent manner (10, 30, 50 and 100 μM) through G2/M phase arrest, which is mediated by down‐regulation of G2/M‐related mRNA and proteins (CDK1 and Cyclin B1). In addition, we found that PAME treatment (2 days) increased p53 and p21 expression, but did not affect the mitochondrial ROS level.

CONCLUSION

PAME inhibits the proliferation of hMSCs through G2/M phase arrest by regulating the p53–p21 signaling pathway.
Effect of Hypoxia and Heat Inactivated Serum on Mesenchymal Stem Cell Migration
Authors: Boomsma Robert A. , Jochems Valerie K. , Spotts Anna R.
Published: November 2023

Previous studies have shown that intravenous injection of mouse bone marrow mesenchymal stem cells (MSC) attenuate the loss of function in the heart after coronary artery occlusion (Boomsma et al, Int J Cardiol 122:17 2007) and secrete paracrine factors that may be responsible for their beneficial effect (Boomsma and Geenen, PLoSONE 7:e3685, 2012). The purpose of the current study was to determine the effect of hypoxia and heat inactivated (HI) serum on MSC migration across surfaces coated with laminin or fibronectin, conditions that better mimic the situation MSC encounter as they migrate in an ischemic heart. MSC were cultured in Mesencult + 20% serum supplement (Stem Cell Technologies) under normoxic (21% O2 + 5% CO2) conditions until confluent. The cells were lifted, placed in Mesencult containing either 20% supplement or 20% HI serum, and transferred to 12 mm Millicell cell culture inserts (PCF membranes, 3 μm pores; 600,000 cells/insert) that were either uncoated, coated with gelatin‐fibronectin, or coated with laminin. The inserts were placed in 24 well plates containing the same media and incubated to allow for cell migration at 37°C in normoxic or hypoxic (1% O2, 5% CO2, 95% N2) conditions. After 24 hours the cells on top of the membranes were removed and the cells on the underside of the membranes were stained with 0.9% crystal violet. The cells were lysed with 10% acetic acid and the absorbance at 560 nm was determined using a plate reader. The amount of migration was calculated as the % change in absorbance from the mean control value. Mean ± SE was calculated and significance was determined by Student's t‐test (p<0.05; n=9). When MSC were cultured in 20% supplement and normoxia, there was no significant change in migration on laminin or fibronectin compared to uncoated membranes. When cultured in HI serum, migration across uncoated membranes was 87% lower compared to supplement. However, migration in HI serum significantly increased 5‐fold on laminin and 9‐fold on fibronectin compared to uncoated membranes returning migration levels back to that found in supplement. Cells in HI serum were also cultured in hypoxic conditions. Hypoxia significantly reduced migration on uncoated (75%) and fibronectin coated (25%) membranes but was unaffected on laminin. These data show that in the absence of serum stimulatory factors, MSC migration is reduced on uncoated membranes, but laminin and fibronectin are able to increase migration in those conditions. Hypoxia reduces migration on fibronectin but not on laminin. Together, these data suggest that MSC migration in the ischemic conditions of the heart is best on fibronectin but is also promoted by laminin.

Support or Funding Information

VanderVelde Research Scholarship from Trinity Christian College
Modeling ion channel function with murine intestinal 3D stem cell cultures
Authors: Sowick Joshua A , Shah Kalpit , Bradbury Neil A.
Published: November 2023

Cystic Fibrosis (CF) is a persistent and progressive genetic disease, manifesting as frequent lung, gastrointestinal, and pancreatic inflammation and chronic infection. The majority of patients with CF have a mutation caused by a single amino acid deletion of phenylalanine (F508del) in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein; this causes an unstable protein to form, with subsequent failure of membrane insertion or lack of function and early degradation. A lack of functional CFTR leads to the production of viscous mucus that is hard to clear from the lungs and gastrointestinal tract, providing a medium for bacterial growth and infection. While treatments for Cystic Fibrosis have made advancements, the median survival age for patients with CF is still 40.

In order to utilize a mouse model for human CF disease, we developed a protocol culturing mouse intestinal crypts into three dimensional organoids. Mature organoids are subsequently dosed with Forskolin, an adenylyl cyclase activator, to elicit ion and fluid transport into the lumen of the organoid, via stimulation of CFTR channels. The unidirectional flow of ions and fluid resulted in the swelling of the organoids as fluid is moved into the lumen. We were able to measure the change in volume and surface area as well as movement of ions across the epithelium using a modified two electrode voltage clamp technique. Forskolin induced swelling was inhibited by the presence of the CFTR inhibitor cftrinh172, consistent with the role of CFTR ion fluid transport. We will present data using intestinal organoids from mice lacking transport proteins, such as LMTK2.

Support or Funding Information

NIH Heart Lung and Blood
Developing Renal Organoids Using Human Embryonic Stem Cells
Authors: Torres Collazo Bryan J. , Cebrian‐Ligero Cristina
Published: November 2023

Stem cells have the remarkable potential to develop into all the different cell types in the body. They are unspecialized cells capable of self‐renewing and, under certain physiologic or experimental conditions, they can be induced to become tissue‐ or organ‐specific cells with unique functions. Human Embryonic Stem Cells (hESCs) are derived from the inner cell mass of a blastocyst and can be induced to differentiate into specialized cell types with defined culture conditions. This ability to grow human tissues from stem cells in culture has the potential to revolutionize the drug discovery process and regenerative medicine. One of these approaches relies in the generation of organoids from hESCs. An organoid is 3‐dimensional structure that mimics the cellular composition and function of the organ of study. The mammalian kidney is derived from the intermediate mesoderm (IM). During embryonic development, the IM differentiates into the ureteric bud(UB) epithelia and into the metanephric mesenchyme. The UB will branch and become the renal collecting system while the mesenchyme will epithelialize and differentiate into nephrons. The primary goal of this work is to differentiate UM63.1 hESCs into renal organoids, specifically organoids composed of UB epithelia. Several signaling pathways have been identified as critical for mouse kidney development, and specifically for UB epithelial growth. We hypothesized that by recapitulating those signaling pathways in culture, UM63.1 cells can be induced to differentiate into kidneys organoids. Our differentiation protocol can be divided into two phases. On phase one cells are grown as a monolayer and treated with decreasing concentrations of an agonist (CHIR99021) to induce intermediate mesoderm. After 7 days in culture, clusters of cells (spheroids) develop. On phase two we growth those spheroids in a 3‐dimensional matrix in the presence of fibroblast growth factors (FGFs) 1 and 9, glial cell‐line derived neurotrophic factor (GDNF) and Retinoic acid. Previous research has shown that these growth factors are involved in mouse embryonic kidney development. After a few days in culture, the spheroids grow into structures resembling kidney organoids. We have imaged the progression of these organoids over time and we have also conducted immunofluorescence assays to detect markers specific for the renal ureteric bud. Our experiments demonstrate that the UM63.1 hESC‐derived organoids are positive for the following UB‐specific markers: TROMA, PAX2 and GATA3 indicating their identity as UB epithelia.
K‐Cl Cotransporter Activity Contributes to Mammalian Cancer Cell Morphology
Authors: Gagnon Kenneth B , Barati Michelle T , Palumbo Alex , Conklin Caryl A , Lederer Eleanor D
Published: November 2023

Changes in cell shape and volume mediated by ion and water flux across cell membranes are an important component of cell migration. Previous studies have demonstrated the importance of K‐Cl cotransporters (KCCs) in a chemically‐induced rat glioblastoma cell line (C6). Inhibition of KCC3 using siRNA reduced C6 cell migration by ~50% in an in vitro scratch wound assay. Dephosphorylation of specific threonine residues within the intracellular cytoplasmic tail of the KCCs by protein phosphatase 1A (PP1A) results in cotransporter activation. The present study aimed to further characterize the expression and regulation of the KCC family of proteins in the motility of mouse melanoma (B16F10) and human breast adenocarcinoma (MDA231) cell lines. B16F10 and MDA231 cells were both grown to confluence in 10 cm diameter cell culture dishes with Dulbecco's minimum essential media including 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified 37°C incubator with 95% O2/5% CO2. Crude membrane preparations were subjected to SDS‐PAGE and immunoblotted for KCC1, KCC3, KCC4, and PP1A proteins. B16F10 and MDA231 cells were also grown to 50% confluence on coverslip glass and immunofluorescently imaged by confocal microscopy for KCC1, KCC3, and KCC4 proteins with and without Calyculin A (PP1A inhibitor) treatment. For both cell lines, western blotting showed KCC3, KCC4, and PP1A expression enriched in crude membrane preparations. We also observed both membrane and intracellular immunofluorescent localization of KCC3 and KCC4 in both cell lines. No WB or immunofluorescent signal was detected for KCC1 in either cell line. Treatment with Calyculin A (5 nM) for 1 h resulted in a morphological change in cell phenotype from flattened with many filopodia to rounded and swollen without visible filopodia. This morphological change was reversible when the Calyculin A was removed and the cells were cultured in control DMEM media for 10 h. These results suggest that mammalian cancer cell lines overexpress KCC3 and KCC4 proteins as well as an important regulator of their activity, PP1A. Based on the Calyculin A studies, we hypothesize that PP1A dephosphorylation of specific K‐Cl cotransporter isoforms is a component of filipodia development and directional cell motility.

Support or Funding Information

Funding for this research was provided by Robley Rex Veterans Affairs Medical Center and University of Louisville School of Medicine (to EDL).
Cardiotoxin III suppresses HGF‐ and EGF‐induced EMT and signaling network in breast cancer MDA‐MB‐231 cells
Authors: Tsai Pei‐Chien , Fu Yaw‐Syan , Chang Long‐Sen , Hsu Sodio C.N. , Lu Tzongshi
Published: November 2023

Introduction and hypothesis

Metastasis causes a poor prognosis and high mortality rate of breast cancer patients. Because the epithelial‐to‐mesenchymal transition (EMT) is the initial step for metastasis of breast cancer, suppression of EMT might improve breast therapy. Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been reported to show anticancer activity. Thus, we investigated the inhibitory activity of CTX III on the HGF‐ and EGF‐induced EMT of breast cancer MDA‐MB‐231 cells.

Methods

CTX III was purified from the venom of Naja naja atra (Taiwan cobra) using chromatographies on Sephadex G‐50 and SP‐Sephadex C‐25 columns. MTT assay, transwell assay and western blotting analyses were employed to study the inhibitory activity of CTX3 on HGF‐ or EGF‐induced cell proliferation, migration, invasion and EMT‐related protein expression.

Results

CTX III treatment resulted in morphological changes of HGF‐ or EGF‐treated cells from elongated and spindle shape to rounded and epithelial‐like shape. Meanwhile, CTXIII induced E‐cadherin upregulation and down‐regulation of MMP‐9, N‐cadherin and vimentin expression, thereby inhibiting migration and invasion of HGF‐ and EGF‐treated cells. Consistently, CTX III suppressed HGF‐ or EGF‐induced expression of transcription factors that regulated the expression of EMT‐related proteins. Moreover, CTX III abrogated phosphorylation of Src, FAK, p130cas, paxillin, c‐Met and EGFR in HGF‐ or EGF‐treated cells. Thus, abolishment of HGF/c‐Met and EGF/EGFR‐mediated activation of ERK1/2 and PI3K/Akt pathways was observed after CTX III treatment.

Conclusions

Our data reveal that CTX III reverses HGF‐ and EGF‐induced epithelial‐to‐mesenchymal transition of MDA‐MB‐231 cells, and suggest that it may be served as an agent for repressing the malignancy‐promoting effect of HGF and EGF on breast cancer.

Support or Funding Information

NSYSUKMU‐104‐P‐001
Microengineered Physiological Biomimicry: Human Organ‐on‐Chips
Authors: Huh Dan Dongeun
Published: November 2023

Human organs are complex living systems in which specialized cells and tissues are assembled in various patterns to carry out integrated functions essential to the survival of the entire organism. A paucity of predictive models that recapitulate the complexity of human organs and physiological systems poses major technical challenges in virtually all areas of life science and technology. This talk will present interdisciplinary research efforts to develop microengineered biomimetic models that reconstitute complex structure, dynamic microenvironment, and physiological function of living human organs. Specifically, I will talk about i) bioinspired microsystems that mimic the structural and functional complexity of the living human lung in health and disease, ii) an organ‐on‐chip microdevice that emulates the ocular surface of the human eye, and iii) microengineered physiological models of human reproductive organs.
The Mechanobiology of Cervical Remodeling: Characterization of Select Cytoskeletal and Rho‐GTPase Signaling Factors in Mice Cervical Tissue During Pregnancy
Authors: Gordon Jacob , Frank Martin , Mowa Chishimba Nathan
Published: November 2023

During pregnancy the developing fetus exerts an increasing gravitational force as it grows in size. However, the effects and underlying molecular mechanism of this process on the cervix is not well known. Our earlier proteomics studies have provided evidence of presence and changes in levels of cytoskeletal mechano‐mediators with advancement of pregnancy. In the present study, we use real time PCR analysis to further characterize expression of select cytoskeletal genes (Filamin A, Gelsolin, Vimentin, Actinin 1, Caveolin 1, Transgelin, Keratin 1, Profilin 1 and Focal Adhesion Kinase) (day 0, day 11 and day 17) to verify findings of the earlier proteomics study. Additionally, we also investigate the expression levels of Rho‐GTPases (RhoA, RhoB and Cdc42), molecules that play cell‐signaling roles upstream of proliferation and cytoskeletal protein organization. The levels of mRNA of all the (12) genes examined increased over the course of pregnancy except 3 that decreased. We conclude that the growing fetus influences cervical remodeling and likely the timing of birth by manipulating mechano‐transduction during pregnancy.

Support or Funding Information

Office of Students Research, Appalachian State University.
H Ficolin: Polymerization and Aggregation from Blood to Cerebrospinal Fluid
Authors: Castillo‐González William , González‐Losada Cristóbal , Rodríguez‐Pérez José Alejandro , Jensenius Jens Christian , Zerr Inga , Schmitz Matthias , Dorta‐Contreras Alberto Juan
Published: November 2023

Ficolins are one of the starters of the lectin pathway It recognizes carbohydrates over the surface of pathogen and active the complement pathway. It plays an important role in the innate immunity.

H Ficolin are produced by hepatic epithelial cells, bile ducts and hepatocyte and was secreted into bile and blood.

The universal validity of the molecular diffusion/cerebrospinal fluid (CSF) flow model is valid for the brain‐derived molecules, is the base for a systematic approach to determine the source of a CSF protein either derived from blood, from brain or from leptomeninges.

Objective

To demonstrate the dynamics of diffusion of H ficolin from blood to CSF over the base of its CSF and serum concentration and the different aggregation forms.

Methods

80 serum and CSF paired samples were obtained from control persons without neuroinflammation process and without oligoclonal IgG bands. H ficolin and albumin were quantified by ELISA and by an immunoturbidimetric analysis. Q albumin=CSF albumin/serum albumin and Q H Ficolin=CSF H Ficolin/serum H ficolin was calculated.

In order to calculate the different aggregation forms was determinate the inflection points of the frequency distribution of H Ficolin vs Q H Ficolin.

Results

Serum and CSF H Ficolin did not follow a normal distribution according to Kolmogorov‐Smirnov test. Mean values of H Ficolin in serum was higher than its mean content in CSF. CSF H Ficolin concentration increased with the increase of Q albumin. H Ficolin is not a brain‐derived protein. Three inflection points was determined. (Table 1)

Conclusions

H ficolin is a blood‐derived protein and the diffusion from blood to CSF indicates three aggregation forms.

H Ficolin aggregation forms.

G1‐H Ficolin
G2‐H Ficolin
G3‐H Ficolin

Q (Quotient ratio)
0,002
0,0002
0,00004

Molecular weight (KDa)
120
800
4500

Aggregation forms
Tetramer (original structure non aggregated)
6 aggregates
24 aggregates
Gene Network and Adipose Transcriptome Analysis of the Northern Elephant Seal During Prolonged Food Deprivation
Authors: Martinez Bridget , Rutherford Kim , Crocker Daniel , Gemmell Neil , Ortiz Rudy M
Published: November 2023

Many animals have been extensively studied for their potential to elucidate cellular pathways that can lead to breakthroughs or progress in the development of therapeutic strategies against human diseases such as cardiovascular disease, metabolic syndrome, and type 2 diabetes mellitus. The northern elephant seal (Mirounga angustirostris) is a natural model for these and other maladies, and its extraordinary physiology has been the subject of substantial examination since their return from the brink of extinction. The most notable contributions have come from the assessment of endocrine changes in peripheral tissues, including adipose and muscle. Yet, the reference sequence for adipose, has not yet been described. Here, we present the first adipose‐derived northern elephant seal transcriptome assembly in response to fasting with RNA from two separate fasting phases. In this de novo assembly, differential expression analysis between fasting periods demonstrated the upregulation of a large number of metabolism‐driving transcripts. This data will provide and serve as a tool in the necessary continuation of future studies elucidating the unique physiology of the northern elephant seal.
Dantrolene Improves Endothelial Function in Type‐1 Diabetic Rats by Decreasing Oxidative Stress
Authors: Roman Marie , Alemar Stephanie , Zack Richard , Matias Jonathan , Morales Myrna , Torres Hector , Quidgley Jose , Crespo Maria J
Published: November 2023

Diabetic patients have a high risk of developing cerebral vasospasms (CVSP). Experimental evidence indicates that the addition of the ryanodine receptor (RyR) blocker dantrolene to standard therapies reduces vasoconstriction in non‐diabetic patients with CVSP. Whether diabetics with CVSP also benefit from this drug, however, is unknown. In this study, we evaluated the effects of a 30‐minute incubation period with dantrolene (50 μM) on acetylcholine (ACh)–induced relaxation in phenylephrine (PHE)‐precontracted aortic rings from streptozotocin (STZ)‐induced Type‐1 diabetic rats. Age‐matched non‐diabetic rats were used as controls (CT). Concentration response curves for the ACh‐induced relaxation from 0.1 nM to 10 μM were performed on aortic rings in the presence and absence of dantrolene. The oxidative‐stress markers malondialdehyde (MDA) and 4‐hydroxyalkenal (4‐HAE) were also measured in aortic homogenates after a 30‐minute incubation period with dantrolene. The EMAX value for the ACh‐induced relaxation was reduced in diabetic rats (40.97 ± 1.92%) when compared to CT (75.74 ± 5.27%) (N=5, P<0.05) without changing the EC50 value. Acute incubation of aortic rings with 50 μM dantrolene increased the EMAX value in diabetic rats to 70.39 ± 13.84 % (N=5, P<0.05), without affecting this parameter in CT. Endothelium‐independent relaxation with 10 μM sodium nitroprusside (SNP) was not affected by dantrolene in either diabetic or CT rats. In addition, dantrolene decreased MDA +4‐HAE (μM/g protein) in the vasculature of diabetic rats from 3.19 ± 1.15 to 1.16 ± 0.10 (N=5, P<0.05). Together, our results suggest that by reducing oxidative stress and improving endothelial function, dantrolene may exert a beneficial effect in diabetic patients with CVSP by reducing arterial tone.

Support or Funding Information

Supported by RCMI Grant G12‐RR03051.
Euglycemic Clamp in the FATZO/Pco Mouse: A New Model of Type 2 Diabetes
Authors: Christe Michael , Konkol Debra , Hawkins Eric , Friedrich Jessica , Jacobs Julie
Published: November 2023

Obesity and metabolicsyndrome are closely linked to the development of Type 2 diabetes. Recently, a new model of polygenic, non‐leptin mediated, obesity (FATZO/Pco) was developed by crossing C57BL6/Jwith AKR/J mice and selectively bred to select for obesity, insulin resistance, and hyperglycemia. Newly published data suggests that the FATZO mouse responds to various anti‐diabetic agents. To extend these findings, we performed aeuglycemic clamp study in age‐matched FATZO mice with low body weight (LBWV; mean BW=34g+3) and high BW (BW=43g+2) to determine the extent and tissue location of insulin resistance. Subgroups of the high BW group were treated with vehicle (HBWV) or rosiglitazone (HBWR; 30mg/kg, po, qd) for 8 days. A euglycemic‐hyperinsulinemic clamp was performed, using a 40mU/kg insulin bolus and 10mU/kg/min infusion and 3‐3H‐glucoseinfusion to estimate endogenous glucose production (EGP). After an overnight fast, the LBWV group was variably hyperglycemic compared to the HBWV group. However, the basal plasma insulin levels of the HBWV were more than twice as high, suggesting significant insulin resistance. The glucose infusion rate(GIR) was reduced nearly 50% in the HBWV compared to the LBWV group. The GIR of the HBWR group was nearly identical to the LBWV group. There was no difference in basal EGP between LBWV and HBWV groups but basal EGP was reduced in the HBWR group. Insulin‐mediated suppression of EGP was 60% in the LBWV group, 5% in the HBWV group, and 75% in the HBWR group. A bolus of 2‐14C‐Deoxyglucose (2‐DG) was subsequently administered to determine tissue glucose uptake. No significant differences between LBWV and HBWV were detected in the sole us muscle or the epididymal fat pad. However, 2‐DG uptake was significantly reduced in the gastrocnemius and quadriceps muscles in the HBWV group. Administration of rosiglitazone increased 2‐DG uptake in all tissues tested. Suppression of plasma free fatty acids during the clamp period was marginally reduced in the HBWV group. In conclusion, these data demonstrate a marked reduction in whole‐body glucose utilization and profound hepatic insulin resistance in the FATZO mice. In addition, the data reveal evidence of significant insulin resistance in peripheral tissues. The FATZO mouse maybe an ideal, non‐leptin model for diabetes/obesity target evaluation and efficacy determination.
Neuronal Inflammation: A Potential Contributing Mechanism to High Fat Diet‐Induced Neuropathy
Authors: Umbaugh David S , Maciejewski J. Claire , Guilford Brianne L.
Published: November 2023

Neuropathy, a debilitating complication of diabetes, has primarily been attributed to poor glycemic control, but has recently been associated with obesity and the metabolic syndrome in nondiabetic individuals. A robust body of evidence indicates that a high fat diet can induce signs of neuropathy in mice but the pathogenesis of high fat diet‐induced neuropathy remains unknown.

PURPOSE

To determine if neuronal inflammation is a potential initiating mechanism for the development of mechanical hypersensitivity and nerve fiber changes (signs of neuropathy) in high fat fed mice.

METHODS

Male C57Bl/6 mice were randomized to a standard (Std, 15% kcal from fat) or high fat diet (HF, 54% kcal from fat) for 2, 4, or 8 wks (n = 11–12 per group). Lumbar dorsal root ganglia were harvested and inflammatory mediators (IL‐1α, IL‐1β, IL‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐10, IL‐12p70, IL‐17, MCP‐1, IFN‐γ, TNF‐α, MIP‐1α, GMCSF, RANTES) were quantified using a Multiplex ELISA and normalized to total protein. Neuropathy was characterized by the von Frey test for mechanical sensitivity at wk 0 and every other week thereafter. Hindpaw foot pad skin was harvested at end study and used to quantify intraepidermal nerve fiber density (IENFD) and a specific population of nociceptive nerve fibers (TrkA) via immunohistochemistry.

RESULTS

After 8 wks, HF had greater bodyweight (33.3 ± 1.0 vs. 26.7 ± 0.5 g, p < 0.001), fasting blood glucose (160.3 ± 9.4 vs. 138.5 ± 3.4 mg/dl, p < 0.05) and insulin (3.58 ± 0.46 vs. 0.82 ± 0.14 μg/L, p < 0.001) compared to Std. IL‐1α and IL‐5 were higher in HF compared to Std after 2 wks and 4 wks, respectively (IL‐1α: 4.8 ± 1.3 vs. 2.9 ± 0.6 pg/mg, p < 0.05; IL‐5: 5.8 ± 0.7 vs. 3.1 ± 0.5 pg/mg, p < 0.05). IENFD and TrkA fiber density were also higher in HF compared to Std after 4 wks (IENFD: 39.4 ± 1.2 vs. 32.2 ± 1.3 fibers/mm, p < 0.001; TrkA: 30.4 ± 1.8 vs. 22.4 ± 1.3 fibers/mm). There were no significant differences in hindpaw sensitivity for Std vs. HF at any time point.

CONCLUSION

Increased neuronal inflammation preceded and accompanied an increase in a specific population of nociceptive nerve fibers (TrkA) in the hindpaw footpads of high fat fed mice. Taken together, these data suggest that a diet high in fat may increase neuronal inflammation and initiate nerve fiber changes responsible for painful neuropathy in nondiabetic and/or diabetic patients.

Support or Funding Information

Supported by Southern Illinois University Edwardsville Seed Grants for Transitional and Exploratory Projects
Heart‐Specific Disruption of Bmal1 Leads to Hepatic Insulin Resistance in Mice
Authors: Nakao Tomomi , Kohsaka Akira , Otsuka Tsuyoshi , Le Hue Thi , Thein Zaw Lin , Sato Fuyuki , Ihara Hayato , Muragaki Yasuteru , Maeda Masanobu
Published: November 2023

Daily balance of glucose metabolism is closely coupled with the function of the circadian clock in mammals. The molecular clock composed of a series of circadian clock genes resides in nearly all cells throughout the body, including cells in both metabolic and non‐metabolic tissues. Disruption of the molecular clock in metabolic tissues such as pancreas was shown to induce glucose intolerance. However, it remains unknown whether the clock in non‐metabolic tissues also plays an important role in maintaining systemic glucose metabolism. By studying mice with heart‐specific deletion of Bmal1, a core circadian clock gene, we demonstrate that clock function in the heart has a great influence on glucose tolerance. Heart‐specific disruption of Bmal1 not only results in a reduction in cardiac function but induces a significant increase in the blood glucose level in an age‐dependent manner. In addition, the insulin tolerance test reveals a decrease in insulin sensitivity in heart‐specific Bmal1 deficient mice, indicating that the increase in the blood glucose level observed in these mice is at least in part due to systemic insulin resistance. Importantly, the levels of mRNA expression of gluconeogenic genes in the liver of heart‐specific Bmal1 deficient animals are not suppressed by systemic administration of insulin, indicating that disruption of the molecular clock in the heart can induce hepatic insulin resistance. Taken together, our results suggest that, in addition to the clock function in major metabolic tissues, the function of the heart clock also has an impact on systemic glucose metabolism by altering hepatic insulin sensitivity.

Support or Funding Information

This work was supported by JSPS KAKENHI Grant Number 25460316 and the Takeda Science Foundation.
The regulation of skeletal muscle insulin‐stimulated signaling by the MEK‐REDD1‐mTOR axis
Authors: Williamson David L , Dungan Cory M
Published: November 2023

Recent findings in adipocytes suggest that mitogen‐activated protein kinase (MAPK)/extracellular‐regulated signaling kinase (ERK) kinase 1/2 (MEK1/2) signaling regulates regulated in development and DNA damage 1 (REDD1) protein expression. Similarly, our previous work show that a lack of REDD1 protein expression, and associated hyperactive basal mechanistic target of rapamycin (mTOR) signaling, limits skeletal muscle's response to insulin. Therefore, we sought to determine: 1) if MEK1/2 inhibition is sufficient to reduce REDD1 protein expression and subsequently insulin receptor substrate‐1 (IRS‐1) tyrosine phosphorylation via negative feedback of hyperactive mTOR in REDD1 wild‐type (WT) mice and 2) if rapamycin‐mediated mTOR inhibition is sufficient to improve IRS‐1 tyrosine phosphorylation in REDD1 knockout (KO) mice. REDD1 WT mice were injected with 10mg/kg BW of the MEK1/2 non‐competitive inhibitor, PD184352, 3 hours prior to acute insulin treatment (0.5 IU/kg BW). In separate studies, REDD1 KO mice were injected with 5mg/kg BW of the mTOR inhibitor, rapamycin, 3 hours prior to acute insulin treatment (0.5 IU/kg BW). Following the inhibitor treatment period, markers of insulin signaling activation (IRS‐1 Y1222, MEK1/2 S217/221, ERK1/2 T202/Y204), REDD1, and mTOR signaling activation (S6K1 T389, rpS6 S240/244) were examined in skeletal muscle collected before and after the 10 minute insulin treatment. PD184352 treatment reduced MEK/ERK phosphorylation and REDD1 protein expression, independent of insulin. This reduction in REDD1 protein expression was associated with elevated basal mTORC1 signaling activation and reduced insulin stimulated IRS‐1 phosphorylation. Conversely, rapamycin inhibited S6K1 and rpS6 activation, and significantly improved IRS‐1 and MEK1/2 phosphorylation in KO mice. These data affirm that REDD1 is required for normal insulin‐stimulated signaling, and that a subtle balance exists between MEK1/2, REDD1, and mTOR for the proper regulation of insulin signaling.

Support or Funding Information

Mark Diamond Foundation
Exercise‐induced Calmodulin dependent protein kinase (CaMK)II activation regulates saturated and unsaturated fatty acids in rat skeletal muscle
Authors: Mukwevho Emmanuel
Published: November 2023

CaMKII regulates many pathways involved in the regulation of various cellular and molecular mechanisms that result in myriad health benefits. Exercise is a key activator of CaMKII, and shown to improve many functional activities in individuals who exercise compared with those who do not exercise, however the mechanism involved not yet fully elucidated. Fatty acids play crucial role in the pathogenesis of metabolic diseases such as type 2 diabetes, obesity, and metabolic syndromes (MetS) (Haffner et al., 1992; Hanson et al., 2002). Since fatty acid have been implicated in the aetiology of metabolic disease, exercise through its activation of CaMKII can prove to be a vital tool in finding therapeutic modalities for treating of the metabolic syndrome.

Lipids were analysed from rats muscle using GC×GC TOFMS. Palmitoleic acid and oleic acid which are monounsaturated fatty acids known to promote insulin sensitivity and improve glycaemic control were investigated. Levels of the exercise group showed ~2.0 fold increase compared with the non‐exercise (control) group. Abolishing CaMKII activity by administration of KN93 significantly decreased exercise‐induced Palmitoleic acid levels. Oleic acid levels of the exercise group were ~ 4.1 folds higher than the non‐exercise group and followed the same pattern as Palmitoleic acid. Lauric acid is a saturated fatty acid, which increases fatty acid needed for better health. The exercise group showed ~ 8.7 fold increase compared with the non‐exercise group of Lauric acid. The exercise + KN93 group significantly reduced induction by ~2.5 fold compared with the exercise group. On the other hand, Myristic acid and palmitic acid which are saturated fatty acids known to increase risk factors of metabolic syndrome. The myristic acid level of the exercise group decreased by ~3.4 fold compared with the control group, whereas the exercise + KN93 group significantly increased by ~4.3 compared with the exercise group.

In conclusion, this study shows that CaMKII activation by exercise regulates saturated and unsaturated fatty acids. Therefore, CaMKII can reduce the risk factors of metabolic syndrome and type 2 diabetes.

Support or Funding Information

National Research Foundation of South Africa
Gut Microbiotal Dysbiosis and Increased Risk for Complex Polygenic Diseases Emerge with Genomic Selection for Low Aerobic Exercise Capacity
Authors: Zhang Youjie , Mell Blair , Kumarasamy Sivarajan , Cheng Xi , Britton Steven L. , Koch Lauren G. , Joe Bina
Published: November 2023

Bi‐directional host‐microbiotal interactions are increasingly being recognized as important factors contributing to the pathophysiology of complex polygenic diseases. While host genomes are inherited, the microbiome is acquired as a result of the composition of the microbiota that choose to reside within the host. The identities of the factors that determine which microbes choose to reside in a given host are unknown. Given the evidence for host‐microbiotal interactions in health and disease, we hypothesized that host genomes exert a selection pressure to influence the types of microbiota that reside in the host and that the resultant microbiotal composition is a pivotal determinant of the divide between health and disease of the host. To test this hypothesis, we developed models of health and disease by inbreeding rats divergent in aerobic exercise capacity. These novel inbred strains were developed by >20 generations of brother‐sister mating of the selectively‐bred low aerobic capacity rats and high aerobic capacity rats and named as LCR/BJ and HCR/BJ, respectively. Inbred HCR/BJ rats had significantly higher aerobic exercise capacity than inbred LCR/BJ rats as recorded by their total running distance to exhaustion of 1151 m vs. 130 m, respectively, p< 0.001. Next‐generation sequencing of the whole genomes of inbred HCR/BJ and LCR/BJ rats revealed considerable differences in their genomic sequences. To test whether this selection for differential genomic sequences resulted in any alterations in microbiota, fecal microbial communities of inbred LCR/BJ and HCR/BJ rats were profiled using 16S rRNA sequences. Interestingly, principal coordinates analysis displayed distinct clustering of the two cohorts (ANOSIM analysis, weighted UniFrac, P = 0.002), indicating significant phylogenetic differences of the microbial community structures between inbred LCR/BJ and HCR/BJ rats. Firmicutes/Bacteriodetes ratio was higher in the LCR/BJ rats compared with HCR/BJ rats suggesting the possibility of the increased risk for diseases in the LCR/BJ rats. Compared with inbred HCR/BJ rats, inbred LCR/BJ rats exhibited elevated blood pressure, increased body weight, lower circulating mean corpuscular hemoglobin, higher neutrophil‐to‐lymphocyte ratio, and higher immobility in the forced swim test. Metabolic phenotyping is currently underway. Overall, these studies provide clear evidence for the ability of the host genome to shape the microbiome and identify an inverse co‐evolutionary relationship between host genomic factors conferring enhanced aerobic exercise capacity and microbiotal dysbiosis driving disease risks.