mRNA Content Research Articles

Translational PK-PD model for invivo CAR-T-cell therapy delivered using CAR mRNA-loaded polymeric nanoparticle vector.

Autologous chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable response rates, yet its widespread implementation is hindered by logistical, financial, and physical constraints. Additionally, challenges such as poor persistence and allorejection are associated with allogeneic cell therapies. An innovative approach involves invivo transduction of endogenous T-cells through the administration of CAR mRNA encapsulated in polymeric nanoparticles (NPs), resulting in transient CAR surface expression on circulating T-cells. This method presents a promising alternative, although the dose-exposure-response relationship of invivo CAR-Ts remains poorly elucidated. The transient nature of CAR expression may necessitate repeated dosing, potentially introducing additional hurdles like cost and patient compliance. To address this issue, we have devised a translational pharmacokinetic-pharmacodynamic (PK-PD) model that characterizes the transient surface CAR expression following mRNA-encapsulated NP administration, leveraging invitro and invivo data alongside critical binding kinetic parameters sourced from literature. Our model adequately captures the transient surface CAR expression in both settings, while incorporating known physiological parameter values and exhibiting precise estimation of unknown parameters (coefficient of variation < 30%). Global sensitivity analyses underscore the significance of intracellular mRNA stability, highlighting the sensitivity of parameters linked to free intracellular mRNA concentration. Model-based simulations indicate that optimizing dose and dosing frequency can achieve sustained CAR expression, despite the transient protein expression characteristic of mRNA-based therapies. This mechanistic PK-PD model holds potential for integration into physiologically-based pharmacokinetic models, facilitating the translation of invivo CAR-T-cell therapies from preclinical studies to human applications.

MRNA compartmentalization via multimodule DNA nanostructure assembly augments the immunogenicity and efficacy of cancer mRNA vaccine.

Messenger RNA (mRNA) vaccine has fueled a great hope for cancer immunotherapy. However, low immunogenicity, caused by inefficient mRNA expression and weak immune stimulation, hampers the efficacy of mRNA vaccines. Here, we present an mRNA compartmentalization-based cancer vaccine, comprising a multimodule DNA nanostructure (MMDNS)-assembled compartment for efficient mRNA translation via in situ localizing mRNA concentration and relevant reaction molecules. The MMDNS is constructed via programmable DNA hybridization chain reaction (HCR)-based strategy, with integrating antigen-coded mRNA, CpG oligodeoxynucleotides (ODNs), acidic-responsive DNA sequence, and dendritic cells targeting aptamer. MMDNS undergoes in situ assembly in acidic lysosomes to form a micro-sized aggregate, inducing an enhanced CpG ODN adjuvant efficacy. Subsequently, the aggregates escape into cytoplasm, providing a moderate compartment which supports the efficient translation of spatially proximal mRNA transcripts via localizing relevant reaction molecules. The mRNA compartmentalization-based vaccine boosts a strong immune response and effectively inhibits tumor growth and metastasis, offering a robust strategy for cancer immunotherapy.

Prediction model of male reproductive function damage caused by CHOP chemotherapy regimen for non-Hodgkin's lymphoma

ObjectiveThe CHOP combined chemotherapy regimen (cyclophosphamide, doxorubicin, vincristine, and prednisone) is commonly used to treat non-Hodgkin Lymphoma (NHL). While these drugs are effective for cancer treatment, they may have side effects on the reproductive system that are poorly studied. This study used a mouse model to investigate the mechanisms of reproductive function impairment induced by the CHOP regimen and developed a predictive model for assessing reproductive damage with a non-invasive procedure.MethodsFrom 2022 to 2023, we statistically analyzed the changes of reproductive function of NHL patients before and after receiving CHOP regimen in the First Affiliated Hospital of Xiamen University. The NHL mouse model was established and divided into CHOP treatment group and control group. The weight of testis and epididymis, sperm quality and motility were compared between the two groups. Histopathological examination of testicular tissue was performed to determine pathological changes. ELISA was used to measure the expression of cytokines and cytokine pathways in serum, protein expression was analyzed by immunohistochemistry, and protein and mRNA levels of cytokines and pathways were evaluated by Western blotting and qPCR. Using stepwise regression method to select important factors, a prediction model of reproductive system damage was constructed.ResultsFifty-two NHL patients included in the questionnaire showed significant reproductive system damage after CHOP regimen treatment. The weight of testis and epididymis, as well as the number and vitality of sperm in the mouse model treatment group were significantly lower than those in the control group. Serum LH, FSH, estradiol and progesterone levels decreased significantly, while inhibin B levels increased significantly. There was no significant change in testosterone or prolactin levels. Inflammatory markers such as CSF-1, IL-1, IL-6, TGF-β1 and GDNF increased significantly, while the level of SOD1 decreased significantly. Immunohistochemical staining analysis showed that CAMP, Caspase3, CSF-1, GDNF, IL-1, IL-6, PRKACB, TGF-β1 and TXNDC5 were all expressed in spermatocytes, and the expression of therapeutic histones was significantly higher than that of the control group. Western blot analysis further detected the protein expression, and QPCR detected the mRNA content. The results showed that the expression of histone and mRNA in the treatment group was significantly higher than that in the control group. Stepwise regression method determined that estradiol (E2) was the most important variable in the prediction model, and the AUC for predicting reproductive damage was 1.ConclusionsThe CHOP regimen induces male reproductive toxicity, potentially mediated through alterations in hormone levels and increased expression of inflammatory cytokines and oxidative stress. Using E2 as the sole predictor in the model accurately predicts the extent of reproductive damage, offering a non-invasive method for detecting reproductive system damage.

Exosomes derived from Umbilical cord mesenchymal stem cell promote hair regrowth in C57BL6 mice through upregulation of the RAS/ERK signaling pathway.

Androgenetic alopecia is one of the common types of hair loss and has become a medical and social problem due to its increasingly young onset. Existing therapies, although effective, have serious side effects and therefore better treatments need to be sought. The aim of this study was to evaluate the efficacy of umbilical cord mesenchymal stem cell-derived exosomes in the treatment of androgenetic alopecia and to investigate the mechanism of exosome regulation of hair growth. First, we randomly divided 20 C57BL/6J mice into blank group, model group, positive control group and exosomal hydrogel group, and mice were treated with hair removal on the back. The mice were injected intraperitoneally with dihydrotestosterone solution except for the blank group. At the end of the experiment, new hairs were collected and the differences in length, diameter and number of hair follicles were compared among the groups; the histopathological changes of hair follicles were observed by HE staining; the expression of androgen receptor mRNA and protein in skin tissues were compared; and the skin tissues were analyzed by real-time PCR, western blotting, immunofluorescence staining and transcriptome sequencing. Finally, the results of transcriptome sequencing experiments were verified by real-time PCR, western blotting and other techniques for the corresponding genes and proteins. Compared with the blank group, mice in the model group had shorter hair length and reduced hair diameter, and pathological observation showed that the total number of hair follicles was significantly reduced and the hair follicles were miniaturized; compared with the model group, mice in the positive control and exosome groups had longer hair length, larger hair diameter and more hair follicles; the androgen receptor mRNA content and protein expression in the skin tissue of mice in the model group were significantly higher than those in the blank group, and the protein expression in the exosome gel group was lower than that in the model group. Similarly, compared with the model group, the expression of stemness-related proteins K15 and CD200 in the skin tissues of mice in the exosome group increased, and the expression of PCNA, a protein related to cell proliferation, increased. The KEGG data showed that the differential genes were mainly enriched in the RAS/ERK pathway. In this study, we demonstrated the therapeutic effect of umbilical cord MSC-derived exosomes on androgenetic alopecia and verified that exosomes regulate hair follicle stem cell stemness through the RAS/ERK pathway to promote hair proliferation and thus hair growth in mice with androgenetic alopecia, providing a potential therapeutic strategy for androgenetic alopecia.

Contractile regulation of the glucocorticoid-sensitive transcriptome in young and aged skeletal muscle.

Elevated glucocorticoids alter the skeletal muscle transcriptome to induce a myopathy characterized by muscle atrophy, muscle weakness, and decreased metabolic function. These effects are more likely to occur and be more severe in aged muscles. Resistance exercise can blunt the development of glucocorticoid myopathy in young muscle, but the potential to oppose the signals initiating myopathy in aged muscle is unknown. To answer this, young (4-mo-old) and aged (24-to 25-mo-old) male C57BL/6 mice were randomized to receive either an intraperitoneal (IP) injection of dexamethasone (DEX; 2 mg/kg) or saline as a control. Two hours postinjections, the tibialis anterior (TA) muscles of mice were subjected to unilateral high-force contractions. Muscles were harvested 4 h later. The glucocorticoid- and contraction-sensitive genes were determined by RNA sequencing. The number of glucocorticoid-sensitive genes was similar between young and aged muscle. Contractions opposed changes to more glucocorticoid-sensitive genes in aged muscle, with this outcome primarily occurring when hormone levels were elevated. Glucocorticoid-sensitive gene programs opposed by contractions were primarily related to metabolism in young mice and muscle size regulation and inflammation in aged mice. In silico analysis implied peroxisome proliferator-activated receptor gamma-1 (PPARG) contributed to the contraction-induced opposition of glucocorticoid-sensitive genes in aged muscle. Increasing PPARG expression in the TA of aged mice using adeno-associated virus serotype 9 partially counteracted the glucocorticoid-induced reduction in runt-related transcription factor 1 (Runx1) mRNA content, recapitulating the effects observed by contractions. Overall, these data contribute to our understanding of the contractile regulation of the glucocorticoid transcriptome in aged skeletal muscle.NEW & NOTEWORTHY We establish the extent to which muscle contractions oppose changes to the glucocorticoid-sensitive transcriptome in both young and aged muscle. We also identify peroxisome proliferator-activated receptor gamma (PPARG) as a transcription factor likely contributing to contraction-induced opposition to the glucocorticoid transcriptome in aged muscle. Overall, these data contribute to our understanding of the contractile regulation of the glucocorticoid transcriptome.

Efficient enzyme-free isolation of brain-derived extracellular vesicles.

Extracellular vesicles (EVs) have gained significant attention as pathology mediators and potential diagnostic tools for neurodegenerative diseases. However, isolation of brain-derived EVs (BDEVs) from tissue remains challenging, often involving enzymatic digestion steps that may compromise the integrity of EV proteins and overall functionality. Here, we describe that collagenase digestion, commonly used for BDEV isolation, produces undesired protein cleavage of EV-associated proteins in brain tissue homogenates and cell-derived EVs. In order to avoid this effect, we studied the possibility of isolating BDEVs with a reduced amount of collagenase or without any protease. Characterization of the isolated BDEVs from mouse and human samples (both female and male) revealed their characteristic morphology and size distribution with both approaches. However, we show that even minor enzymatic digestion induces 'artificial' proteolytic processing in key BDEV markers, such as Flotillin-1, CD81, and the cellular prion protein (PrPC), whereas avoiding enzymatic treatment completely preserves their integrity. We found no major differences in mRNA and protein content between non-enzymatically and enzymatically isolated BDEVs, suggesting that the same BDEV populations are purified with both approaches. Intriguingly, the lack of Golgi marker GM130 signal, often referred to as contamination indicator (or negative marker) in EV preparations, seems to result from enzymatic digestion rather than from its actual absence in BDEV samples. Overall, we show that non-enzymatic isolation of EVs from brain tissue is possible and avoids artificial pruning of proteins while achieving an overall high BDEV yield and purity. This protocol will help to understand the functions of BDEV and their associated proteins in a near-physiological setting, thus opening new research approaches.

Evaluation of cosmetic efficacy of lychee seed fermentation liquid

BackgroundLychee seeds were fermented by three kinds of bacteria (Lactobacillus plantarum, Saccharomyces cerevillus and ganoderma lucidum mycelium), and two effective strains were selected by two indexes of activity content and antioxidant, so as to further verify whether lychee seeds have waste multiplication effect and can protect cells damaged by oxidation from anti-inflammatory, anti-aging and safety.ResultsThe contents of polyphenols, flavonoids and proteins in the solution fermented by Ganoderma lucidum mycelium did not increase, thus affecting the antioxidant capacity of the solution was far less than that of the water extract. The active content of the other two fermentation solutions was higher than that of the water extract, and the ability of scavenging free radicals of the two solutions increased with the increase of the volume fraction. At the cellular level, the two fermentation solutions showed repair effects on UVA-induced damaged cells. The contents of type I collagen (COL-1), total antioxidant capacity and ELN were increased, the contents of reactive oxygen species and MDA were decreased, and the expressions of inflammatory factors IL-6, TNF-a, iNOS and COX-2 were decreased in HaCaT cells. From the gene level, the mRNA contents of IL-6, TNF-a, Caspase-3, Caspase-9, Bax and Bcl were significantly decreased. The test of chick embryo chorioallantoic membrane (HTET CAM) showed that there was no bleeding and litchi seed fermentation liquid was not irritating.ConclusionsTherefore, two kinds of litchi seed fermentation can be used as natural plant raw materials for cosmetics, and have strong antioxidant, anti-inflammatory and anti-aging functions on skin, and also have good human safety.Graphical

Loss of Y-chromosomal genes in macrophages induces paracrine phenotypic alterations in cardiac cells

Abstract Background At the intersection of gender medicine and ageing, mosaic loss of the Y chromosome (mLoY) in hematopoietic cells has emerged as a novel cardiovascular risk factor. As the most prevalent chromosomal aberration in men, mLoY is age-associated and characterised by interindividual varying frequencies of LoY cells in blood. It is associated with an increased risk for cardiovascular disease and mortality. A murine model of mLoY demonstrates myocardial fibrosis and reduced lifespan. However, the interactions between LoY hematopoietic and cardiac cells remain elusive. It is uncertain, whether the loss of single genes or combinations of multiple genes drive the observed effects, impeding the development of targeted therapies. Purpose We aimed to examine the impact of individual Y-chromosomal genes in macrophages concerning their paracrine effects on cardiac cells types. Methods and results To identify target genes, we analysed two single-nuclei RNA sequencing datasets of patient-derived PBMCs and THP-1 monocytic cells for their expression of Y-chromosomal genes. We identified nine genes that exhibited robust and concordant expression in both datasets. Seven were protein coding (RPS4Y, DDX3Y, EIF1AY, ZFY, UTY, USP9Y and KDM5D) and two were non-coding (TTTY14 and LINC00278). Using siRNA pools, we achieved &amp;gt;50% reductions in mRNA content in THP-1 M0 macrophages for all genes (p&amp;lt;0.05) and collected conditioned media (CM). As previous data suggested increased myocardial fibrosis in mLoY, we assessed the impact of CM on primary human cardiac fibroblasts. Notably, treatment with CM from ZFY, TTTY14 or LINC00278 silenced macrophages induced collagen 1A1 protein expression by 1.44-, 1.30- and 1.86-fold, respectively (all p&amp;lt;0.05), suggesting that loss of these genes might contribute to myocardial fibrosis. To explore potential effects on cardiomyocytes, neonatal rat cardiomyocytes were exposed to CM. We observed reduced beating frequencies upon treatment with CM from ZFY, TTTY14 or UTY silenced macrophages (relative fold-changes 0.45, 0.40, 0.53, respectively; all p&amp;lt;0.05). While suggestive of cardiomyocyte stress, we found no changes in the expression of the stress markers Nppa or Nppb, possibly indicating direct effects on ion transporters. Given the strong association of cardiovascular disease with endothelial cell dysfunction, we investigated the effects of CM on human umbilical vein endothelial cells (HUVEC). CM from ZFY and KDM5D silenced macrophages induced ICAM protein expression in HUVEC by 1.9- and 1.6-fold, respectively (both p&amp;lt;0.05). Conclusion Loss of individual Y-chromosomal genes in monocytic cells elicits distinct paracrine effects on various cardiac cell types. Particularly, silencing of ZFY in macrophages demonstrated profound effects on all investigated cell types, warranting further investigation. Additionally, two long non-coding RNAs, previously unexplored in cardiovascular disease, influenced paracrine activity of macrophages.

Enhancing RNA encapsulation quantification in lipid nanoparticles: Sustainable alternatives to Triton X-100 in the RiboGreen assay

To quantify concentration and encapsulation efficiency (EE) of mRNA in lipid nanoparticles (LNPs) the RiboGreen assay is extensively used. As part of this assay, a surfactant is used to release mRNA from LNPs for detection with the RiboGreen dye. So far, the surfactant of choice has been Triton X-100, which is harmful to human health and the environment. Alternatives to Triton X-100 are therefore needed, but surprisingly no such effort has yet been described in the literature. Here we show how three, less harmful, surfactants (Brij 93, Zwittergent 3–14 and Tween 20) compare to Triton X-100 for releasing mRNA from LNPs for detection with the RiboGreen assay. We found that Zwittergent 3–14 and Tween 20 at high concentrations (0.5 %) are at the minimum as effective as Triton X-100 at high concentration (0.5 %) across three different mRNA-LNP formulations. Interestingly, Tween 20 was the most effective at releasing mRNA from LNPs, across all concentration ranges explored (0.0025 %, 0.01 %, 0.1 % and to 0.5 % (v/v)) highlighting its potency at solubilizing the three different LNP formulations. Our results show that Tween 20 can be used as an alternative to Triton X-100 in the RiboGreen assay, resulting in more accurate quantification of the total mRNA concentration and EE%, as well as making the assay more environmentally friendly. Such improvement could potentially increase the likelihood of identifying therapeutically attractive hard-to-solubilize LNP-mRNA formulations that would be discharged when using Triton X-100 due to their apparent low EE values, as well as ensure more accurate mRNA dosing in both in vitro and in vivo studies.

Comparison of direct cDNA and PCR-cDNA Nanopore sequencing of RNA from Escherichia coli isolates.

Whole-transcriptome (long-read) RNA sequencing (Oxford Nanopore Technologies, ONT) holds promise for reference-agnostic analysis of differential gene expression in pathogenic bacteria, including for antimicrobial resistance genes (ARGs). However, direct cDNA ONT sequencing requires large concentrations of polyadenylated mRNA, and amplification protocols may introduce technical bias. Here we evaluated the impact of direct cDNA- and cDNA PCR-based ONT sequencing on transcriptomic analysis of clinical Escherichia coli. Four E. coli bloodstream infection-associated isolates (n=2 biological replicates per isolate) were sequenced using the ONT Direct cDNA Sequencing SQK-DCS109 and PCR-cDNA Barcoding SQK-PCB111.24 kits. Biological and technical replicates were distributed over eight flow cells using 16 barcodes to minimize batch/barcoding bias. Reads were mapped to a transcript reference and transcript abundance was quantified after in silico depletion of low-abundance and rRNA genes. We found there were strong correlations between read counts using both kits and when restricting the analysis to include only ARGs. We highlighted that correlations were weaker for genes with a higher GC content. Read lengths were longer for the direct cDNA kit compared to the PCR-cDNA kit whereas total yield was higher for the PCR-cDNA kit. In this small but methodologically rigorous evaluation of biological and technical replicates of isolates sequenced with the direct cDNA and PCR-cDNA ONT sequencing kits, we demonstrated that PCR-based amplification substantially improves yield with largely unbiased assessment of core gene and ARG expression. However, users of PCR-based kits should be aware of a small risk of technical bias which appears greater for genes with an unusually high (>52%)/low (<44%) GC content.

Impact of an extended light regimen imposed during nursery period on the performance and lipid metabolism of weanling pigs.

This study aimed to assess the impact of a prolonged photoperiod on the growth performance and lipid metabolism of weaned piglets. Twenty-four piglets weaned at 28 days of age were randomly dichotomized into two groups that were alternatively subjected to either long photoperiod (LP) group (16L:8D) or short photoperiod (SP) group (10L:14D) for 42days. Four replicates of three animals per replicates were used per experimental treatment. Our results demonstrated that prolonged photoperiod increased piglet body weight, average daily weight gain (ADG), backfat thickness (BF), backfat index during the nursery period, and increased ADG, average daily feed intake (ADFI), and decreased the F/G of piglets during the experiment days 29 to 42. Meanwhile, we observed LP piglets' plasma melatonin, growth hormone and serotonin levels were decreased at 14 d and 42 d compared to SP piglets. Moreover, up-regulated mRNA or protein expression of PPARγ and CEBPα, and lower mRNA or protein expression of MTR1, ATGL, HSL, PPARα, and CPT1α, were observed in back subcutaneous fat (BSF) of LP group compared with that of SP group. Significant increases were observed in the mRNA or protein contents of lipogenic genes, including C/EBPα, SREBP-1c, ACCα, and FAS, in the liver of LP piglets, whereas CPT1α and ACOX1 mRNA levels and PPARα and MTR1 protein expression were significantly down-regulated in LP group compared to SP group. Extended photoperiod also increased lipid content in longissimus dorsi muscle (LDM) that was associated with higher mRNA or protein levels of SREBP-1c, ACCα, FAS, Pref1, and LPL, decreased mRNA or protein contents of LeptinR, MTR1, HSL, and ACOX1. Together, these findings suggest that there is an advantage, in terms of growth performance and fat deposition, in imposing a prolonged light program (16-h light/day) on nursery piglets to alleviate the negative aspects of weaning stress.

Spatial mRNA expression patterns of orexin receptors in the dorsal hippocampus.

Orexins are wake-promoting neuropeptides that originate from hypothalamic neurons projecting to widespread brain areas throughout the central nervous system. They modulate various physiological functions via their orexin 1 (OXR1) and 2 (OXR2) receptors, including sleep-wake rhythm but also cognitive functions such as memory formation. Here, we provide a detailed analysis of OXR1 and OXR2 mRNA expression profiles in the dorsal hippocampus as a key region for memory formation, using RNAscope multiplex in situ hybridization. Interconnected subareas relevant for cognition and memory such as the medial prefrontal cortex and the nucleus reuniens of the thalamus were assessed as well. Both receptor types display distinct profiles, with the highest percentage of OXR1 mRNA-positive cells in the hilus of the dentate gyrus. Here, the content of OXR1 mRNA per cell was slightly modulated at selected time points over a 12h light/ 12 dark light phase. Using RNAScope and quantitative polymerase chain reaction approaches, we began to address a cell-type specific expression of OXR1 in hilar GABAergic interneurons. The distinct expression profiles of both receptor subtypes within hippocampal subareas and circuits provide an interesting basis for future interventional studies on orexin receptor function in spatial and contextual memory.

Inhibiting the Cholesterol Storage Enzyme ACAT1/SOAT1 in Myelin Debris-Treated Microglial Cell Lines Activates the Gene Expression of Cholesterol Efflux Transporter ABCA1

Aging is the major risk factor for Alzheimer’s disease (AD). In the aged brain, myelin debris accumulates and is cleared by microglia. Phagocytosed myelin debris increases neutral lipid droplet content in microglia. Neutral lipids include cholesteryl esters (CE) and triacylglycerol (TAG). To examine the effects of myelin debris on neutral lipid content in microglia, we added myelin debris to human HMC3 and mouse N9 cells. The results obtained when using 3H-oleate as a precursor in intact cells reveal that myelin debris significantly increases the biosynthesis of CE but not TAG. Mass analyses have shown that myelin debris increases both CE and TAG. The increase in CE biosynthesis was abolished using inhibitors of the cholesterol storage enzyme acyl-CoA:cholesterol acyltransferase 1 (ACAT1/SOAT1). ACAT1 inhibitors are promising drug candidates for AD treatment. In myelin debris-loaded microglia, treatment with two different ACAT1 inhibitors, K604 and F12511, increased the mRNA and protein content of ATP-binding cassette subfamily A1 (ABCA1), a protein that is located at the plasma membrane and which controls cellular cholesterol disposal. The effect of the ACAT1 inhibitor on ABCA1 was abolished by preincubating cells with the liver X receptor (LXR) antagonist GSK2033. We conclude that ACAT1 inhibitors prevent the accumulation of cholesterol and CE in myelin debris-treated microglia by activating ABCA1 gene expression via the LXR pathway.

12439 Investigating The Regulation Of Islet Alpha-cell Function Mediated By The ISL1 And LDB1 Transcriptional Complex

Abstract Disclosure: I. Deeba: None. S. Poole: None. Y. Liu: None. C. Hunter: None. Glucose homeostasis is maintained by the action of insulin-secreting β-cells and glucagon-producing α-cells in the pancreatic islet. Diabetes, traditionally considered a dysglycemia centered on the β-cell, also has clear contributions by dysregulated α-cells. However, little is known of the key transcriptional regulators, including transcription factors (TFs) and interacting co-regulators, driving α-cell development and function. A published pancreas-wide knockout (KO) of the LIM-Homeodomain Islet-1 (ISL1) TF in mice led to a dominant β-cell phenotype, including glucose intolerance and reduced insulin secretion. However, significant reductions of glucagon (Gcg) and Arx mRNA and pancreatic GCG content were also noted, suggesting α-cell impacts. Furthermore, we demonstrated that pancreatic ISL1 activity involves interactions with the LDB1 scaffolding co-regulator. However, nothing is known of the comparative α-cell-specific target genes and functions of these protein partners. To first assess ISL1:LDB1 complexes in α-cells, we performed co-IP and proximity ligation assay (PLA) in mouse α-cell lines and found low-glucose stimulated ISL1 and LDB1 interactions. These observations led us to hypothesize that ISL1 and LDB1 complexes regulate transcription of target genes to drive α-cell function. To assess comparative DNA occupancy, we performed ChIP and observed significant ISL1 and LDB1 binding in conserved Gcg promoter and intronic domains. siRNA-mediated Isl1 or Ldb1 knockdown followed by RNA analysis revealed a similar differential expression of α-cell mRNAs (e.g., Gcg, IL6ra, Sox8, Cox5a, Atp5k, Atp5e), suggesting roles in α-cell function. To compare ISL1 and LDB1 importance in vivo, we developed two novel mouse α-cell ISL1 or LDB1 KO models using Gcg-Cre, termed Isl1Δα and Ldb1Δα, respectively. ISL1 and LDB1 loss was confirmed in α-cells by immunofluorescence. Significant fasting hypoglycemia and hypoglucagonemia were found in postnatal Isl1Δα and Ldb1Δα mice, along with decreased GCG+ cell numbers in Ldb1Δα mice, compared to controls. These findings underscore the crucial roles of these factors in regulating GCG production and secretion. Immunostaining for the MAFB α-cell TF in Ldb1Δα islets revealed potential heterogeneous MAFB expression, marked by MAFB+ cells lacking GCG, or co-expressing INS, each indicating a potential loss of α-cell identity. Our lineage tracing analysis revealed α-cell lineage labeled tomato+ cells in Isl1Δα mice appearing to co-express INS, suggesting transdifferentiation. Currently, inducible α-cell-specific KO mice are in development to investigate the role of ISL1 and LDB1 in adult α-cells. In the future, we will also explore the effects of α-cell-specific ISL1 or LDB1 deficiency during embryogenesis. Overall, our study will benefit efforts in identifying new molecular targets and cell-based therapies to combat diabetes. Presentation: 6/1/2024

12248 Leptin Signaling Deficiency At The Central Nervous System Reduces Hepatic Glucagon Sensibility Disturbing Liver Amino Acid Metabolism Leading To Hyperaminoacidemia And Hyperglucagonemia In Male Adult Rats

Abstract Disclosure: C. Pintado Losa: None. L. Mazuecos Fernández-Pacheco: None. Ó. Gómez Torres: None. S. Artigas Jerónimo: None. E. Burgos Ramos: None. I. Ramos: None. J. Poveda Colado: None. C. Arribas Mocoroa: None. A. Andres: None. N. Gallardo: None. Leptin regulates glucagon secretion, but the contribution of central leptin resistance to circulating glucagon levels and hepatic glucagon signaling is poorly studied. To analyze the effects of deficient hypothalamic leptin signaling on glucagon levels and hepatic glucagon signaling, we infused intracerebroventricularly a rat-specific leptin receptor antagonist SLA (0,2 µg/day) for 21 days to male 3-month-old Wistar rats (n=12) or PBS (n=12). At day 21 of treatment and 30 min before sacrifice, a glucagon challenge (100 µg/kg) was performed in 16h fasted rats. After sacrifice, blood and liver samples were collected for analysis. Serum hormones, urea, and total and branched-chain amino acids (BCAA) levels were determined using commercial kits and by RP-HPLC. Hepatic neutral lipid was extracted and quantified using an enzymatic kit and the hepatic content of mRNA and proteins involved in lipid and amino acid metabolism were measured by RT-PCR and Western-Blot. Additionally, the HOMA-IR and glucagon-alanine index were determined in all rats. Blocking central leptin signaling led to impaired fat oxidation and triglyceride export capacity in the liver, resulting in a 50% increase in hepatic triglyceride content in SLA-treated rats. Glucagonemia increased in SLA-treated rats, indicating impaired hepatic glucagon sensitivity. In fact, in response to exogenous glucagon, the hepatic phosphorylation of CREB was reduced by 55% in SLA-infused compared to control rats. Importantly, SLA treatment significantly decreases the expression and the activity of BCKDH (branched-chain alpha-keto acid dehydrogenase) in the liver, leading to a reduction in the BCAA degradation in SLA-treated rats, hyperaminoacidemia, and decreased serum urea levels. In conclusion, deficient hypothalamic leptin signaling alters glucose, lipid, and amino acid homeostasis, highlighting the relevance of central leptin action in the control of peripheral metabolism and glucagon sensitivity. Funding: Work supported by Ministerio de Ciencia, Innovación y Universidades under project grants RTI2018-098643-B-I00, PID2021-128243OB-I00, and from the University of Castilla-La Mancha and the European Regional Development Fund (FEDER) under project grant 2023- GRIN-34280. Presentation: 6/1/2024

Delaying post-exercise carbohydrate intake impairs next-day exercise capacity but not muscle glycogen or molecular responses.

To investigate how delayed post-exercise carbohydrate intake affects muscle glycogen, metabolic- and mitochondrial-related molecular responses, and subsequent high-intensity interval exercise (HIIE) capacity. In a double-blind cross-over design, nine recreationally active men performed HIIE (10 × 2-min cycling, ~94% W˙peak) in the fed state, on two occasions. During 0-3 h post-HIIE, participants drank either carbohydrates ("Immediate Carbohydrate" [IC], providing 2.4 g/kg) or water ("Delayed Carbohydrate" [DC]); total carbohydrate intake over 24 h post-HIIE was matched (~7 g/kg/d). Skeletal muscle (sampled pre-HIIE, post-HIIE, +3 h, +8 h, +24 h) was analyzed for whole-muscle glycogen and mRNA content, plus signaling proteins in cytoplasmic- and nuclear-enriched fractions. After 24 h, participants repeated the HIIE protocol until failure, to test subsequent HIIE capacity; blood lactate, heart rate, and ratings of perceived effort (RPE) were measured throughout. Muscle glycogen concentrations, and relative changes, were similar between conditions throughout (p > 0.05). Muscle glycogen was reduced from baseline (mean ± SD mmol/kg dm; IC: 409 ± 166; DC: 352 ± 76) at post-HIIE (IC: 253 ± 96; DC: 214 ± 82), +3 h (IC: 276 ± 62; DC: 269 ± 116) and + 8 h (IC: 321 ± 56; DC: 269 ± 116), returning to near-baseline by +24 h. Several genes (PGC-1ɑ, p53) and proteins (p-ACCSer79, p-P38 MAPKThr180/Tyr182) elicited typical exercise-induced changes irrespective of condition. Delaying carbohydrate intake reduced next-day HIIE capacity (5 ± 3 intervals) and increased RPE (~2 ratings), despite similar physiological responses between conditions. Molecular responses to HIIE (performed in the fed state) were not enhanced by delayed post-exercise carbohydrate intake. Our findings support immediate post-exercise refueling if the goal is to maximize next-day HIIE capacity and recovery time is ≤24 h.

Responses to Exercise with Low Carbohydrate Availability on Muscle Glycogen and Cell Signaling: A Systematic Review and Meta-analysis.

The growing interest in how exercise and carbohydrate (CHO) restriction may modify molecular responses that promote endurance adaptations has led to many interesting controversies. We conducted a systematic review and a meta-analysis regarding the effect of low-carbohydrate availability (LOW) pre-, during, or post-exercise, on the mRNA content of commonly measured genes involved in mitochondrial biogenesis (PGC-1α, TFAM mRNA) and metabolism (PDK4, UCP3 and GLUT4 mRNA), and on muscle glycogen levels, compared with a high-CHO (CON) condition. MEDLINE, Scopus, and Web of Science databases were searched following the PRISMA 2020 guidelines (with an end date of November 2023). In total, 19 randomized-controlled studies were considered for inclusion. We evaluated the methodological quality of all studies using the Cochrane Risk of Bias tool for randomized clinical studies. A meta-analysis was performed using a random effects model to calculate the standardized mean difference (SMD), estimated by Hedges' g, and 95% confidence intervals (CIs). The LOW condition was associated with an increased mRNA content of several genes during the early recovery period post-exercise, such as PDK4 (SMD 1.61; 95% CI 0.80-2.42), GLUT4 (SMD 1.38; 95% CI 0.46-2.30), and UCP3 (SMD 2.05; 95% CI 0.40-3.69). However, overall, there was no significant effect on the mRNA content of PGC-1α or TFAM. Finally, CHO restriction and exercise significantly reduced muscle glycogen levels (SMD 3.69; 95% CI 2.82-5.09). A meta-analysis of subgroups from studies with a difference in muscle glycogen concentration of > 200mmolkgdw-1 between the LOW and CON conditions showed an increase in exercise-induced PGC-1α mRNA (SMD 2.08; 95% CI 0.64-3.52; p = 0.005; I2 = 75%) and a greater effect in PDK4 and GLUT4 mRNA. The meta-analysis results show that CHO restriction was associated with an increase in the exercise-induced mRNA content of PDK4, UCP3, and GLUT4, but not the exercise-induced mRNA content of PGC-1ɑ and TFAM. However, when there were substantial differences in glycogen depletion between CON and LOW CHO conditions (> 200mmolkgdw-1), there was a greater effect of CHO restriction on the exercise-induced mRNA content of metabolic genes, and an increase in exercise-induced PGC-1α mRNA.

Mitochondrial antioxidant SkQ1 attenuates C26 cancer-induced muscle wasting in males and improves muscle contractility in female tumor-bearing mice.

Mitochondrial dysfunction is a hallmark of cancer cachexia (CC). Mitochondrial reactive oxygen species (ROS) are elevated in muscle shortly after tumor onset. Targeting mitochondrial ROS may be a viable option to prevent CC. The aim of this study was to evaluate the efficacy of a mitochondria-targeted antioxidant, SkQ1, to mitigate CC in both biological sexes. Male and female Balb/c mice were injected bilaterally with colon 26 adenocarcinoma (C26) cells (total 1 × 106 cells) or PBS (equal volume control). SkQ1 was dissolved in drinking water (∼250 nmol/kg body wt/day) and administered to mice beginning 7 days following tumor induction, whereas control groups consumed normal drinking water. In vivo muscle contractility of dorsiflexors, deuterium oxide-based protein synthesis, mitochondrial respiration and mRNA content of mitochondrial, protein turnover, and calcium channel-related markers were assessed at endpoint (25 days following tumor induction). Two-way ANOVAs, followed by Tukey's post hoc test when interactions were significant (P ≤ 0.05), were performed. SkQ1 attenuated cancer-induced atrophy, promoted protein synthesis, and abated Redd1 and Atrogin induction in gastrocnemius of C26 male mice. In female mice, SkQ1 decreased muscle mass and increased catabolic signaling in the plantaris of tumor-bearing mice, as well as reduced mitochondrial oxygen consumption, regardless of tumor. However, in females, SkQ1 enhanced muscle contractility of the dorsiflexors with concurrent induction of Ryr1, Serca1, and Serca2a in TA. In conclusion, the mitochondria-targeted antioxidant SkQ1 may attenuate CC-induced muscle loss in males, while improving muscle contractile function in tumor-bearing female mice, suggesting sexual dimorphism in the effects of this mitochondrial therapy in CC.NEW & NOTEWORTHY Herein, we assess the efficacy of the mitochondria-targeted antioxidant SkQ1 to mitigate cancer cachexia (CC) in both biological sexes. We demonstrate that SkQ1 administration attenuates muscle wasting induced by C26 tumors in male, but not female, mice. Conversely, we identify that in females, SkQ1 improves muscle contractility. These phenotypic adaptations to SkQ1 are aligned with respective responses in muscle protein synthesis, mitochondrial respiration, and mRNA content of protein turnover, as well as mitochondrial and calcium handling-related markers.

Potential and Metabolic Impacts of Double Enrichments of Docosahexaenoic Acid and 25-Hydroxy Vitamin D3 in Tissues of Broiler Chickens

BackgroundChicken may be enriched with 25-hydroxy D3 [25(OH)D3] and docosahexaenoic acid (DHA) to enhance the dietary intake of the public. ObjectivesTwo experiments (Expt.) were conducted to determine the potential and metabolic impacts of enriching both DHA and 25(OH)D3 in the tissues of broiler chickens. MethodsIn Expt. 1, 144 chicks (6 cages/treatment and 6 birds/cage) were fed a corn–soybean meal basal diet (BD), BD + 10,000 IU 25(OH)D3/kg [BD + 25(OH)D3], BD + 1% DHA-rich Aurantiochytrium (1.2 g DHA/kg; BD + DHA), or BD + 25(OH)D3+DHA for 6 wk. In Expt. 2, 180 chicks were fed the BD, BD + DHA-rich microalgal oil (1.5–3.0 g DHA/kg, BD + DHA), BD + DHA + eicosapentaenoic acid (EPA)-rich microalgae (0.3–0.6 g EPA/kg, BD + DHA + EPA), BD + DHA + 25(OH)D3 [6000 to 12,000 IU/kg diet; BD + DHA + 25(OH)D3], and BD + DHA + EPA + 25(OH)D3 for 6 wk. ResultsSupranutrition of these 2 nutrients resulted in 57–62 mg DHA and 1.9–3.3 μg of 25(OH)D3/100 g of breast or thigh muscles. The DHA enrichment was independent of dietary EPA or 25(OH)D3, but that of 25(OH)D3 in the liver was decreased (68%, P < 0.05) by dietary DHA in Expt. 1. Compared with BD, BD + 25(OH)D3 enhanced (P < 0.05) gene expression related to D3 absorption (scavenger receptor class B type 1 and Niemann-pick c1 like 1) in the liver and D3 degradation (cytochrome P450 24A1) in the breast, and decreased mRNA or protein concentrations of vitamin D binding protein in the adipose tissue or thigh muscle. Supranutrition of DHA decreased mRNA concentrations of lipid metabolism-related genes (fatty acid desaturase 1,2, ELOVL fatty acid elongase 5, fatty acid desaturase 2, fatty acid synthase, and sterol regulatory element-binding protein 1). ConclusionsBoth DHA and 25(OH)D3 were enriched in the muscles up to meeting 50%–100% of the suggested intakes of these nutrients by consuming 2 servings of 100 g of fortified chicken. The enrichments altered gene expression related to lipid biosynthesis and vitamin D transport or storage.

Structural and network analysis of ADAM9 and ADAM10 and its transcript expression in the male reproductive tract of Bos Taurus

A disintegrin and metalloproteinases (ADAMs) are a family of proteins expressed as transmembrane and secreted proteins in tissues. This study investigated the expression of glycoproteins and transcripts of ADAM9 and ADAM10 in different regions of the bovine male reproductive tract (MRT). Fresh bovine samples were collected from the testis, epididymis, and vas deferens of MRT for this study. Special stains were utilized for identifying glycoproteins depending on the type of mucopolysaccharides in the seminiferous tubules of the testis and epithelial cells of the epididymis and vas deferens. Quantitative real-time PCR (qPCR) was used to assess the mRNA concentrations of ADAMs9 and 10. Additionally, structural and network analysis was performed to investigate the similarity between human and bovine ADAM9 and ADAM10. Variable glycoprotein (acidic and neutral mucopolysaccharides) expression was observed in the seminiferous tubules of the testis and epithelial cells of the epididymis and vas deferens. ADAM9 and 10 mRNA were detected in the testis epididymis and vas deferens, with ADAM9 expression being significantly higher than ADAM10 mRNA expression in the testis and tail of epididymis. Significant similarity between the protein sequence of human and bovine ADAM9 (85.75%) and ADAM10 (96.66%) was observed, while the similarity between protein sequence of ADAM 9 and ADAM10 in humans and bovine was &lt;26%. Bovine ADAM9 showed 4 binding sites, while bovine ADAM10, human ADAM9, and human ADAM10 showed 7 binding pockets, each with considerable variations in the affinity of specific antibody sequences to these binding pockets. Network analysis confirmed that ADAM9 and ADAM10 have critical roles in physiological processes involving cell migration, protease activity, and secretase-related functions. In addition, they have large numbers of hydrogen bonds to most other members of the ADAM family. Our study reports similarities in the expression of glycoproteins and ADAM9 and ADAM10 mRNA in the MRT of Bos Taurus. Structural and network analysis of ADAM9 and ADAM10 suggested functional similarities in these proteins, which are likely to be in facilitating spermatogenesis, transcervical sperm migration, egg-sperm fusion, and fertilization. Further, the similarity between human and bovine ADAM9 and ADAM10 proteins suggests that bovine sperms can be considered as a model system for comparative medicine studies.