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Biologics : Targets & Therapy 2024Gene expression, morphology, and electrophysiological combination are essential for assessing the dynamic development of human induced pluripotent stem cell-derived...
Gene Expression, Morphology, and Electrophysiology During the Dynamic Development of Human Induced Pluripotent Stem Cell-Derived Atrial- and Ventricular-Like Cardiomyocytes.
BACKGROUND AND OBJECTIVES
Gene expression, morphology, and electrophysiological combination are essential for assessing the dynamic development of human induced pluripotent stem cell-derived atrial- and ventricular-like cardiomyocytes (iPS-AM and iPS-VM, respectively).
METHODS
For iPS-AM/VM differentiation, we performed the small molecule-based temporal modulation of the retinoic acid and bone morphogenetic protein signaling pathways. We investigated the gene expression and morphology using immunofluorescence, quantitative real-time polymerase chain reaction, flow cytometry, and transmission electron microscopy as well as registered electrophysiological functions using a whole-cell patch clamp on days 20, 30, and 60 post-differentiations.
RESULTS
Pan-cardiomyocyte marker, including troponin T2 () and alpha-actinin-2 (), expressions increased both in iPS-AMs and iPS-VMs. Similarly, the mRNA expression of both iPS-AM-specific markers, ie, natriuretic peptide A (), myosin light chain 7 (), and K+ channel Kir3.4 (), and iPS-VM-specific markers, ie, gap junction α-1 (), myosin light chain 2 (), and alpha-1-subunit of a voltage-dependent L-type calcium channel (), increased from 0 to 20 days, and then decreased from 30 to 60 days. Concerning morphology, cardiac troponin-T (cTnT) arrangement was progressively organized and developed from a disorderly myofibrillar distribution to an organized sarcomere pattern both in iPS-AMs and iPS-VMs. Mitochondrial numbers gradually increased and those of lipid droplets decreased during dynamic development. Regarding physiological function, the resting and action potential amplitudes remained statistically indifferent in both cell types, and the action potential duration was prolonged during the development.
CONCLUSION
IPS-AMs/VMs displayed dynamic development concerning their gene expression, morphology, and electrophysiological function. The discoveries of this study could provide novel insights into heart development and encourage further research.
PubMed: 38746773
DOI: 10.2147/BTT.S448054 -
Frontiers in Endocrinology 2024Diabetic kidney disease (DKD), a significant complication associated with diabetes mellitus, presents limited treatment options. The progression of DKD is marked by... (Review)
Review
Diabetic kidney disease (DKD), a significant complication associated with diabetes mellitus, presents limited treatment options. The progression of DKD is marked by substantial lipid disturbances, including alterations in triglycerides, cholesterol, sphingolipids, phospholipids, lipid droplets, and bile acids (BAs). Altered lipid metabolism serves as a crucial pathogenic mechanism in DKD, potentially intertwined with cellular ferroptosis, lipophagy, lipid metabolism reprogramming, and immune modulation of gut microbiota (thus impacting the liver-kidney axis). The elucidation of these mechanisms opens new potential therapeutic pathways for DKD management. This research explores the link between lipid metabolism disruptions and DKD onset.
Topics: Humans; Diabetic Nephropathies; Lipid Metabolism; Animals; Lipid Metabolism Disorders; Gastrointestinal Microbiome
PubMed: 38742197
DOI: 10.3389/fendo.2024.1336402 -
Fly Dec 2024To identify genes required for brain growth, we took an RNAi knockdown reverse genetic approach in . One potential candidate isolated from this effort is the...
To identify genes required for brain growth, we took an RNAi knockdown reverse genetic approach in . One potential candidate isolated from this effort is the anti-lipogenic gene (). Adp has an established role in the negative regulation of lipogenesis in the fat body of the fly and adipose tissue in mammals. While fat is key to proper development in general, has not been investigated during brain development. Here, we found that RNAi knockdown of in neuronal stem cells and neurons results in reduced brain lobe volume and sought to replicate this with a mutant fly. We generated a novel mutant that acts as a loss-of-function mutant based on buoyancy assay results. We found that despite a change in fat content in the body overall and a decrease in the number of larger (>5 µm) brain lipid droplets, there was no change in the brain lobe volume of mutant larvae. Overall, our work describes a novel mutant that can functionally replace the long-standing mutant and shows that the gene has no obvious involvement in brain growth.
Topics: Animals; Drosophila Proteins; Brain; Drosophila melanogaster; Loss of Function Mutation; RNA Interference; Neurons; Larva; Neural Stem Cells; Drosophila; Adipose Tissue; Mutation
PubMed: 38741287
DOI: 10.1080/19336934.2024.2352938 -
Molecular Metabolism Jul 2024Sulfonylureas (SUs) are still among the mostly prescribed antidiabetic drugs with an established mode of action: release of insulin from pancreatic β-cells. In...
OBJECTIVE
Sulfonylureas (SUs) are still among the mostly prescribed antidiabetic drugs with an established mode of action: release of insulin from pancreatic β-cells. In addition, effects of SUs on adipocytes by activation of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) have been described, which might explain their insulin-sensitizing potential observed in patients. However, there is a discrepancy between the impact of SUs on antidiabetic action and their rather moderate in vitro effect on PPARγ transcriptional activity. Recent studies have shown that some PPARγ ligands can improve insulin sensitivity by blocking PPARγ Ser-273 phosphorylation without having full agonist activity. It is unknown if SUs elicit their antidiabetic effects on adipocytes by inhibition of PPARγ phosphorylation. Here, we investigated if binding of SUs to PPARγ can interfere with PPARγ Ser-273 phosphorylation and determined their antidiabetic actions in vitro in primary human white adipocytes and in vivo in high-fat diet (HFD) obese mice.
METHODS
Primary human white preadipocytes were differentiated in the presence of glibenclamide, glimepiride and PPARγ ligands rosiglitazone and SR1664 to compare PPARγ Ser-273 phosphorylation, glucose uptake and adipokine expression. Transcriptional activity at PPARγ was determined by luciferase assays, quantification of PPARγ Ser-273 phosphorylation was determined by Western blotting and CDK5 kinase assays. In silico modelling was performed to gain insight into the binding characteristics of SUs to PPARγ. HFD mice were administered SUs and rosiglitazone for 6 days. PPARγ Ser-273 phosphorylation in white adipose tissue (WAT), body composition, glucose tolerance, adipocyte morphology and expression levels of genes involved in PPARγ activity in WAT and brown adipose tissue (BAT) were evaluated.
RESULTS
SUs inhibit phosphorylation of PPARγ at Ser-273 in primary human white adipocytes and exhibit a positive antidiabetic expression profile, which is characterized by up regulation of insulin-sensitizing and down regulation of insulin resistance-inducing adipokines. We demonstrate that SUs directly bind to PPARγ by in silico modelling and inhibit phosphorylation in kinase assays to a similar extend as rosiglitazone and SR1664. In HFD mice SUs reduce PPARγ phosphorylation in WAT and have comparable effects on gene expression to rosiglitazone. In BAT SUs increase UCP1 expression and reduce lipid droplets sizes.
CONCLUSIONS
Our findings indicate that a part of SUs extra-pancreatic effects on adipocytes in vitro and in vivo is probably mediated via their interference with PPARγ phosphorylation rather than via classical agonistic activity at clinical concentrations.
Topics: PPAR gamma; Animals; Phosphorylation; Hypoglycemic Agents; Mice; Sulfonylurea Compounds; Humans; Adipocytes; Male; Serine; Mice, Inbred C57BL; Diet, High-Fat; Cells, Cultured; Insulin Resistance
PubMed: 38735390
DOI: 10.1016/j.molmet.2024.101956 -
Ultrasonics Sonochemistry Jun 2024Partial coalescence is a key factor contributing to the instability of crystalline oil-in-water emulsions in products like dressings and sauces, reducing shelf life. The...
Ultrasound treatment of crystalline oil-in-water emulsions stabilized by sodium caseinate: Impact on emulsion stability through altered crystallization behavior in the oil globules.
Partial coalescence is a key factor contributing to the instability of crystalline oil-in-water emulsions in products like dressings and sauces, reducing shelf life. The intrinsic characteristics of semi-crystalline droplets, including solid fat content, fat crystal arrangement, and polymorphism, play a pivotal role in influencing partial coalescence, challenging prevention efforts even with emulsifiers like amphiphilic proteins. High-intensity ultrasound (HIU) has emerged as an efficient and cost-effective technology for manipulating bulk fat crystallization, thereby enhancing physical properties. This study specifically investigates the impact of HIU treatment on fat crystallization on protein-stabilized crystalline emulsions, utilizing palm olein stearin (POSt) as the lipid phase and sodium caseinate (NaCas) as the surfactant under various HIU powers (100, 150, 200, 300, and 400 W). Results show that increasing HIU power maintained the interfacial potential (-20 mV) provided by NaCas in the emulsions without significant differences. Higher HIU power induced the most stable polymorphic form (β) in the emulsions. Engagingly, the emulsions at 200 W exhibited better storage stability and slower partial coalescence kinetics. Semi-crystalline globules had more uniform and integral crystal clusters that were distributed tangentially near the droplet boundary, perhaps attributed to intermediate subcooling (40.4 °C) at 200 W. The acoustic energy of HIU significantly translates into thermal effects, influencing subcooling degrees as a dominant factor affecting crystallisation in the emulsions. This study establishes ultrasonic crystallization as a novel strategy for modifying the stability of emulsions containing fat crystals.
PubMed: 38735250
DOI: 10.1016/j.ultsonch.2024.106897 -
Biochimica Et Biophysica Acta.... Aug 2024Lipid droplets (LD) are highly dynamic organelles specialized for the regulation of energy storage and cellular homeostasis. LD consist of a neutral lipid core...
Lipid droplets (LD) are highly dynamic organelles specialized for the regulation of energy storage and cellular homeostasis. LD consist of a neutral lipid core surrounded by a phospholipid monolayer membrane with embedded proteins, most of which are involved in lipid homeostasis. In this study, we focused on one of the major LD proteins, sterol C24-methyltransferase, encoded by ERG6. We found that the absence of Erg6p resulted in an increased accumulation of yeast perilipin Pet10p in LD, while the disruption of PET10 was accompanied by Erg6p LD over-accumulation. An observed reciprocal enrichment of Erg6p and Pet10p in pet10Δ and erg6Δ mutants in LD, respectively, was related to specific functional changes in the LD and was not due to regulation on the expression level. The involvement of Pet10p in neutral lipid homeostasis was observed in experiments that focused on the dynamics of neutral lipid mobilization as time-dependent changes in the triacylglycerols (TAG) and steryl esters (SE) content. We found that the kinetics of SE hydrolysis was reduced in erg6Δ cells and the mobilization of SE was completely lost in mutants that lacked both Erg6p and Pet10p. In addition, we observed that decreased levels of SE in erg6Δpet10Δ was linked to an overexpression of steryl ester hydrolase Yeh1p. Lipid analysis of erg6Δpet10Δ showed that PET10 deletion altered the composition of ergosterol intermediates which had accumulated in erg6Δ. In conclusion, yeast perilipin Pet10p functionally interacts with Erg6p during the metabolism of ergosterol.
Topics: Ergosterol; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Perilipin-1; Lipid Droplets; Methyltransferases; Lipid Metabolism; Triglycerides
PubMed: 38734059
DOI: 10.1016/j.bbalip.2024.159506 -
Nutrients Apr 2024Obesity can lead to excessive lipid accumulation in non-adipose tissues, such as the liver and skeletal muscles, leading to ectopic lipid deposition and damaging target...
Obesity can lead to excessive lipid accumulation in non-adipose tissues, such as the liver and skeletal muscles, leading to ectopic lipid deposition and damaging target organ function through lipotoxicity. FGF-21 is a key factor in regulating lipid metabolism, so we aim to explore whether FGF-21 is involved in improving ectopic lipid deposition. We observed the characteristics of ectopic lipid deposition in the liver and skeletal muscles of obesity-resistant mice, detected the expression of FGF-21 and perilipin, and found that obesity-resistant mice showed a decrease in ectopic lipid deposition in the liver and skeletal muscles and increased expression of FGF-21. After inhibiting the expression of FGF-21, a more severe lipid deposition in liver cells and skeletal muscle cells was found. The results indicate that inhibiting FGF-21 can exacerbate ectopic lipid deposition via regulating lipid droplet synthesis and decomposition, as well as free fatty acid translocation and oxidation. In conclusion, FGF-21 is involved in improving ectopic lipid deposition caused by obesity in the liver and skeletal muscles.
Topics: Animals; Fibroblast Growth Factors; Muscle, Skeletal; Liver; Lipid Metabolism; Mice; Obesity; Male; Mice, Inbred C57BL; Perilipin-1; Lipid Droplets
PubMed: 38732501
DOI: 10.3390/nu16091254 -
Nanomaterials (Basel, Switzerland) Apr 2024Nicotine activates nicotinic acetylcholine receptors (nAChRs), which are overexpressed in numerous cancer types, leading to signaling pathways that increase lung cancer...
Nicotine activates nicotinic acetylcholine receptors (nAChRs), which are overexpressed in numerous cancer types, leading to signaling pathways that increase lung cancer invasiveness and resistance to chemotherapeutic agents. In this study, the effects of APS12-2, a synthetic analog of marine sponge toxin that acts as an antagonist of nAChRs, was investigated in vitro on A549 human lung adenocarcinoma cells and non-tumorigenic human lung epithelial BEAS-2B cells. In addition, gelatin nanoparticles (GNPs) loaded with APS12-2 (APS12-2-GNPs) were prepared and their effects were compared with those of free APS12-2. Nicotine reduced cytotoxicity, the formation of reactive oxygen species, and the formation of lipid droplets caused by cisplatin on A549 cells. The effects of nicotine on the decreased efficacy of cisplatin were reduced by APS12-2 and APS12-2-GNPs. APS12-2-GNPs showed a substantial advantage compared with free APS12-2; the cytotoxicity of APS12-2 on BEAS-2B cells was greatly reduced when APS12-2 was loaded in GNPs, whereas the cytotoxicity on A549 cells was only slightly reduced. Our results suggest that both APS12-2 and APS12-2-GNPs hold promise as supportive agents in the cisplatin-based chemotherapy of lung cancer.
PubMed: 38727371
DOI: 10.3390/nano14090777 -
Cells Apr 2024SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in host cells, which may contribute to the pathogenesis of COVID-19....
BACKGROUND
SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in host cells, which may contribute to the pathogenesis of COVID-19. To understand the complex interplay between SARS-Co-V2 infection and UPR signaling, we examined the effects of acute pre-existing ER stress on SARS-Co-V2 infectivity.
METHODS
Huh-7 cells were treated with Tunicamycin (TUN) and Thapsigargin (THA) prior to SARS-CoV-2pp transduction (48 h p.i.) to induce ER stress. Pseudo-typed particles (SARS-CoV-2pp) entry into host cells was measured by Bright Glo luciferase assay. Cell viability was assessed by cell titer Glo luminescent assay. The mRNA and protein expression was evaluated by RT-qPCR and Western Blot.
RESULTS
TUN (5 µg/mL) and THA (1 µM) efficiently inhibited the entry of SARS-CoV-2pp into host cells without any cytotoxic effect. TUN and THA's attenuation of virus entry was associated with differential modulation of ACE2 expression. Both TUN and THA significantly reduced the expression of stress-inducible ER chaperone GRP78/BiP in transduced cells. In contrast, the IRE1-XBP1s and PERK-eIF2α-ATF4-CHOP signaling pathways were downregulated with THA treatment, but not TUN in transduced cells. Insulin-mediated glucose uptake and phosphorylation of Ser IRS-1 and downstream p-AKT were enhanced with THA in transduced cells. Furthermore, TUN and THA differentially affected lipid metabolism and apoptotic signaling pathways.
CONCLUSIONS
These findings suggest that short-term pre-existing ER stress prior to virus infection induces a specific UPR response in host cells capable of counteracting stress-inducible elements signaling, thereby depriving SARS-Co-V2 of essential components for entry and replication. Pharmacological manipulation of ER stress in host cells might provide new therapeutic strategies to alleviate SARS-CoV-2 infection.
Topics: Humans; Apoptosis; COVID-19; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Proto-Oncogene Proteins c-akt; SARS-CoV-2; Signal Transduction; Thapsigargin; Tunicamycin; Unfolded Protein Response; Virus Internalization
PubMed: 38727305
DOI: 10.3390/cells13090769 -
Cells Apr 2024The antipsychotic drug clozapine demonstrates superior efficacy in treatment-resistant schizophrenia, but its intracellular mode of action is not completely understood....
The antipsychotic drug clozapine demonstrates superior efficacy in treatment-resistant schizophrenia, but its intracellular mode of action is not completely understood. Here, we analysed the effects of clozapine (2.5-20 µM) on metabolic fluxes, cell respiration, and intracellular ATP in human HL60 cells. Some results were confirmed in leukocytes of clozapine-treated patients. Neuroreceptor inhibition under clozapine reduced Akt activation with decreased glucose uptake, thereby inducing ER stress and the unfolded protein response (UPR). Metabolic profiling by liquid-chromatography/mass-spectrometry revealed downregulation of glycolysis and the pentose phosphate pathway, thereby saving glucose to keep the electron transport chain working. Mitochondrial respiration was dampened by upregulation of the F0F1-ATPase inhibitory factor 1 (IF1) leading to 30-40% lower oxygen consumption in HL60 cells. Blocking IF1 expression by cotreatment with epigallocatechin-3-gallate (EGCG) increased apoptosis of HL60 cells. Upregulation of the mitochondrial citrate carrier shifted excess citrate to the cytosol for use in lipogenesis and for storage as triacylglycerol in lipid droplets (LDs). Accordingly, clozapine-treated HL60 cells and leukocytes from clozapine-treated patients contain more LDs than untreated cells. Since mitochondrial disturbances are described in the pathophysiology of schizophrenia, clozapine-induced mitohormesis is an excellent way to escape energy deficits and improve cell survival.
Topics: Humans; Clozapine; Mitochondria; HL-60 Cells; Antipsychotic Agents; Apoptosis; Adenosine Triphosphate; Schizophrenia; Leukocytes; Endoplasmic Reticulum Stress; Cellular Reprogramming; Metabolic Reprogramming
PubMed: 38727298
DOI: 10.3390/cells13090762