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Journal of Biological Engineering May 2024Adiponectin (ADPN) plays a critical role in endocrine and cardiovascular functions, but traditional production methods, such as Escherichia coli and mammalian systems,...
BACKGROUND
Adiponectin (ADPN) plays a critical role in endocrine and cardiovascular functions, but traditional production methods, such as Escherichia coli and mammalian systems, have faced challenges in generating sufficiently active middle molecular weight (MMW) and high molecular weight (HMW) forms of recombinant human ADPN (hADPN). In our previous study, we proposed genome-edited chickens as an efficient platform for producing multimeric hADPN. However, the consistency of multimeric hADPN expression in this system across generations had not been further investigated.
RESULTS
In this study, subsequent generations of ovalbumin (OVA) ADPN knock-in chickens showed stable multimeric hADPN production, yielding ~ 26% HMW ADPN (0.59 mg/mL) per hen. Comparative analysis revealed that egg white (EW)-derived hADPN predominantly consisted of hexameric and HMW forms, similar to serum-derived hADPN. In contrast, hADPN obtained from human embryonic kidney (HEK) 293 and High-Five (Hi-5) cells also exhibited the presence of trimers, indicating variability across different production systems. Furthermore, transcriptional expression analysis of ADPN multimerization-associated endoplasmic reticulum chaperone genes (Ero1-Lα, DsbA-L, ERP44, and PDI) indicated upregulation in the oviduct magnum of ADPN KI hens, suggesting the chicken oviduct magnum as the optimal site for HMW ADPN production. Lastly, the functional analysis demonstrated that EW-derived hADPN significantly reduced lipid droplets and downregulated lipid accumulation-related genes (LOX-1, AT1R, FAS, and FABP4) in human umbilical vein endothelial cells (HUVECs).
CONCLUSION
In summary, stable and functional multimeric hADPN can be produced in genome-edited chickens even after generations. This highlights the potential of using chicken bioreactor for producing various high-value proteins.
PubMed: 38715027
DOI: 10.1186/s13036-024-00427-2 -
Communications Biology May 2024Intervertebral disc degeneration (IDD) is a highly prevalent musculoskeletal disorder affecting millions of adults worldwide, but a poor understanding of its...
Intervertebral disc degeneration (IDD) is a highly prevalent musculoskeletal disorder affecting millions of adults worldwide, but a poor understanding of its pathogenesis has limited the effectiveness of therapy. In the current study, we integrated untargeted LC/MS metabolomics and magnetic resonance spectroscopy data to investigate metabolic profile alterations during IDD. Combined with validation via a large-cohort analysis, we found excessive lipid droplet accumulation in the nucleus pulposus cells of advanced-stage IDD samples. We also found abnormal palmitic acid (PA) accumulation in IDD nucleus pulposus cells, and PA exposure resulted in lipid droplet accumulation and cell senescence in an endoplasmic reticulum stress-dependent manner. Complementary transcriptome and proteome profiles enabled us to identify solute carrier transporter (SLC) 43A3 involvement in the regulation of the intracellular PA level. SLC43A3 was expressed at low levels and negatively correlated with intracellular lipid content in IDD nucleus pulposus cells. Overexpression of SLC43A3 significantly alleviated PA-induced endoplasmic reticulum stress, lipid droplet accumulation and cell senescence by inhibiting PA uptake. This work provides novel integration analysis-based insight into the metabolic profile alterations in IDD and further reveals new therapeutic targets for IDD treatment.
Topics: Nucleus Pulposus; Endoplasmic Reticulum Stress; Palmitic Acid; Cellular Senescence; Intervertebral Disc Degeneration; Humans; Lipid Droplets; Male; Female; Adult; Middle Aged
PubMed: 38714886
DOI: 10.1038/s42003-024-06248-9 -
PLoS Biology May 2024Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and...
Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii.
Topics: Toxoplasma; Protozoan Proteins; Endoplasmic Reticulum; rab2 GTP-Binding Protein; Secretory Pathway; Protein Domains; Protein Transport; Lipid Droplets; Animals; Humans
PubMed: 38713739
DOI: 10.1371/journal.pbio.3002634 -
Molecular Metabolism Jun 2024Lipid metabolism plays an important role in early pregnancy, but its effects on decidualization are poorly understood. Fatty acids (FAs) must be esterified by fatty...
OBJECTIVE
Lipid metabolism plays an important role in early pregnancy, but its effects on decidualization are poorly understood. Fatty acids (FAs) must be esterified by fatty acyl-CoA synthetases to form biologically active acyl-CoA in order to enter the anabolic and/or catabolic pathway. Long-chain acyl-CoA synthetase 4 (ACSL4) is associated with female reproduction. However, whether it is involved in decidualization is unknown.
METHODS
The expression of ACSL4 in human and mouse endometrium was detected by immunohistochemistry. ACSL4 levels were regulated by the overexpression of ACSL4 plasmid or ACSL4 siRNA, and the effects of ACSL4 on decidualization markers and morphology of endometrial stromal cells (ESCs) were clarified. A pregnant mouse model was established to determine the effect of ACSL4 on the implantation efficiency of mouse embryos. Modulation of ACSL4 detects lipid anabolism and catabolism.
RESULTS
Through examining the expression level of ACSL4 in human endometrial tissues during proliferative and secretory phases, we found that ACSL4 was highly expressed during the secretory phase. Knockdown of ACSL4 suppressed decidualization and inhibited the mesenchymal-to-epithelial transition induced by MPA and db-cAMP in ESCs. Further, the knockdown of ACSL4 reduced the efficiency of embryo implantation in pregnant mice. Downregulation of ACSL4 inhibited FA β-oxidation and lipid droplet accumulation during decidualization. Interestingly, pharmacological and genetic inhibition of lipid droplet synthesis did not affect FA β-oxidation and decidualization, while the pharmacological and genetic inhibition of FA β-oxidation increased lipid droplet accumulation and inhibited decidualization. In addition, inhibition of β-oxidation was found to attenuate the promotion of decidualization by the upregulation of ACSL4. The decidualization damage caused by ACSL4 knockdown could be reversed by activating β-oxidation.
CONCLUSIONS
Our findings suggest that ACSL4 promotes endometrial decidualization by activating the β-oxidation pathway. This study provides interesting insights into our understanding of the mechanisms regulating lipid metabolism during decidualization.
Topics: Female; Coenzyme A Ligases; Animals; Mice; Humans; Endometrium; Fatty Acids; Pregnancy; Lipid Droplets; Oxidation-Reduction; Decidua; Adult; Lipid Metabolism; Embryo Implantation; Stromal Cells
PubMed: 38710444
DOI: 10.1016/j.molmet.2024.101953 -
Journal of Pharmacy & Pharmaceutical... 2024Unhealthy sources of fats, ultra-processed foods with added sugars, and a sedentary lifestyle make humans more susceptible to developing overweight and obesity. While... (Review)
Review
Unhealthy sources of fats, ultra-processed foods with added sugars, and a sedentary lifestyle make humans more susceptible to developing overweight and obesity. While lipids constitute an integral component of the organism, excessive and abnormal lipid accumulation that exceeds the storage capacity of lipid droplets disrupts the intracellular composition of fatty acids and results in the release of deleterious lipid species, thereby giving rise to a pathological state termed lipotoxicity. This condition induces endoplasmic reticulum stress, mitochondrial dysfunction, inflammatory responses, and cell death. Recent advances in omics technologies and analytical methodologies and clinical research have provided novel insights into the mechanisms of lipotoxicity, including gut dysbiosis, epigenetic and epitranscriptomic modifications, dysfunction of lipid droplets, post-translational modifications, and altered membrane lipid composition. In this review, we discuss the recent knowledge on the mechanisms underlying the development of lipotoxicity and lipotoxic cardiometabolic disease in obesity, with a particular focus on lipotoxic and diabetic cardiomyopathy.
Topics: Humans; Obesity; Diabetic Cardiomyopathies; Animals; Lipid Metabolism
PubMed: 38706718
DOI: 10.3389/jpps.2024.12568 -
Nature Communications May 2024Tools for accessing and studying organelles remain underdeveloped. Here, we present a method by which giant organelle vesicles (GOVs) are generated by submitting cells...
Tools for accessing and studying organelles remain underdeveloped. Here, we present a method by which giant organelle vesicles (GOVs) are generated by submitting cells to a hypotonic medium followed by plasma membrane breakage. By this means, GOVs ranging from 3 to over 10 µm become available for micromanipulation. GOVs are made from organelles such as the endoplasmic reticulum, endosomes, lysosomes and mitochondria, or in contact with one another such as giant mitochondria-associated ER membrane vesicles. We measure the mechanical properties of each organelle-derived GOV and find that they have distinct properties. In GOVs procured from Cos7 cells, for example, bending rigidities tend to increase from the endoplasmic reticulum to the plasma membrane. We also found that the mechanical properties of giant endoplasmic reticulum vesicles (GERVs) vary depending on their interactions with other organelles or the metabolic state of the cell. Lastly, we demonstrate GERVs' biochemical activity through their capacity to synthesize triglycerides and assemble lipid droplets. These findings underscore the potential of GOVs as valuable tools for studying the biophysics and biology of organelles.
Topics: Animals; Chlorocebus aethiops; COS Cells; Endoplasmic Reticulum; Intracellular Membranes; Cell Membrane; Mitochondria; Organelles; Lipid Droplets; Triglycerides; Humans; Lysosomes
PubMed: 38704407
DOI: 10.1038/s41467-024-48086-7 -
NPJ Science of Food May 2024Cultivated meat (CM) offers a sustainable and ethical alternative to conventional animal agriculture, involving cell maturation in a controlled environment. To emulate...
Cultivated meat (CM) offers a sustainable and ethical alternative to conventional animal agriculture, involving cell maturation in a controlled environment. To emulate the structural complexity of traditional meat, the development of animal-free and edible scaffolds is crucial, providing vital physical and biological support during tissue development. The aligned vascular bundles of the decellularised asparagus scaffold were selected to facilitate the attachment and alignment of murine myoblasts (C2C12) and porcine adipose-derived mesenchymal stem cells (pADMSCs). Muscle differentiation was assessed through immunofluorescence staining with muscle markers, including Myosin heavy chain (MHC), Myogenin (MYOG), and Desmin. The metabolic activity of Creatine Kinase in C2C12 differentiated cells significantly increased compared to proliferated cells. Quantitative PCR analysis revealed a significant increase in Myosin Heavy Polypeptide 1 (MYH1) and MYOG expression compared to Day 0. These results highlight the application of decellularised plant scaffold (DPS) as a promising, edible material conducive to cell attachment, proliferation, and differentiation into muscle tissue. To create a CM prototype with biological mimicry, pADMSC-derived muscle and fat cells were also co-cultured on the same scaffold. The co-culture was confirmed through immunofluorescence staining of muscle markers and LipidTOX staining, revealing distinct muscle fibres and adipocytes containing lipid droplets respectively. Texture profile analysis conducted on uncooked CM prototypes and pork loin showed no significant differences in textural values. However, the pan-fried CM prototype differed significantly in hardness and chewiness compared to pork loin. Understanding the scaffolds' textural profile enhances our insight into the potential sensory attributes of CM products. DPS shows potential for advancing CM biomanufacturing.
PubMed: 38702314
DOI: 10.1038/s41538-024-00262-1 -
Research Square Apr 2024Metabolic homeostasis within cells and tissues requires engagement of catabolic and anabolic pathways consuming nutrients needed to generate energy to drive these and...
Metabolic homeostasis within cells and tissues requires engagement of catabolic and anabolic pathways consuming nutrients needed to generate energy to drive these and other subcellular processes. However, the current understanding of cell homeostasis and metabolism, including how cells utilize nutrients, comes largely from tissue and cell models analyzed after fractionation. These bulk strategies do not reveal the spatial characteristics of cell metabolism at the single cell level, and how these aspects relate to the location of cells and organelles within the complexity of the tissue they reside within. Here we pioneer the use of high-resolution electron and stable isotope microscopy (MIMS-EM) to quantitatively map the fate of nutrient-derived C atoms at subcellular scale. When combined with machine-learning image segmentation, our approach allows us to establish the cellular and organellar spatial pattern of glucose C flux in hepatocytes . We applied network analysis algorithms to chart the landscape of organelle-organelle contact networks and identified subpopulations of mitochondria and lipid droplets that have distinct organelle interactions and C enrichment levels. In addition, we revealed a new relationship between the initiation of glycogenesis and proximity of lipid droplets. Our results establish MIMS-EM as a new tool for tracking and quantifying nutrient metabolism at the subcellular scale, and to identify the spatial channeling of nutrient-derived atoms in the context of organelle-organelle interactions .
PubMed: 38699373
DOI: 10.21203/rs.3.rs-4096781/v1 -
Biomolecules & Biomedicine Apr 2024Bile leakage is a common complication following liver surgeries, particularly in the cases of liver hydatid cyst operations. Currently, there is no adequate method which...
Bile leakage is a common complication following liver surgeries, particularly in the cases of liver hydatid cyst operations. Currently, there is no adequate method which could be utilized to prevent this complication effectively. Our study aimed to assess the efficacy of the biliary lipid test (BLT) in reducing biliary complications after hydatid cyst surgery. We retrospectively included patients who underwent open liver hydatid cyst surgery between January 2011 and January 2024. The study encompassed 122 patients, with 41 males and 81 females, ranging in age from 18 to 79 years. In the BLT group, a lipid solution was injected transcystically after cholecystectomy. The BLT was performed on 65 patients, while 57 patients did not undergo the test. Following the transcystic injection of the lipid solution, orifices at the site of lipid droplets that became visible were ligated with 5.0 prolene sutures. A total of 200 leak sites were sutured. Remarkably, none of the patients in the BLT group experienced postoperative bile leakage lasting longer than one week. Consequently, a shorter hospital stay was observed in this group. Transcystic injection of the lipid solution with distal clamping effectively demonstrated leak sites, and suturing these sites prevented postoperative bile leakage. Our study demonstrates the effectiveness of the LIpid test in LIver Hydatid Cyst Surgery (Lili-Hics) in reducing biliary complications following hydatid cyst surgery.
PubMed: 38696705
DOI: 10.17305/bb.2024.10371 -
Nature Communications May 2024Only ~20% of heavy drinkers develop alcohol cirrhosis (AC). While differences in metabolism, inflammation, signaling, microbiome signatures and genetic variations have...
Only ~20% of heavy drinkers develop alcohol cirrhosis (AC). While differences in metabolism, inflammation, signaling, microbiome signatures and genetic variations have been tied to the pathogenesis of AC, the key underlying mechanisms for this interindividual variability, remain to be fully elucidated. Induced pluripotent stem cell-derived hepatocytes (iHLCs) from patients with AC and healthy controls differ transcriptomically, bioenergetically and histologically. They include a greater number of lipid droplets (LDs) and LD-associated mitochondria compared to control cells. These pre-pathologic indicators are effectively reversed by Aramchol, an inhibitor of stearoyl-CoA desaturase. Bioenergetically, AC iHLCs have lower spare capacity, slower ATP production and their mitochondrial fuel flexibility towards fatty acids and glutamate is weakened. MARC1 and PNPLA3, genes implicated by GWAS in alcohol cirrhosis, show to correlate with lipid droplet-associated and mitochondria-mediated oxidative damage in AC iHLCs. Knockdown of PNPLA3 expression exacerbates mitochondrial deficits and leads to lipid droplets alterations. These findings suggest that differences in mitochondrial bioenergetics and lipid droplet formation are intrinsic to AC hepatocytes and can play a role in its pathogenesis.
Topics: Humans; Hepatocytes; Induced Pluripotent Stem Cells; Energy Metabolism; Lipid Droplets; Liver Cirrhosis, Alcoholic; Lipase; Mitochondria; Male; Membrane Proteins; Female; Middle Aged; Adult; Oxidative Stress; Acyltransferases; Phospholipases A2, Calcium-Independent
PubMed: 38693144
DOI: 10.1038/s41467-024-47085-y