OBJECTIVE: To explore the inhibitory effects of Nardostachys Jatamansi DC. volatile oil (NJVO) on psychological factors substance P (SP)/cortisol (CORT)-induced hyperpigmentation. METHODS: The model of psychologically-induced hyperpigmentation of B16F10 cells was created using SP (10 nmol/L) + CORT (10 µmol/L) for 72 h. The levels of melanin content, tyrosinase (TYR) activity using NaOH lysis and L-dihydroxyphenylalanine (L-DOPA) oxidation methods were assessed, respectively. The effect of NJVO on SP/CORT-induced normal human skin tissue pigmentation was detected by Masson staining. Protein expressions of tyrosinase-related protein 1 (TRP-1), tyrosinase-relative protein 2 (DCT), and microphthalmia-associated transcription factor were determined using Western blot. The melanosome number, maturation, and melanosomal structure changes were detected through transmission electron microscopy and immunofluorescence experiments. In vivo, zebrafish pigment content was evaluated in SP/CORT-induced zebrafish hyperpigmentation model. RESULTS: NJVO significantly reduced the melanin content (P<0.01) and inhibited tyrosinase activity (P<0.01), the pigmentation of the normal skin tissue in the NJVO group was significantly lower than that in the SP/CORT group (P<0.05). And NJVO considerably downregulated expressions of melanogenesis-related proteins (TYR, TRP-1, DCT) in cells (P<0.01). In addition, the number of melanosomes was decreased and the dentrites formation of B16F10 cells was inhibited after NJVO treatment (P<0.01). In vivo, NJVO significantly reduced the pigment content in the zebrafish body (P<0.01). CONCLUSION NJVO effectively reversed SP/CORT-induced hyperpigmentation by suppressing the activity and expression of TYR and TRPs and inhibiting melanosome maturation in mouse B16F10 melanoma cells.
Animals ; Hyperpigmentation/psychology* ; Zebrafish ; Oils, Volatile/therapeutic use* ; Melanins/metabolism* ; Humans ; Monophenol Monooxygenase/metabolism* ; Mice ; Nardostachys/chemistry* ; Substance P ; Hydrocortisone ; Skin Pigmentation/drug effects* ; Cell Line, Tumor ; Melanosomes/ultrastructure* ; Microphthalmia-Associated Transcription Factor/metabolism* ; Melanoma, Experimental ; Oxidoreductases/metabolism* ; Intramolecular Oxidoreductases/metabolism*

OBJECTIVES: To investigate the subcellular localization and biological functions of transmembrane protein 240 (TMEM240). METHODS: NCBI BLAST and TMHMM bioinformatics software were used for protein sequence analysis and prediction of transmembrane domain of TMEM240. Brain tissues from male C57BL/6 mice (18-20 days old) were examined for distribution of TMEM240 using in situ hybridization, and qPCR and Western blotting were used to detect TMEM240 expression in different mouse tissues and in cortical neurons at different time points (n=3). In the in vitro experiment, HepG2 and Neuro-2a cells were transfected with plasmids for overexpression of TMEM240, and subcellular localization of TMEM240 was analyzed using cell imaging. In primary cultures of cortical neurons isolated from C57BL/6 mice, TMEM240 expression and its biological functions were investigated using qPCR, Western blotting, and immunofluorescence staining. RESULTS: Human and mouse TMEM240 proteins share a 97.69% similarity in the protein sequences, and both are transmembrane proteins with two transmembrane domains. TMEM240 mRNA and protein were highly expressed in mouse brain tissues and cortical neurons. In isolated mouse cortical neurons, TMEM240 expression reached the peak level after primary culture for 9 days and distributed in scattered spots within the cells. In HepG2 cells, TMEM240 was characterized as intracellular membrane structures and showed 80% colocalization with peroxisomes. In Neuro-2a cells, TMEM240 overexpression caused significant enhancement of the expressions of Rho GDP dissociation inhibitor β (ARHGDIB) at both the mRNA and protein levels. CONCLUSIONS TMEM240 is a novel intracellular subcellular structure specifically expressed in neurons with significant potential for targeted cellular function regulation.
Animals ; Humans ; Mice ; Peroxisomes/metabolism* ; Membrane Proteins/genetics* ; Mice, Inbred C57BL ; Neurons/metabolism* ; Male ; rho-Specific Guanine Nucleotide Dissociation Inhibitors ; Hep G2 Cells ; Brain/metabolism*

Membrane-bound organelles and membraneless organelles (MLOs) coordinate various biological processes within eukaryotic cells. Among these, stress granules (SGs) are significant cytoplasmic MLOs that form in response to cellular stress, exhibiting liquid-like properties alongside stable substructures. SGs interact with diverse organelles, thereby influencing cellular pathways that are critical in both health and disease contexts. This review discusses the interplay between SGs and organelles and explores the methodologies employed to analyze interactions between SGs and other MLOs. Furthermore, it highlights the pivotal roles SGs play in regulating cellular responses and the pathogenesis of amyotrophic lateral sclerosis. Gaining insights into these interactions is essential for deciphering the mechanisms underlying both physiological processes and pathological conditions.
Humans ; Stress Granules/pathology* ; Organelles/metabolism* ; Amyotrophic Lateral Sclerosis/pathology* ; Animals ; Stress, Physiological ; Cytoplasmic Granules/metabolism*

Stress granules (SGs) are cytoplasmic ribonucleoprotein assemblies formed under stress conditions and are related to various biological processes and human diseases. Previous studies have reported the regulatory role of some proteins and linear RNAs in SG assembly. However, the relationship between circular RNAs (circRNAs) and SGs has not been discovered. Here, we screened both linear RNAs and circRNAs in SGs using improved total RNA sequencing of purified SG cores in mammalian cells and identified circular transcripts specifically localized in SGs. circRNAs with higher SG-related RNA-binding protein (RBP) binding abilities are more likely to be enriched in SGs. Furthermore, some SG-enriched circRNAs are differentially expressed in hepatocellular carcinoma (HCC) and adjacent tissues. These results suggest the regulatory role of circRNAs in SG formation and provide insights into the biological function of circRNAs and SGs in HCC.
Animals ; Humans ; RNA, Circular/metabolism* ; Carcinoma, Hepatocellular/metabolism* ; Stress Granules ; Cytoplasmic Granules/metabolism* ; Liver Neoplasms/metabolism* ; RNA/metabolism* ; Stress, Physiological/genetics* ; Mammals/genetics*

The nucleocapsid (N) protein of SARS-CoV-2 has been reported to have a high ability of liquid-liquid phase separation, which enables its incorporation into stress granules (SGs) of host cells. However, whether SG invasion by N protein occurs in the scenario of SARS-CoV-2 infection is unknow, neither do we know its consequence. Here, we used SARS-CoV-2 to infect mammalian cells and observed the incorporation of N protein into SGs, which resulted in markedly impaired self-disassembly but stimulated cell cellular clearance of SGs. NMR experiments further showed that N protein binds to the SG-related amyloid proteins via non-specific transient interactions, which not only expedites the phase transition of these proteins to aberrant amyloid aggregation in vitro, but also promotes the aggregation of FUS with ALS-associated P525L mutation in cells. In addition, we found that ACE2 is not necessary for the infection of SARS-CoV-2 to mammalian cells. Our work indicates that SARS-CoV-2 infection can impair the disassembly of host SGs and promote the aggregation of SG-related amyloid proteins, which may lead to an increased risk of neurodegeneration.
Amyloidogenic Proteins/metabolism* ; Amyotrophic Lateral Sclerosis/genetics* ; Animals ; COVID-19 ; Cytoplasmic Granules/metabolism* ; Mammals ; SARS-CoV-2 ; Stress Granules

BACKGROUND: Dysregulation of hepatic glucose production (HGP) contributes to the development of type 2 diabetes mellitus. Telmisartan, an angiotensin II type 1 receptor blocker (ARB), has various ancillary effects in addition to common blood pressure-lowering effects. The effects and mechanism of telmisartan on HGP have not been fully elucidated and, therefore, we investigated these phenomena in hyperglycemic HepG2 cells and high-fat diet (HFD)-fed mice.METHODS: Glucose production and glucose uptake were measured in HepG2 cells. Expression levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase α (G6Pase-α), and phosphorylation levels of insulin receptor substrate-1 (IRS-1) and protein kinase C ζ (PKCζ) were assessed by western blot analysis. Animal studies were performed using HFD-fed mice.RESULTS: Telmisartan dose-dependently increased HGP, and PEPCK expression was minimally increased at a 40 μM concentration without a change in G6Pase-α expression. In contrast, telmisartan increased phosphorylation of IRS-1 at Ser302 (p-IRS-1-Ser302) and decreased p-IRS-1-Tyr632 dose-dependently. Telmisartan dose-dependently increased p-PKCζ-Thr410 which is known to reduce insulin action by inducing IRS-1 serine phosphorylation. Ectopic expression of dominant-negative PKCζ significantly attenuated telmisartan-induced HGP and p-IRS-1-Ser302 and -inhibited p-IRS-1-Tyr632. Among ARBs, including losartan and fimasartan, only telmisartan changed IRS-1 phosphorylation and pretreatment with GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist, did not alter this effect. Finally, in the livers from HFD-fed mice, telmisartan increased p-IRS-1-Ser302 and decreased p-IRS-1-Tyr632, which was accompanied by an increase in p-PKCζ-Thr410.CONCLUSION: These results suggest that telmisartan increases HGP by inducing p-PKCζ-Thr410 that increases p-IRS-1-Ser302 and decreases p-IRS-1-Tyr632 in a PPARγ-independent manner.
Animals ; Blotting, Western ; Diabetes Mellitus, Type 2 ; Diet, High-Fat ; Ectopic Gene Expression ; Glucose ; Glucose-6-Phosphatase ; Hep G2 Cells ; Insulin Receptor Substrate Proteins ; Insulin ; Liver ; Losartan ; Mice ; Peroxisomes ; Phosphoenolpyruvate ; Phosphorylation ; Protein Kinase C ; Protein Kinases ; Receptor, Angiotensin, Type 1 ; Receptor, Insulin ; Serine

Differentiation of preadipocyte, also named adipogenesis, leads to the phenotype of mature adipocyte that is filled with many lipid droplets. Excessive lipid accumulation in adipocytes leads to the development of obesity. In this study, we investigated the effect of 11 different natural compounds on lipid accumulation during the differentiation of 3T3-L1 preadipocytes into 3T3-L1 adipocytes. Strikingly, among the natural compounds, cryptotanshinone at 10 µM most strongly reduced triglyceride (TG) contents in 3T3-L1 cells after 8 days of the differentiation. Furthermore, cryptotanshinone at 10 µM significantly suppressed lipid accumulation in 3T3-L1 cells after 8 days of the differentiation. Cryptotanshinone at 1 to 10 µM tested did not affect the survival of 3T3-L1 cells after 8 days of the differentiation. On mechanistic levels, cryptotanshinone time-differentially decreased the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during the 3T3-L1 cell differentiation. Taken together, these findings demonstrate that cryptotanshinone inhibits lipid accumulation in differentiating 3T3-L1 cells, which appears to be mediated through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, Perilipin A, and STAT-3.
3T3-L1 Cells ; Adipocytes ; Adipogenesis ; Lipid Droplets ; Obesity ; Peroxisomes ; Phenotype ; Phosphorylation ; Transducers ; Triglycerides

Luffa cylindrica (LC) is a very fast-growing climber and its fruit have been considered as agricultural wastes. We conducted to check the comparative qualities of ethanol solvent extraction (LCE) and supercritical carbon dioxide extraction (LCS) of L. cylindrica fruit and seed. LCS had higher antioxidant and polyphenol contents than LCE. LCS were significantly increased peroxisome proliferator-activated receptor (PPAR)-a and involucrin expression as epidermal differentiation marker in 3D skin equivalent model. LCS also showed antimicrobial activity against Staphylococcus aureus, a causative bacteria in atopic dermatitis. In addition, LCS inhibited the adipocyte differentiation of 3T3-L1 cells. When treated with the extract at a concentration of 100 µg/mL, the Wnt/β-catenin pathway reporter luciferase activity of HEK 293-TOP cells was increased approximately by 2-folds compared to that of the untreated control group. These results indicate that L. cylindrica supercritical carbon dioxide extract may serve as a cosmeceutical for improving skin barrier function and the treatment of obesity.
3T3-L1 Cells ; Adipocytes ; Bacteria ; Carbon Dioxide ; Carbon ; Dermatitis, Atopic ; Ethanol ; Fruit ; Luciferases ; Luffa ; Obesity ; Peroxisomes ; Skin ; Staphylococcus aureus

BACKGROUND/OBJECTIVES: Docosahexaenoic acid (DHA), an n-3 long chain polyunsaturated fatty acid (LCPUFA), is acquired by dietary intake or the in vivo conversion of α-linolenic acid. Many enzymes participating in LCPUFA synthesis are regulated by peroxisome proliferator-activated receptor alpha (PPARα). Therefore, it was hypothesized that the tissue accretion of endogenously synthesized DHA could be modified by PPARα. MATERIALS/METHODS: The tissue DHA concentrations and mRNA levels of genes participating in DHA biosynthesis were compared among PPARα homozygous (KO), heterozygous (HZ), and wild type (WT) mice (Exp I), and between WT mice treated with clofibrate (PPARα agonist) or those not treated (Exp II). In ExpII, the expression levels of the proteins associated with DHA function in the brain cortex and retina were also measured. An n3-PUFA depleted/replenished regimen was applied to mitigate the confounding effects of maternal DHA. RESULTS: PPARα ablation reduced the hepatic Acox, Fads1, and Fads2 mRNA levels, as well as the DHA concentration in the liver, but not in the brain cortex. In contrast, PPARα activation increased hepatic Acox, Fads1, Fads2 and Elovl5 mRNA levels, but reduced the DHA concentrations in the liver, retina, and phospholipid of brain cortex, and decreased mRNA and protein levels of the brain-derived neurotrophic factor in brain cortex. CONCLUSIONS: LCPUFA enzyme expression was altered by PPARα. Either PPARα deficiency or activation-decreased tissue DHA concentration is a stimulus for further studies to determine the functional significance.
Animals ; Brain ; Brain-Derived Neurotrophic Factor ; Clofibrate ; Docosahexaenoic Acids ; Fatty Acid Desaturases ; Liver ; Mice ; Peroxisomes ; PPAR alpha ; Retina ; RNA, Messenger

Fumigaclavine C (FC), an active indole alkaloid, is obtained from endophytic Aspergillus terreus (strain No. FC118) by the root of Rhizophora stylosa (Rhizophoraceae). This study is designed to evaluate whether FC has anti-adipogenic effects in 3T3-L1 adipocytes and whether it ameliorates lipid accumulation in high-fat diet (HFD)-induced obese mice. FC notably increased the levels of glycerol in the culture supernatants and markedly reduced lipid accumulation in 3T3-L1 adipocytes. FC differentially inhibited the expressions of adipogenesis-related genes, including the peroxisome proliferator-activated receptor proteins, CCAAT/enhancer-binding proteins, and sterol regulatory element-binding proteins. FC markedly reduced the expressions of lipid synthesis-related genes, such as the fatty acid binding protein, lipoprotein lipase, and fatty acid synthase. Furthermore, FC significantly increased the expressions of lipolysis-related genes, such as the hormone-sensitive lipase, Aquaporin-7, and adipose triglyceride lipase. In HFD-induced obese mice, intraperitoneal injections of FC decreased both the body weight and visceral adipose tissue weight. FC administration significantly reduced lipid accumulation. Moreover, FC could dose-dependently and differentially regulate the expressions of lipid metabolism-related transcription factors. All these data indicated that FC exhibited anti-obesity effects through modulating adipogenesis and lipolysis.
Adipocytes ; Adipogenesis ; Animals ; Aspergillus ; Body Weight ; Carrier Proteins ; Diet, High-Fat ; Glycerol ; Injections, Intraperitoneal ; Intra-Abdominal Fat ; Lipase ; Lipolysis ; Lipoprotein Lipase ; Mice ; Mice, Obese ; Peroxisomes ; Rhizophoraceae ; Sterol Esterase ; Transcription Factors