Accumulation Of Intracellular Lipid Droplets Research Articles

Effect of MEHP on testosterone synthesis via Sirt1/Foxo1/Rab7 signaling pathway inhibition of lipophagy in TM3 cells

Mono-2-ethylhexyl phthalic acid (MEHP) is the most toxic metabolite of the plasticizer di-2-ethylhexyl phthalic acid (DEHP), and studies have shown that MEHP causes serious reproductive effects. However, its exact mechanisms of action remain elusive. In this study, we aimed to investigate the reproductive effects of MEHP and preliminarily explore its underlying molecular mechanisms. We found that TM3 cells gradually secreted less testosterone and intracellular free cholesterol with increasing MEHP exposure. MEHP exposure inhibited lipophagy and the Sirt1/Foxo1/Rab7 signaling pathway in TM3 cells, causing aberrant accumulation of intracellular lipid droplets. Addition of the Sirt1 agonist SRT1720 and Rab7 agonist ML-098 alleviated the inhibition of lipophagy and increased free cholesterol and testosterone contents in TM3 cells. SRT1720 alleviated the inhibitory effect of MEHP on the Sirt1/Foxo1/Rab7 signaling pathway, whereas ML-098 only alleviated the inhibition of Rab7 protein expression by MEHP and had no effect on Sirt1 and Foxo1 protein expression. This suggests that MEHP inhibits lipophagy in TM3 cells by suppressing the Sirt1/Foxo1/Rab7 signaling pathway, ultimately leading to a further decrease in cellular testosterone secretion. This study improves our current understanding of the toxicity and molecular mechanisms of action of MEHP and provides new insights into the reproductive effects of phthalic acid esters.

FOXO3: at the crossroads of metabolic, inflammatory, and tumorigenic remodeling in the colon.

The Forkhead box O3 (FOXO3) transcription factor regulates the expression of genes critical for diverse cellular functions in homeostasis. Diminished FOXO3 activity is associated with human diseases such as obesity, metabolic diseases, inflammatory diseases, and cancer. In the mouse colon, FOXO3 deficiency leads to an inflammatory immune landscape and dysregulated molecular pathways, which, under various insults, exacerbates inflammation and tumor burden, mimicking characteristics of human diseases. This deficiency also results in dysregulated lipid metabolism, and consequently, the accumulation of intracellular lipid droplets (LDs) in colonic epithelial cells and infiltrated immune cells. FOXO3 and LDs form a self-reinforcing negative regulatory loop in colonic epithelial cells, neutrophils, and macrophages, which is associated with inflammatory bowel disease and colon cancer, particularly in the context of obesity.

Lysine tRNA fragments and miR-194-5p co-regulate hepatic steatosis via β-Klotho and perilipin 2

ObjectiveNon-alcoholic fatty liver disease (NAFLD) involves hepatic accumulation of intracellular lipid droplets via incompletely understood processes. Here, we report distinct and cooperative NAFLD roles of LysTTT-5′tRF transfer RNA fragments and microRNA miR-194-5p. MethodsCombined use of diet induced obese mice with human-derived oleic acid-exposed Hep G2 cells revealed new NAFLD roles of LysTTT-5′tRF and miR-194-5p. ResultsUnlike lean animals, dietary-induced NAFLD mice showed concurrent hepatic decrease of both LysTTT-5′tRF and miR-194-5p levels, which were restored following miR-132 antisense oligonucleotide treatment which suppresses hepatic steatosis. Moreover, exposing human-derived Hep G2 cells to oleic acid for 7 days co-suppressed miR-194-5p and LysTTT-5′tRF levels while increasing lipid accumulation. Inversely, transfecting fattened cells with a synthetic LysTTT-5′tRF mimic elevated mRNA levels of the metabolic regulator β-Klotho while decreasing triglyceride amounts by 30% within 24 h. In contradistinction, antisense suppression of miR-194-5p induced accumulation of its novel target, the NAFLD-implicated lipid droplet-coating PLIN2 protein. Further, two out of 15 steatosis-alleviating screened drug-repurposing compounds, Danazol and Latanoprost, elevated miR-194-5p or LysTTT-5′tRF levels. ConclusionOur findings highlight the different yet complementary roles of miR-194-5p and LysTTT-5′tRF and offer new insights into the complex roles of small non-coding RNAs and the multiple pathways involved in NAFLD pathogenesis.

A positive feedback loop between ZEB2 and ACSL4 regulates lipid metabolism to promote breast cancer metastasis

Lipid metabolism plays a critical role in cancer metastasis. However, the mechanisms through which metastatic genes regulate lipid metabolism remain unclear. Here, we describe a new oncogenic–metabolic feedback loop between the epithelial–mesenchymal transition transcription factor ZEB2 and the key lipid enzyme ACSL4 (long-chain acyl-CoA synthetase 4), resulting in enhanced cellular lipid storage and fatty acid oxidation (FAO) to drive breast cancer metastasis. Functionally, depletion of ZEB2 or ACSL4 significantly reduced lipid droplets (LDs) abundance and cell migration. ACSL4 overexpression rescued the invasive capabilities of the ZEB2 knockdown cells, suggesting that ACSL4 is crucial for ZEB2-mediated metastasis. Mechanistically, ZEB2-activated ACSL4 expression by directly binding to the ACSL4 promoter. ACSL4 binds to and stabilizes ZEB2 by reducing ZEB2 ubiquitination. Notably, ACSL4 not only promotes the intracellular lipogenesis and LDs accumulation but also enhances FAO and adenosine triphosphate production by upregulating the FAO rate-limiting enzyme CPT1A (carnitine palmitoyltransferase 1 isoform A). Finally, we demonstrated that ACSL4 knockdown significantly reduced metastatic lung nodes in vivo. In conclusion, we reveal a novel positive regulatory loop between ZEB2 and ACSL4, which promotes LDs storage to meet the energy needs of breast cancer metastasis, and identify the ZEB2–ACSL4 signaling axis as an attractive therapeutic target for overcoming breast cancer metastasis.

Clearance of intracellular lipids by lipophagy impaired in APOE ε4 human astrocytes

AbstractBackgroundApolipoprotein E (APOE) ɛ4 is the strongest known genetic risk factor for Alzheimer’s disease. APOE ɛ4 has been found to impair lipid metabolism in human glia causing accumulation of intracellular lipid droplets. We hypothesized that this phenotype in human APOE ɛ4/ɛ4 astrocytes was due to defective clearance via lipophagy.MethodGlobal transcriptomic analyses were performed on astrocytes derived from isogenic and population model human induced pluripotent stem cells (iPSCs). Fluorescent reporters, immunocytochemistry, and western blotting were used to quantify lipid droplets and autophagic machinery in iPSC‐based models.ResultGlobal transcriptomic analyses reveal that APOE ε4 drives autophagy dysregulation in astrocytes from human iPSCs. High‐content imaging of fluorescent autophagy reporters and immunocytochemistry revealed accumulation of lipid droplets and autophagy machinery in APOE ɛ4/ɛ4 astrocytes which was ameliorated by induction of autophagy.ConclusionHuman iPSC‐derived APOE ɛ4/ɛ4 astrocytes exhibit impaired lipophagy.

Transcriptional landscape of human trophoblast cells treated with calcitriol and TGF-β1

Calcitriol and transforming growth factor beta 1 (TGF-β1) are unrelated molecules that regulate biological processes according to the genetic target, cell type, and context. Several studies have shown independent effects of calcitriol and TGF-βs on the placenta, but there is no information regarding the impact of their combination on these cells. Therefore, this study analyzed the effects of calcitriol, TGF-β1, and their combination in primary cultures of human trophoblast cells using a whole genome expression microarray. Data analysis revealed a set of differentially expressed genes induced by each treatment. Enrichment pathway analysis identified modulatory effects of calcitriol on genes related to metabolic processes such as vitamin D, steroid, and fat-soluble vitamins as well as antimicrobial and immune responses. In relation to TGF-β1, the analysis showed a few differentially expressed genes that were mainly associated with the neutrophil immune response. Lastly, the analysis revealed that the combination of calcitriol and TGF-β1 up-regulated genes involving both immunologic processes and the biosynthesis of unsaturated fatty acids, eicosanoids, and lipoxins, among others. In contrast, pathways down-regulated by the combination were mostly associated with the catabolic process of acylglycerols and peptides, PPAR signaling pathway, cellular response to low-density lipoprotein stimulus, renin angiotensin system and digestion, mobilization and transport of lipids. Consistent with these results, the combined treatment on human trophoblast cells induced the accumulation of intracellular neutral lipid droplets and stimulated both gene and protein expression of 15-hydroxyprostaglandin dehydrogenase. In conclusion, the results revealed that differentially expressed genes induced by the combination modified the transcriptional landscape compared to each treatment alone, mainly altering the storage, activity and metabolism of lipids, which might have an impact on placental development.

Imbalanced lipid homeostasis caused by membrane αKlotho deficiency contributes to the acute kidney injury to chronic kidney disease transition

After acute kidney injury (AKI), renal tubular epithelial cells (RTECs) are pathologically characterized by intracellular lipid droplet (LD) accumulation, which are involved in RTEC injury and kidney fibrosis. However, its pathogenesis remains incompletely understood. The protein, αKlotho, primarily expressed in RTECs, is well known as an anti-aging hormone wielding versatile functions, and its membrane form predominantly acts as a co-receptor for fibroblast growth factor 23. Here, we discovered a connection between membrane αKlotho and intracellular LDs in RTECs. Fluorescent fatty acid (FA) pulse-chase assays showed that membrane αKlotho deficiency in RTECs, as seen in αKlotho homozygous mutated (kl/kl) mice or in mice with ischemia-reperfusion injury (IRI)-induced AKI, inhibited FA mobilization from LDs by impairing adipose triglyceride lipase (ATGL)-mediated lipolysis and lipophagy. This resulted in LD accumulation and FA underutilization. IRI-induced alterations were more striking in αKlotho deficiency. Mechanistically, membrane αKlotho deficiency promoted E3 ligase peroxin2 binding to ubiquitin-conjugating enzyme E2 D2, resulting in ubiquitin-mediated degradation of ATGL which is a common molecular basis for lipolysis and lipophagy. Overexpression of αKlotho rescued FA mobilization by preventing ATGL ubiquitination, thereby lessening LD accumulation and fibrosis after AKI. This suggests that membrane αKlotho is indispensable for the maintenance of lipid homeostasis in RTECs. Thus, our study identified αKlotho as a critical regulator of lipid turnover and homeostasis in AKI, providing a viable strategy for preventing tubular injury and the AKI-to-chronic kidney disease transition.

Porcine reproductive and respiratory syndrome virus upregulates SMPDL3B to promote viral replication by modulating lipid metabolism

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a severe threat to the health of pigs globally. Host factors play a critical role in PRRSV replication. Using PRRSV as a model for genome-scale CRISPR knockout (KO) screening, we identified a host factor critical to PRRSV infection: sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B). Our findings show that SMPDL3B restricted PRRSV attachment, entry, replication, and secretion and that its depletion significantly inhibited PRRSV proliferation, indicating that SMPDL3B plays a positive role in PRRSV replication. Our data also show that SMPDL3B deficiency resulted in an accumulation of intracellular lipid droplets (LDs). The expression level of key genes (ACC, SCD-1, and FASN) involved in lipogenesis was increased, whereas the fundamental lipolysis gene, ATGL, was inhibited when SMPDL3B was knocked down. Overall, our findings suggest that SMPDL3B deficiency can effectively inhibit viral infection through the modulation of lipid metabolism.

CircDOCK7 facilitates the proliferation and adipogenic differentiation of chicken abdominal preadipocytes through the gga-miR-301b-3p/ACSL1 axis

BackgroundAbdominal fat deposition depends on both the proliferation of preadipocytes and their maturation into adipocytes, which is a well-orchestrated multistep process involving many regulatory molecules. Circular RNAs (circRNAs) have emergingly been implicated in mammalian adipogenesis. However, circRNA-mediated regulation in chicken adipogenesis remains unclear. Our previous circRNA sequencing data identified a differentially expressed novel circRNA, 8:27,886,180|27,889,657, during the adipogenic differentiation of chicken abdominal preadipocytes. This study aimed to investigate the regulatory role of circDOCK7 in the proliferation and adipogenic differentiation of chicken abdominal preadipocytes, and explore its molecular mechanisms of competing endogenous RNA underlying chicken adipogenesis.ResultsOur results showed that 8:27,886,180|27,889,657 is an exonic circRNA derived from the head-to-tail splicing of exons 19–22 of the dedicator of cytokinesis 7 (DOCK7) gene, abbreviated as circDOCK7. CircDOCK7 is mainly distributed in the cytoplasm of chicken abdominal preadipocytes and is stable because of its RNase R resistance and longer half-life. CircDOCK7 is significantly upregulated in the abdominal fat tissues of fat chickens compared to lean chickens, and its expression gradually increases during the proliferation and adipogenic differentiation of chicken abdominal preadipocytes. Functionally, the gain- and loss-of-function experiments showed that circDOCK7 promoted proliferation, G0/G1- to S-phase progression, and glucose uptake capacity of chicken abdominal preadipocytes, in parallel with adipogenic differentiation characterized by remarkably increased intracellular lipid droplet accumulation and triglyceride and acetyl coenzyme A content in differentiated chicken abdominal preadipocytes. Mechanistically, a pull-down assay and a dual-luciferase reporter assay confirmed that circDOCK7 interacted with gga-miR-301b-3p, which was identified as an inhibitor of chicken abdominal adipogenesis. Moreover, the ACSL1 gene was demonstrated to be a direct target of gga-miR-301b-3p. Chicken ACSL1 protein is localized in the endoplasmic reticulum and mitochondria of chicken abdominal preadipocytes and acts as an adipogenesis accelerator. Rescue experiments showed that circDOCK7 could counteract the inhibitory effects of gga-miR-301b-3p on ACSL1 mRNA abundance as well as the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.ConclusionsCircDOCK7 serves as a miRNA sponge that directly sequesters gga-miR-301b-3p away from the ACSL1 gene, thus augmenting adipogenesis in chickens. These findings may elucidate a new regulatory mechanism underlying abdominal fat deposition in chickens.

FOXO3 Deficiency in Neutrophils Drives Colonic Inflammation and Tumorigenesis.

Inflammatory bowel disease (IBD), characterized by infiltration of polymorphonuclear neutrophils (PMNs), increases the risk of colon cancer. PMN activation corresponds to the accumulation of intracellular Lipid Droplets (LDs). As increased LDs are negatively regulated by transcription factor Forkhead Box O3 (FOXO3), we aim to determine the significance of this regulatory network in PMN-mediated IBD and tumorigenesis. Affected tissue of IBD and colon cancer patients, colonic and infiltrated immune cells, have increased LDs' coat protein, PLIN2. Mouse peritoneal PMNs with stimulated LDs and FOXO3 deficiency have elevated transmigratory activity. Transcriptomic analysis of these FOXO3-deficient PMNs showed differentially expressed genes (DEGs; FDR < 0.05) involved in metabolism, inflammation, and tumorigenesis. Upstream regulators of these DEGs, similar to colonic inflammation and dysplasia in mice, were linked to IBD and human colon cancer. Additionally, a transcriptional signature representing FOXO3-deficient PMNs (PMN-FOXO3389) separated transcriptomes of affected tissue in IBD (p = 0.00018) and colon cancer (p = 0.0037) from control. Increased PMN-FOXO3389 presence predicted colon cancer invasion (lymphovascular p = 0.015; vascular p = 0.046; perineural p = 0.03) and poor survival. Validated DEGs from PMN-FOXO3389 (P2RX1, MGLL, MCAM, CDKN1A, RALBP1, CCPG1, PLA2G7) are involved in metabolism, inflammation, and tumorigenesis (p < 0.05). These findings highlight the significance of LDs and FOXO3-mediated PMN functions that promote colonic pathobiology.

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment.

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 µm < d ≤ 3 µm), intermediate vesicles (3 µm < d ≤ 9 µm), and macrovesicles (d > 9 µm), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

Short-chain fatty and carboxylic acid changes associated with fecal microbiota transplant communally influence microglial inflammation

The intestinal microbiota has been proposed to influence human mental health and cognition through the gut-brain axis. Individuals experiencing recurrent Clostridioides difficile infection (rCDI) frequently report depressive symptoms, which are improved after fecal microbiota transplantation (FMT); however, mechanisms underlying this association are poorly understood. Short-chain fatty acids and carboxylic acids (SCCA) produced by the intestinal microbiota cross the blood brain barrier and have been proposed to contribute to gut-brain communication. We hypothesized that changes in serum SCCA measured before and after successful FMT for rCDI influences the inflammatory response of microglia, the resident immune cells of the central nervous system. Serum SCCA were quantified using gas chromatography-mass spectroscopy from 38 patients who participated in a randomized trial comparing oral capsule-vs colonoscopy-delivered FMT for rCDI, and quality of life was assessed by SF-36 at baseline, 4, and 12 weeks after FMT treatment. Successful FMT was associated with improvements in mental and physical health, as well as significant changes in a number of circulating SCCA, including increased butyrate, 2-methylbutyrate, valerate, and isovalerate, and decreased 2-hydroxybutyrate. Primary cultured microglia were treated with SCCA and the response to a pro-inflammatory stimulus was measured. Treatment with a combination of SCCA based on the post-FMT serum profile, but not single SCCA species, resulted in significantly reduced inflammatory response including reduced cytokine release, reduced nitric oxide release, and accumulation of intracellular lipid droplets. This suggests that both levels and diversity of SCCA may be an important contributor to gut-brain communication.

Tussilagone ameliorates high-fat diet-induced hepatic steatosis by enhancing energy metabolism and antioxidant activity.

Non-alcoholic fatty liver disease (NAFLD) is a major health problem. However, no effective treatments are currently available. Thus, there is a critical need to develop novel drugs that can prevent and treat NAFLD with few side effects. In this study, Tussilagone (TUS), a natural sesquiterpene isolated from Tussilago farfara L, was explored in vitro and in vivo for its potential to treat NAFLD. Our results showed that in vitro TUS reduced oleic acid palmitate acid-induced triglyceride and cholesterol synthesis in HepG2cells, reduced intracellular lipid droplet accumulation, improved glucose metabolism disorders and increased energy metabolism and reduced oxidative stress levels. In vivo, TUS significantly reduced fat accumulation and improved liver injury in high-fat diet (HFD)-induced mice. TUS treatment significantly increased liver mitochondrial counts and antioxidant levels compared to the HFD group of mice. In addition, TUS was found to reduce the expression of genes involved in lipid synthesis sterol regulatory element binding protein-1 (SREBP1), fatty acid synthase (FASN), and stearoy-CoA desaturase 1 (SCD1) in vitro and in vivo. Our results suggest that TUS may be helpful in the treatment of NAFLD, suggesting that TUS is a promising compound for the treatment of NAFLD. Our findings provided novel insights into the application of TUS in regulating lipid metabolism.

Palmitate-Induced Cardiac Lipotoxicity Is Relieved by the Redox-Active Motif of SELENOT through Improving Mitochondrial Function and Regulating Metabolic State.

Cardiac lipotoxicity is an important contributor to cardiovascular complications during obesity. Given the fundamental role of the endoplasmic reticulum (ER)-resident Selenoprotein T (SELENOT) for cardiomyocyte differentiation and protection and for the regulation of glucose metabolism, we took advantage of a small peptide (PSELT), derived from the SELENOT redox-active motif, to uncover the mechanisms through which PSELT could protect cardiomyocytes against lipotoxicity. To this aim, we modeled cardiac lipotoxicity by exposing H9c2 cardiomyocytes to palmitate (PA). The results showed that PSELT counteracted PA-induced cell death, lactate dehydrogenase release, and the accumulation of intracellular lipid droplets, while an inert form of the peptide (I-PSELT) lacking selenocysteine was not active against PA-induced cardiomyocyte death. Mechanistically, PSELT counteracted PA-induced cytosolic and mitochondrial oxidative stress and rescued SELENOT expression that was downregulated by PA through FAT/CD36 (cluster of differentiation 36/fatty acid translocase), the main transporter of fatty acids in the heart. Immunofluorescence analysis indicated that PSELT also relieved the PA-dependent increase in CD36 expression, while in SELENOT-deficient cardiomyocytes, PA exacerbated cell death, which was not mitigated by exogenous PSELT. On the other hand, PSELT improved mitochondrial respiration during PA treatment and regulated mitochondrial biogenesis and dynamics, preventing the PA-provoked decrease in PGC1-α and increase in DRP-1 and OPA-1. These findings were corroborated by transmission electron microscopy (TEM), revealing that PSELT improved the cardiomyocyte and mitochondrial ultrastructures and restored the ER network. Spectroscopic characterization indicated that PSELT significantly attenuated infrared spectral-related macromolecular changes (i.e., content of lipids, proteins, nucleic acids, and carbohydrates) and also prevented the decrease in membrane fluidity induced by PA. Our findings further delineate the biological significance of SELENOT in cardiomyocytes and indicate the potential of its mimetic PSELT as a protective agent for counteracting cardiac lipotoxicity.

Quercetin and Its Derivative Counteract Palmitate-Dependent Lipotoxicity by Inhibiting Oxidative Stress and Inflammation in Cardiomyocytes.

Cardiac lipotoxicity plays an important role in the pathogenesis of obesity-related cardiovascular disease. The flavonoid quercetin (QUE), a nutraceutical compound that is abundant in the "Mediterranean diet", has been shown to be a potential therapeutic agent in cardiac and metabolic diseases. Here, we investigated the beneficial role of QUE and its derivative Q2, which demonstrates improved bioavailability and chemical stability, in cardiac lipotoxicity. To this end, H9c2 cardiomyocytes were pre-treated with QUE or Q2 and then exposed to palmitate (PA) to recapitulate the cardiac lipotoxicity occurring in obesity. Our results showed that both QUE and Q2 significantly attenuated PA-dependent cell death, although QUE was effective at a lower concentration (50 nM) when compared with Q2 (250 nM). QUE decreased the release of lactate dehydrogenase (LDH), an important indicator of cytotoxicity, and the accumulation of intracellular lipid droplets triggered by PA. On the other hand, QUE protected cardiomyocytes from PA-induced oxidative stress by counteracting the formation of malondialdehyde (MDA) and protein carbonyl groups (which are indicators of lipid peroxidation and protein oxidation, respectively) and intracellular ROS generation, and by improving the enzymatic activities of catalase and superoxide dismutase (SOD). Pre-treatment with QUE also significantly attenuated the inflammatory response induced by PA by reducing the release of key proinflammatory cytokines (IL-1β and TNF-α). Similar to QUE, Q2 (250 nM) also significantly counteracted the PA-provoked increase in intracellular lipid droplets, LDH, and MDA, improving SOD activity and decreasing the release of IL-1β and TNF-α. These results suggest that QUE and Q2 could be considered potential therapeutics for the treatment of the cardiac lipotoxicity that occurs in obesity and metabolic diseases.

Pre-Diabetes-Linked miRNA miR-193b-3p Targets PPARGC1A, Disrupts Metabolic Gene Expression Profile and Increases Lipid Accumulation in Hepatocytes: Relevance for MAFLD.

Distinct plasma microRNA profiles associate with different disease features and could be used to personalize diagnostics. Elevated plasma microRNA hsa-miR-193b-3p has been reported in patients with pre-diabetes where early asymptomatic liver dysmetabolism plays a crucial role. In this study, we propose the hypothesis that elevated plasma hsa-miR-193b-3p conditions hepatocyte metabolic functions contributing to fatty liver disease. We show that hsa-miR-193b-3p specifically targets the mRNA of its predicted target PPARGC1A/PGC1α and consistently reduces its expression in both normal and hyperglycemic conditions. PPARGC1A/PGC1α is a central co-activator of transcriptional cascades that regulate several interconnected pathways, including mitochondrial function together with glucose and lipid metabolism. Profiling gene expression of a metabolic panel in response to overexpression of microRNA hsa-miR-193b-3p revealed significant changes in the cellular metabolic gene expression profile, including lower expression of MTTP, MLXIPL/ChREBP, CD36, YWHAZ and GPT, and higher expression of LDLR, ACOX1, TRIB1 and PC. Overexpression of hsa-miR-193b-3p under hyperglycemia also resulted in excess accumulation of intracellular lipid droplets in HepG2 cells. This study supports further research into potential use of microRNA hsa-miR-193b-3p as a possible clinically relevant plasma biomarker for metabolic-associated fatty liver disease (MAFLD) in dysglycemic context.

MiR-328-5p inhibits the adipogenic differentiation of hMSCs by targeting fatty acid synthase.

Adipogenesis, a highly coordinated process regulated by numerous effectors, is largely responsible for the quantity and size of adipocytes. Attenuation of adipocyte differentiation has been proposed as a viable technique for reducing obesity and its associated diseases. MicroRNAs play an important role in human bone marrow mesenchymal stem cells (hMSCs) adipogenic differentiation. However, there is a lack of clarity regarding the role of miR-328-5p in adipogenesis. Using the lentiviral vectors to overexpress fatty acid synthase (FASN) and miR-328-5p, RT-qPCR and Western blotting were carried out to assess RNA expression and protein levels of FASN and adipogenic marker factors. Meanwhile, Oil red O staining and lipid quantification was performed to evaluate the accumulation of intracellular lipid droplets. Additionally, the validity of FASN as a potential target gene for miR-328-5p was carried out using a luciferase reporter assay. Our data showed that hMSCs adipogenic differentiation was associaed with the reduced miR-328-5p expression, while an elevated expression of the underlined miRNA attenuated adipogenesis and the expression of adipogenic marker genes. Luciferase reporter assay validated FASN as a target gene of miR-328-5p, and an elevated FASN expression reversed the anti-adipogenic effects of miR-328-5p. The results revealed that miR-328-5p inhibits hMSCs adipogenic differentiation by targeting FASN. These findings contribute to our understanding of obesity-related disease development.

Rewiring Lipid Metabolism by Targeting PCSK9 and HMGCR to Treat Liver Cancer.

Alterations in lipid handling are an important hallmark in cancer. Our aim here is to target key metabolic enzymes to reshape the oncogenic lipid metabolism triggering irreversible cell breakdown. We targeted the key metabolic player proprotein convertase subtilisin/kexin type 9 (PCSK9) using a pharmacological inhibitor (R-IMPP) alone or in combination with 3-hydroxy 3-methylglutaryl-Coenzyme A reductase (HMGCR) inhibitor, simvastatin. We assessed the effect of these treatments using 3 hepatoma cell lines, Huh6, Huh7 and HepG2 and a tumor xenograft in chicken choriorallantoic membrane (CAM) model. PCSK9 deficiency led to dose-dependent inhibition of cell proliferation in all cell lines and a decrease in cell migration. Co-treatment with simvastatin presented synergetic anti-proliferative effects. At the metabolic level, mitochondrial respiration assays as well as the assessment of glucose and glutamine consumption showed higher metabolic adaptability and surge in the absence of PCSK9. Enhanced lipid uptake and biogenesis led to excessive accumulation of intracellular lipid droplets as revealed by electron microscopy and metabolic tracing. Using xenograft experiments in CAM model, we further demonstrated the effect of anti-PCSK9 treatment in reducing tumor aggressiveness. Targeting PCSK9 alone or in combination with statins deserves to be considered as a new therapeutic option in liver cancer clinical applications.

Prognosis and pain dissection of novel signatures in kidney renal clear cell carcinoma based on fatty acid metabolism-related genes.

Renal cell carcinoma (RCC) is a malignant tumor that is characterized by the accumulation of intracellular lipid droplets. The prognostic value of fatty acid metabolism-related genes (FMGs) in RCC remains unclear. Alongside this insight, we collected data from three RCC cohorts, namely, The Cancer Genome Atlas (TCGA), E-MTAB-1980, and GSE22541 cohorts, and identified a total of 309 FMGs that could be associated with RCC prognosis. First, we determined the copy number variation and expression levels of these FMGs, and identified 52 overall survival (OS)-related FMGs of the TCGA-KIRC and the E-MTAB-1980 cohort data. Next, 10 of these genes-FASN, ACOT9, MID1IP1, CYP2C9, ABCD1, CPT2, CRAT, TP53INP2, FAAH2, and PTPRG-were identified as pivotal OS-related FMGs based on least absolute shrinkage and selection operator and Cox regression analyses. The expression of some of these genes was confirmed in patients with RCC by immunohistochemical analyses. Kaplan-Meier analysis showed that the identified FMGs were effective in predicting the prognosis of RCC. Moreover, an optimal nomogram was constructed based on FMG-based risk scores and clinical factors, and its robustness was verified by time-dependent receiver operating characteristic analysis, calibration curve analysis, and decision curve analysis. We have also described the biological processes and the tumor immune microenvironment based on FMG-based risk score classification. Given the close association between fatty acid metabolism and cancer-related pain, our 10-FMG signature may also serve as a potential therapeutic target with dual effects on ccRCC prognosis and cancer pain and, therefore, warrants further investigation.

Overexpression of goat STEAP4 promotes the differentiation of subcutaneous adipocytes.

Objective: The focus of this study was the six-transmembrane epithelial antigen of the prostate 4 (STEAP4) gene, on the basis of the cloned goat STEAP4 gene sequence. Its molecular and expression characteristics were analyzed, and its influence on the differentiation of goat subcutaneous adipocytes was explored through overexpression. Method: Reverse-transcription PCR (RT-PCR) was used to clone the goat STEAP4 sequence, and online tools were used to analyze the molecular characteristic. Real-time quantitative PCR (qPCR) was used to detect the expression level of STEAP4 in goat tissues and subcutaneous adipocyte differentiation. Liposome transfection, BODIPY, Oil Red O staining, and qPCR were used to explore the effect of overexpression of STEAP4 on adipocyte differentiation. Results: The cloned goat STEAP4 gene sequence was 1388 bp, and the complete coding sequence (CDS) region was 1197 bp, which encoded a total of 398 amino acids. Compared with the predicted sequence (XM_005679300.3), there were three base mutations in the CDS region of goat STEAP4, A188G, T281C, and A507G. Among them, A507G changed the amino acid at position 170 from Ile to Val. Analysis of the physical and chemical properties of the protein showed that STEAP4 was a stable hydrophilic basic protein. STEAP4 gene expression level was highest in goat liver tissue ( ), followed by lung and back subcutaneous adipose tissue. STEAP4 showed different expression levels in goat subcutaneous adipocytes at different times during the induction of differentiation. The expression in the late stage of differentiation was higher than that before differentiation and lowest at 12 h ( ). Overexpression of STEAP4 promoted the accumulation of intracellular lipid droplets; C/EBP (CCAAT enhancer binding protein) was extremely significantly up-regulated ( ), and aP2 (fatty acid binding protein) was significantly up-regulated ( ). Conclusion: Overexpression of STEAP4 could promote the differentiation of goat subcutaneous preadipocytes. This study lays the foundation for an in-depth study of the role of STEAP4 in goat lipid deposition.