Adipose tissue peroxisomal lipid synthesis orchestrates obesity and insulin resistance through LXR-dependent lipogenesis.

OBJECTIVE: Adipose tissue mass is maintained by a balance between lipolysis and lipid storage. The contribution of adipose tissue lipogenesis to fat mass, especially in the setting of high-fat feeding, is considered minor. Here we investigated the effect of adipose-specific inactivation of the peroxisomal lipid synthetic protein PexRAP on fatty acid synthase (FASN)-mediated lipogenesis and its impact on adiposity and metabolic homeostasis. METHODS: To explore the role of PexRAP in adipose tissue, we metabolically phenotyped mice with adipose-specific knockout of PexRAP. Bulk RNA sequencing was used to determine transcriptomic responses to PexRAP deletion and 14C-malonyl CoA allowed us to measure de novo lipogenic activity in adipose tissue of these mice. In vitro cell culture models were used to elucidate the mechanism of cellular responses to PexRAP deletion. RESULTS: Adipose-specific PexRAP deletion promoted diet-induced obesity and insulin resistance through activation of de novo lipogenesis. Mechanistically, PexRAP inactivation inhibited the flux of carbons to ethanolamine plasmalogens. This increased the nuclear PC/PE ratio and promoted cholesterol mislocalization, resulting in activation of liver X receptor (LXR), a nuclear receptor known to be activated by increased intracellular cholesterol. LXR activation led to increased expression of the phospholipid remodeling enzyme LPCAT3 and induced FASN-mediated lipogenesis, which promoted diet-induced obesity and insulin resistance. CONCLUSIONS: These studies reveal an unexpected role for peroxisome-derived lipids in regulating LXR-dependent lipogenesis and suggest that activation of lipogenesis, combined with dietary lipid overload, exacerbates obesity and metabolic dysregulation.

Mefloquine enhances the efficacy of anti-PD-1 immunotherapy via IFN-γ-STAT1-IRF1-LPCAT3-induced ferroptosis in tumors.

BACKGROUND: Ferroptosis plays an important role in enhancing the efficacy of anti-programmed cell death 1 (PD-1) immunotherapy; however, the molecular mechanisms by which tumor ferroptosis sensitizes melanoma and lung cancer to anti-PD-1 immunotherapy have not been elucidated. METHODS: Cytotoxicity assays, colony formation assays, flow cytometry and animal experiments were used to evaluate the effects of mefloquine (Mef) on survival and ferroptosis in melanoma and lung cancer. RNA sequencing, Real-time quantitative PCR (qRT-PCR), western blotting, chromatin immunoprecipitation-qPCR and flow cytometry were used to determine the molecular mechanisms by which Mef regulates lysophosphatidylcholine acyltransferase 3 (LPCAT3). The relationship between LPCAT3 and the efficacy of anti-PD-1 immunotherapy was verified via a clinical database and single-cell RNA sequencing (ScRNA-Seq). RESULTS: In this study, we discovered that Mef induces ferroptosis. Furthermore, treatment with Mef in combination with T-cell-derived interferon-γ (IFN-γ) enhanced tumor ferroptosis and sensitized melanoma and lung cancer cells to anti-PD-1 immunotherapy. Mechanistically, Mef upregulated the expression of LPCAT3, a key gene involved in lipid peroxidation, by activating IFN-γ-induced STAT1-IRF1 signaling, and knocking down LPCAT3 impaired the induction of ferroptosis by Mef+IFN-γ. Clinically, analysis of the transcriptome and single-cell sequencing results in patients with melanoma showed that LPCAT3 expression was significantly lower in patients with melanoma than in control individuals, and LPCAT3 expression was positively correlated with the efficacy of anti-PD-1 immunotherapy. CONCLUSIONS: In conclusion, our study demonstrated a novel mechanism by which LPCAT3 is regulated, and demonstrated that Mef is a highly promising new target that can be utilized to enhance the efficacy of anti-PD-1 immunotherapy.

Inhibition of the MALT1-LPCAT3 axis protects cartilage degeneration and osteoarthritis.

The proinflammatory cytokines and arachidonic acid (AA)-derived eicosanoids play a key role in cartilage degeneration in osteoarthritis (OA). The lysophosphatidylcholine acyltransferase 3 (LPCAT3) preferentially incorporates AA into the membranes. Our recent studies showed that MALT1 [mucosa-associated lymphoid tissue lymphoma translocation protein 1]) plays a crucial role in propagating inflammatory signaling triggered by IL-1ß and other inflammatory mediators in endothelial cells. The present study shows that LPCAT3 expression was up-regulated in both human and mice articular cartilage of OA, and correlated with severity of OA. The IL-1ß-induces cell death via upregulation of LPCAT3, MMP3, ADAMTS5, and eicosanoids via MALT1. Gene silencing or pharmacological inhibition of LPCAT3 or MALT1 in chondrocytes and human cartilage explants notably suppressed the IL-1ß-induced cartilage catabolism through inhibition of expression of MMP3, ADAMTS5, and also secretion of cytokines and eicosanoids. Mechanistically, overexpression of MALT1 in chondrocytes significantly upregulated the expression of LPCAT3 along with MMP3 and ADAMTS5 via c-Myc. Inhibition of c-Myc suppressed the IL-1ß-MALT1-dependent upregulation of LPCAT3, MMP3 and ADAMTS5. Consistent with the in vitro data, pharmacological inhibition of MALT1 or gene silencing of LPCAT3 using siRNA-lipid nanoparticles suppressed the synovial articular cartilage erosion, pro-inflammatory cytokines, and eicosanoids such as PGE2, LTB4, and attenuated osteoarthritis induced by the destabilization of the medial meniscus in mice. Overall, our data reveal a previously unrecognized role of the MALT1-LPCAT3 axis in osteoarthritis. Targeting the MALT1-LPCAT3 pathway with MALT1 inhibitors or siRNA-liposomes of LPCAT3 may become an effective strategy to treat OA by suppressing eicosanoids, matrix-degrading enzymes, and proinflammatory cytokines.

LPCAT1 promotes melanoma cell proliferation via Akt signaling.

Melanoma is the most lethal type of skin cancer with an increasing cutaneous cancer­related mortality rate worldwide. Despite therapeutic advances in targeted therapy and immunotherapy, the overall survival of patients with melanoma remains unsatisfactory. Thus, a further understanding of the pathogenesis of melanoma may aid towards the development of therapeutic strategies. Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a key enzyme that converts lysophosphatidylcholine into phosphatidylcholine in lipid remodeling. In the present study, LPCAT1 was found to play a pro­proliferative role in melanoma. Firstly, the expression of LPCAT1 was found to be upregulated in tissues from patients with melanoma compared with that in benign nevi. Subsequently, LPCAT1 knockdown was performed, utilizing short hairpin RNA, which induced melanoma cell cycle arrest at the G1/S transition and promoted cell death. Moreover, LPCAT1 facilitated melanoma cell growth in an Akt­dependent manner. In summary, the results of the present study indicate that targeting LPCAT1 may impede cell proliferation by inhibiting Akt signaling, thus providing a promising therapeutic strategy for melanoma in clinical practice.

Research progress, challenges and perspectives of phospholipids metabolism in the LXR­LPCAT3 signaling pathway and its relation to NAFLD (Review).

Phospholipids (PLs) are principle constituents of biofilms, with their fatty acyl chain composition significantly impacting the biophysical properties of membranes, thereby influencing biological processes. Recent studies have elucidated that fatty acyl chains, under the enzymatic action of lyso­phosphatidyl­choline acyltransferases (LPCATs), expedite incorporation into the sn­2 site of phosphatidyl­choline (PC), profoundly affecting pathophysiology. Accumulating evidence suggests that alterations in LPCAT activity are implicated in various diseases, including non­alcoholic fatty liver disease (NAFLD), hepatitis C, atherosclerosis and cancer. Specifically, LPCAT3 is instrumental in maintaining systemic lipid homeostasis through its roles in hepatic lipogenesis, intestinal lipid absorption and lipoprotein secretion. The liver X receptor (LXR), pivotal in lipid homeostasis, modulates cholesterol, fatty acid (FA) and PL metabolism. LXR's capacity to modify PL composition in response to cellular sterol fluctuations is a vital mechanism for protecting biofilms against lipid stress. Concurrently, LXR activation enhances LPCAT3 expression on cell membranes and elevates polyunsaturated PL levels. This activation can ameliorate saturated free FA effects in vitro or endoplasmic reticulum stress in vivo due to lipid accumulation in hepatic cells. Pharmacological interventions targeting LXR, LPCAT and membrane PL components could offer novel therapeutic directions for NAFLD management. The present review primarily focused on recent advancements in understanding the LPCAT3 signaling pathway's role in lipid metabolism related to NAFLD, aiming to identify new treatment targets for the disease.

[Effect and mechanism of Jiedu Huoxue Decoction in regulating YAP/ACSL4 pathway to inhibit ferroptosis in treatment of acute kidney injury].

Jiedu Huoxue Decoction(JDHX), first recorded in the Correction on Errors in Medical Works by WANG Qing-ren, is an effective formula screened out from ancient formulas by the traditional Chinese medicine(TCM) master ZHANG Qi to treat acute kidney injury(AKI) caused by heat, toxicity, stasis, and stagnation. This paper elucidated the therapeutic effect of JDHX on AKI and probed into the potential mechanism from ferroptosis. Thirty-two male C57BL/6 mice were randomized into four groups(n=8): normal, model, and low-and high-dose JDHX. Since the clinical treatment of AKI depends on supportive or alternative therapies and there is no specific drug, this study did not include a positive drug group. The low dose of JDHX corresponded to half of clinically equivalent dose, while the high dose corresponded to the clinically equivalent dose. Mice were administrated with JDHX by gavage daily for 7 consecutive days, while those in the normal group and the model group were administered with the corresponding volume of distilled water. On day 5 of drug administration, mice in other groups except the normal group were injected intraperitoneally with cisplatin solution at a dose of 20 mg·kg~(-1) to induce AKI, and the normal group was injected with saline. All of the mice were sacrificed 72 h after modeling, blood and kidney samples were collected for subsequent analysis. The levels of serum creatine(Scr) and blood urea nitrogen(BUN) were measured by the commercial kits. The expression level of kidney injury molecule 1(KIM-1) in the serum was measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin(HE) staining, periodic acid-Schiff(PAS) staining, and Prussian blue staining were employed to observe the pathological changes, glycogen deposition, and iron deposition, respectively, in the renal tissue. In addition, the levels of glutathione(GSH), superoxide dismutase(SOD), and catalase(CAT) in the renal tissue were examined by biochemical colorimetry. Western blot was performed to determine the protein levels of acyl-CoA synthetase long chain family member 4(ACSL4), lysophosphatidylcholine acyltransferase 3(LPCAT3), and Yes-associated protein(YAP, a key molecule in the Hippo pathway) in the renal tissue. Immunohistochemistry was then employed to detect the location and expression of YAP in the renal tissue. Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR) was performed to measure the mRNA levels of ACSL4 and glutathione peroxidase 4(GPX4). Compared with the normal group, the model group showed elevated serum levels of Scr, BUN, and KIM-1. In the AKI model group, the tubular epithelial cells underwent atrophy and necrotic detachment, disappearance of brush border, and some tubules became protein tubules or experienced vacuole-like degeneration. In addition, this group presented widening of the interstitium or even edema, increased renal tubule injury score, and obvious glycogen and iron deposition in parts of the renal tissue. Moreover, the model group had lower GSH, SOD, and CAT levels, higher ASCL4 and LPCAT3 levels, and lower GPX4 expression and higher YAP expression than the normal group. Compared with the model group, high dose of JDHX effectively protected renal function, lowered the levels of Scr, BUN and KIM-1, alleviated renal pathological injury, reduced glycogen and iron deposition, and elevated the GSH, SOD, and CAT levels in the renal tissue. Furthermore, JDHX down-regulated the protein levels of ACSL4, LPCAT3, and YAP and up-regulated the level of GPX4, compared with the model group. In conclusion, JDHX can protect mice from cisplatin-induced AKI by inhibiting ferroptosis via regulating the YAP/ACSL4 signaling pathway.

Icariin, astragaloside a and puerarin mixture attenuates cognitive impairment in APP/PS1 mice via inhibition of ferroptosis-lipid peroxidation.

Alzheimer's disease (AD) is a neurodegenerative disease that seriously threatens the quality of life of the elderly. Its pathogenesis has not yet been fully elucidated. Ferroptosis, a cell death caused by excessive accumulation of iron-dependent lipid peroxides, has been implicated in the pathogenesis of AD. Uncontrolled lipid peroxidation is the core process of ferroptosis, and inhibiting lipid peroxidation of ferroptosis may be an important therapeutic target for AD. Based on previous studies, we mixed standards of icariin, astragaloside IV, and puerarin, named the standard mixture YHG, and investigated the effect of YHG on ferroptosis -lipid peroxidation in APP/PS1 mice. DFX, a ferroptosis inhibitor, was used as a control drug. In this study, APP/PS1 mice were used as an AD animal model, and behavioral experiments, iron level detection, Transmission electron microscopy (TEM) observation, lipid peroxidation level detection, antioxidant capacity detection, immunofluorescence, Western blot and real-time qPCR were performed. It was found that YHG could reduce body weight, significantly improve abnormal behaviors and the ultrastructure of hippocampal neurons in APP/PS1 mice. The results of biochemical tests showed that YHG reduced the contents of iron, malondialdehyde (MDA) and lipid peroxide (LPO) in brain tissue and serum, and increased the levels of superoxide dismutase (SOD) and reduced glutathione (GSH). Immunofluorescence, WesternBlot and real-time qPCR results showed that YHG could promote the expression of solute carrier family 7 member 11 (SLC7A11), solute carrier family 3 member 2 (SLC3A2) and glutathione peroxidase 4(GPX4). Inhibited the expression of long-chain acyllipid coenzyme a synthetase 4(ACSL4) and lysophosphatidyltransferase 3 (LPCAT3). This study suggests that the mechanism by which YHG improves cognitive dysfunction in APP/PS1 mice may be related to the inhibition of ferroptosis-lipid peroxidation.

Using multi-scale genomics to associate poorly annotated genes with rare diseases.

BACKGROUND: Next-generation sequencing (NGS) has significantly transformed the landscape of identifying disease-causing genes associated with genetic disorders. However, a substantial portion of sequenced patients remains undiagnosed. This may be attributed not only to the challenges posed by harder-to-detect variants, such as non-coding and structural variations but also to the existence of variants in genes not previously associated with the patient's clinical phenotype. This study introduces EvORanker, an algorithm that integrates unbiased data from 1,028 eukaryotic genomes to link mutated genes to clinical phenotypes. METHODS: EvORanker utilizes clinical data, multi-scale phylogenetic profiling, and other omics data to prioritize disease-associated genes. It was evaluated on solved exomes and simulated genomes, compared with existing methods, and applied to 6260 knockout genes with mouse phenotypes lacking human associations. Additionally, EvORanker was made accessible as a user-friendly web tool. RESULTS: In the analyzed exomic cohort, EvORanker accurately identified the "true" disease gene as the top candidate in 69% of cases and within the top 5 candidates in 95% of cases, consistent with results from the simulated dataset. Notably, EvORanker outperformed existing methods, particularly for poorly annotated genes. In the case of the 6260 knockout genes with mouse phenotypes, EvORanker linked 41% of these genes to observed human disease phenotypes. Furthermore, in two unsolved cases, EvORanker successfully identified DLGAP2 and LPCAT3 as disease candidates for previously uncharacterized genetic syndromes. CONCLUSIONS: We highlight clade-based phylogenetic profiling as a powerful systematic approach for prioritizing potential disease genes. Our study showcases the efficacy of EvORanker in associating poorly annotated genes to disease phenotypes observed in patients. The EvORanker server is freely available at https://ccanavati.shinyapps.io/EvORanker/ .

Overexpression of lysophospholipid acyltransferase, LPLAT10/LPCAT4/LPEAT2, in the mouse liver increases glucose-stimulated insulin secretion.

Postprandial hyperglycemia is an early indicator of impaired glucose tolerance that leads to type 2 diabetes mellitus (T2DM). Alterations in the fatty acid composition of phospholipids have been implicated in diseases such as T2DM and nonalcoholic fatty liver disease. Lysophospholipid acyltransferase 10 (LPLAT10, also called LPCAT4 and LPEAT2) plays a role in remodeling fatty acyl chains of phospholipids; however, its relationship with metabolic diseases has not been fully elucidated. LPLAT10 expression is low in the liver, the main organ that regulates metabolism, under normal conditions. Here, we investigated whether overexpression of LPLAT10 in the liver leads to improved glucose metabolism. For overexpression, we generated an LPLAT10-expressing adenovirus (Ad) vector (Ad-LPLAT10) using an improved Ad vector. Postprandial hyperglycemia was suppressed by the induction of glucose-stimulated insulin secretion in Ad-LPLAT10-treated mice compared with that in control Ad vector-treated mice. Hepatic and serum levels of phosphatidylcholine 40:7, containing C18:1 and C22:6, were increased in Ad-LPLAT10-treated mice. Serum from Ad-LPLAT10-treated mice showed increased glucose-stimulated insulin secretion in mouse insulinoma MIN6 cells. These results indicate that changes in hepatic phosphatidylcholine species due to liver-specific LPLAT10 overexpression affect the pancreas and increase glucose-stimulated insulin secretion. Our findings highlight LPLAT10 as a potential novel therapeutic target for T2DM.

Molecular cloning, tissue expression pattern, responses to different fatty acids and potential functions of lysophosphatidylcholine acyltransferase 1 (LPCAT1) in large yellow croaker (Larimichthys crocea).

In farmed fish, diets rich in palm oil have been observed to promote abnormal lipid build-up in the liver, subsequently leading to physiological harm and disease onset. Emerging research suggests that integrating phospholipids into the feed could serve as a potent countermeasure against hepatic impairments induced by vegetable oil consumption. Phosphatidylcholine is the most abundant type among phospholipids. In the metabolic processes of mammal, lysophosphatidylcholine acyltransferase 1 (LPCAT1), crucial for phosphatidylcholine remodeling, demonstrates a marked affinity towards palmitic acid (PA). Nonetheless, aspects concerning the cloning, tissue-specific distribution, and affinity of the LPCAT1 gene to diverse oil sources have yet to be elucidated in the large yellow croaker (Larimichthys crocea). Within the scope of this study, we successfully isolated and cloned the cDNA of the LPCAT1 gene from the large yellow croaker. Subsequent analysis revealed distinct gene expression patterns of LPCAT1 across ten different tissues of the species. The fully sequenced coding DNA sequence (CDS) of LPCAT1 spans 1503 bp and encodes a sequence of 500 amino acids. Comparative sequence alignment indicates that LPCAT1 shares a 69.75 % amino acid similarity with its counterparts in other species. Although LPCAT1 manifests across various tissues of the large yellow croaker, its predominance is markedly evident in the liver and gills. Furthermore, post exposure of the large yellow croaker's hepatocytes to varied fatty acids, PA has a strong response to LPCAT1. Upon the addition of appropriate lysolecithin to palm oil feed, the mRNA expression of LPCAT1 in the liver cells of the large yellow croaker showed significant variations compared to other subtypes. Concurrently, the mRNA expression of pro-inflammatory genes il-1ß, il-6, il-8, tnf-α and ifn-γ in the liver tissue of the large yellow croaker decreased. Interestingly, they exhibit the same trend of change. In conclusion, we have cloned the LPCAT1 gene on fish successfully and find the augmented gene response of LPCAT1 in hepatocytes under PA treatment first. The results of this study suggest that LPCAT1 may be associated with liver inflammation in fish and offer new insights into mitigating liver diseases in fish caused by palm oil feed.

Di-(2-ethylhexyl) phthalate induces ferroptosis in prepubertal mouse testes via the lipid metabolism pathway.

Di-(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, has been shown to cause reproductive toxicity, but the precise mechanism remains unclear. This study aimed to investigate the possible molecular mechanism of DEHP-induced testicular damage. In vivo study, we administered different doses of DEHP (0, 250, and 500 mg/kg/day) to male C57BL/6 mice from 22 and 35 days after birth. We found that DEHP exposure induced histopathological alterations in prepubertal testes, and testicular lipidomics indicated notable alterations in lipid metabolism and significant enrichment of ferroptosis. Further tests showed that ferrous iron (Fe2+ ) and malondialdehyde (MDA) levels significantly increased after DEHP exposure. Western blotting revealed that DEHP exposure reduced glutathione peroxidase 4 (GPX4) expression, and elevated acyl coenzyme A synthetase long-chain member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) expression. The in vitro results were consistent with the in vivo results. When Leydig cells and Sertoli cells were treated with ferrostatin-1 and monoethylhexyl phthalate (MEHP), MEHP-induced increases in Fe2+ and MDA levels, accumulation of lipid reactive oxygen species, downregulation of GPX4, and upregulation of ACSL4 and LPCAT3 were reversed. Collectively, our findings suggested that aberrant lipid metabolism and ferroptosis may be involved in prepubertal DEHP exposure-induced testicular damage.

Methylation Regulation of LPCAT3 Improves Osteoarthritis by Regulating ACSL4 to Inhibit Chondrocyte Ferroptosis.

With the increasing aging population in China, the incidence rate of knee osteoarthritis is expected to rise annually. Therefore, we conducted a study to investigate the crucial role of LPCAT3 in osteoarthritis and its underlying mechanisms. We collected samples from normal volunteers (n = 12) and patients with osteoarthritis (n = 12) at our hospital. It was observed that LPCAT3 mRNA expression was reduced and positively correlated with IL-1ß mRNA expression in patients with osteoarthritis. In a mouse model, LPCAT3 mRNA and protein expression were found to be suppressed. Furthermore, in an in vitro model, the enrichment level of LPCAT3 mRNA was inhibited by a specific m6A antibody through si-METTL3. Si-METTL3 also reduced the stability of LPCAT3 mRNA in the in vitro model. The inhibition of LPCAT3 was found to exacerbate osteoarthritis in the mouse model. Additionally, LPCAT3 was shown to reduce inflammation in the in vitro model. It was also observed that LPCAT3 reduced chondrocyte ferroptosis by inhibiting mitochondrial damage. LPCAT3 protein was found to interact with ACSL4 protein, and its up-regulation suppressed ACSL4 expression in the in vitro model. ACSL4 was identified as a target of LPCAT3 for suppressing mitochondrial damage in the in vitro model. In conclusion, this study demonstrates that LPCAT3 improves osteoarthritis by regulating ACSL4 to inhibit chondrocyte ferroptosis, thus providing a novel target for the treatment of osteoarthritis.

LPCAT3/LPLAT12 deficiency in the liver ameliorates acetaminophen-induced acute liver injury.

Acetaminophen (APAP) is a double-edged sword, mainly depending on the dosage. A moderate dose of APAP is effective for fever and pain relief; however, an overdose induces acute liver injury. The mechanism underlying APAP-induced acute liver failure is unclear, and its treatment is limited. A recent report has shown that several oxidized phospholipids are associated with APAP-induced acute liver failure. Lysophosphatidylcholine acyltransferase 3 (Lpcat3, Lplat12), which is highly expressed in the liver, preferentially catalyzes the incorporation of arachidonate into lysophospholipids (PLs). In the present study, we investigated the roles of Lpcat3 on APAP-induced acute liver injury using liver-specific Lpcat3-knockout mice. Hepatic Lpcat3 deficiency reduced the degree of APAP-induced necrosis of hepatocytes around Zone 3 and ameliorated the elevation of hepatic injury serum marker levels, and prolonged survival. Lipidomic analysis showed that the accumulation of oxidized and hydroperoxidized phospholipids was suppressed in Lpcat3-knockout mice. The amelioration of APAP-induced acute liver injury was due not only to the reduction in the lipid synthesis of arachidonic acid PLs because of Lpcat3 deficiency, but also to the promotion of the APAP detoxification pathway by facilitating the conjugation of glutathione and N-acetyl-p-benzoquinone imine. Our findings suggest that Lpcat3 is a potential therapeutic target for treating APAP-induced acute liver injury.

Inhibition of the skeletal muscle Lands cycle ameliorates weakness induced by physical inactivity.

BACKGROUND: Lipid hydroperoxides (LOOH) have been implicated in skeletal muscle atrophy with age and disuse. Lysophosphatidylcholine acyltransferase 3 (LPCAT3), an enzyme of the Lands cycle, conjugates a polyunsaturated fatty acyl chain to a lysophospholipid to form a polyunsaturated fatty acid containing phospholipid (PUFA-PL) molecule, providing substrates for LOOH propagation. Previous studies suggest that inhibition of the Lands cycle is an effective strategy to suppress LOOH. Mice with skeletal muscle-specific tamoxifen-inducible knockout of LPCAT3 (LPCAT3-MKO) were utilized to determine if muscle-specific attenuation of LOOH may alleviate muscle atrophy and weakness with disuse. METHODS: LPCAT3-MKO and control mice underwent 7 days of sham or hindlimb unloading (HU model) to study muscle mass and force-generating capacity. LOOH was assessed by quantifying 4-hydroxynonenal (4-HNE)-conjugated peptides. Quantitative PCR and lipid mass spectrometry were used to validate LPCAT3 deletion. RESULTS: Seven days of HU was sufficient to induce muscle atrophy and weakness concomitant to a ~2-fold increase in 4-HNE (P = 0.0069). Deletion of LPCAT3 reversed HU-induced increase in muscle 4-HNE (P = 0.0256). No difference was found in body mass, body composition, or caloric intake between genotypes. The soleus (SOL) and plantaris (PLANT) muscles of the LPCAT3-MKO mice experienced ~15% and ~40% less atrophy than controls, respectively. (P = 0.0011 and P = 0.0265). Type I and IIa SOL myofibers experienced a ~40% decrease in cross sectional area (CSA), which was attenuated to only 15% in the LPCAT3-MKO mice (P = 0.0170 and P = 0.0411, respectively). Strikingly, SOL muscles were fully protected and extensor digitorum longus (EDL) muscles experienced a ~35% protection from HU-induced reduction in force-generating capacity in the LPCAT3-MKO mice compared with controls (P < 0.0001 for both muscles). CONCLUSIONS: Our findings demonstrate that attenuation of skeletal muscle lipid hydroperoxides is sufficient to restore its function, in particular a protection from reduction in muscle specific force. Our findings suggest muscle lipid peroxidation contributes to atrophy and weakness induced by disuse in mice.

Targeting phospholipid remodeling pathway improves insulin resistance in diabetic mouse models.

Previous studies have revealed that membrane phospholipid composition controlled by lysophosphatidylcholine acyltransferase 3 (LPCAT3) is involved in the development of insulin resistance in type 2 diabetes. In this study, we aimed to investigate the therapeutic potential of targeting Lpcat3 in the treatment of insulin resistance in diabetic mouse models. Lpcat3 expression was suppressed in the whole body by antisense oligonucleotides (ASO) injection or in the liver by adeno-associated virus (AAV)-encoded Cre in high-fat diet (HFD)-induced and genetic ob/ob type 2 diabetic mouse models. Glucose tolerance test (GTT), insulin tolerance test (ITT), fasting blood glucose, and insulin levels were used to assess insulin sensitivity. Lipid levels in the liver and serum were measured. The expression of genes involved in de novo lipogenesis was analyzed by real-time RT-PCR. Metabolic rates were measured by indirect calorimetry using the Comprehensive Lab Animal Monitoring System (CLAMS). Our data demonstrate that acute knockout of hepatic Lpcat3 by AAV-Cre improves both hyperglycemia and hypertriglyceridemia in HFD-fed mice. Similarly, whole-body ablation of Lpcat3 by ASO administration improves obesity and insulin resistance in both HFD-fed and ob/ob mice. These findings demonstrate that targeting LPCAT3 could be a novel therapy for insulin resistance.

Hyperbaric oxygen improves cerebral ischemia-reperfusion injury in rats via inhibition of ferroptosis.

BACKGROUND: Our previous study found that hyperbaric oxygen (HBO) attenuated cognitive impairment in mice induced by cerebral ischemia-reperfusion injury (CIRI). However, its mechanism of action is not fully understood. In this study, we aimed to establish a rat model of cerebral ischemia-reperfusion, explore the possible role of ferroptosis in the pathogenesis of CIRI, and observe the effect of HBO on ferroptosis-mediated CIRI. METHODS: Sprague Dawley (SD) rats were randomly divided into control, model, Ferrostatin-1 (Fer-1), HBO and Fer-1+ HBO groups. Morris water maze, myelin basic protein (MBP) and ß-tubulin immunoreactivity were assessed to evaluate the neuroprotective effects of HBO on cerebral ischemia reperfusion injury. Ferroptosis were examined to investigate the mechanism underlying the effects of HBO. RESULTS: Our result showed that Fer-1 and HBO improved learning and memory ability in the navigation trail and probe trail of the Morris water maze and increased MBP and ß-tubulin immunoreactivity of the cortex in the model rats. The levels of ferritin, malondialdehyde (MDA) and glutathione (GSH) in the serum were also reversed by Fer-1 and HBO treatment. Mitochondrial cristae dissolution and vacuolization were observed in the model group by transmission electron microscopy and these conditions were improved in the Fer-1 and HBO groups. Furthermore, Fer-1 and HBO treatment reversed Prostaglandin-Endoperoxide Synthase 2 (PTGS2), Iron Responsive Element Binding Protein 2 (IREB2), acyl-CoA synthetase long chain family member 4 (ACSL4) and Solute Carrier Family 7 Member 11 (SLC7A11) mRNA levels and Transferrin Receptor 1 (TFR1), ferritin light chain (FTL), ferritin heavy chain 1 (FTH1), glutathione peroxidase 4 (GPX4), Nuclear factor E2-related factor 2 (Nrf2), lysophosphatidylcholine acyltransferase 3 (LPCAT3), c-Jun N-terminal kinase (JNK), phosphorylated c-Jun N-terminal kinase (P-JNK) phosphorylated Extracellular signal-regulated protein kinase (P-ERK) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) protein levels. The above changes were more pronounced in Fer-1+ HBOGroup. DISCUSSION: The results of the present study indicated that HBO improves cerebral ischemia-reperfusion injury in rats, which may be related to inhibition of ferroptosis. This also means that ferroptosis may become a new target of HBO against CIRI.

Suberosin Alleviates Thiazolidinedione-Induced Cardiomyopathy in Diabetic Rats by Inhibiting Ferroptosis via Modulation of ACSL4-LPCAT3 and PI3K-AKT Signaling Pathways.

Thiazolidinediones are useful antidiabetic medications. However, their use is associated with adverse side effects like edema, heart failure and bone fractures. In this study, we investigated the anti-ferroptosis effects of suberosin (SBR; a prenylated coumarin) in diabetic Sprague Dawley rats. Further, we assessed the effects of co-administration of SBR (30 and 90 mg/kg/day) with thiazolidinedione (TZ at 15 mg/kg) to mitigate TZ-induced cardiomyopathy in diabetic rats. Our results showed that cardiac output, stroke volume, left ventricle systolic and diastolic pressures were aggravated in diabetic rats treated with TZ alone after 4 weeks. TZ treatments induced ferroptosis as well as marked histoarchitecture disarrangements in rat cardiomyocytes. The study found that optimizing volume overload alleviated cardiac hypertrophy and mitigated left ventricular dysfunction in diabetic rats co-treated with SBR. SBR co-administration with TZ reduced MDA levels in heart tissue and serum iron concentration (biomarkers of ferroptosis), downregulated mRNA expressions of LOX, ACSL4, LPCAT3, and promoted GPX4 activity as well as upregulated mRNA levels of AKT/PI3K/GSK3ß as compared to the group administered with TZ at 15 mg/kg. SBR co-administration also helped to retain the normal histoarchitecture of cardiomyocytes in diabetic rats. Hence, our results suggested that SBR is an effective supplement and could be prescribed to diabetic patients along with TZ but this requires further clinical trials.

Ferroptosis-related genes LPCAT3 and PGD are potential diagnostic biomarkers for osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is the most common chronic joint disease and how ferroptosis contributes to OA has garnered much attention recently. Bioinformatics promoted the discovery of ferroptosis-related biomarkers for OA. But since OA is a whole-joint disease, sensitive biomarkers for OA are still limited. We herein focused on subchondral bone, a joint component often-ignored by existing bioinformatic reports, to identify ferroptosis-related diagnostic biomarkers for OA. METHOD: Microarray datasets GSE51588 and GSE55457 were downloaded from Gene Expression Omnibus database. Ferroptosis-related differential expression genes (Ferr-DEGs) between OA and normal samples were identified and their functional enrichment was analyzed. Common genes for OA diagnosis were selected from Ferr-DEGs using the combination of SVM-RFE, LASSO regression, and RandomForest machine learning algorithms. Common genes' diagnostic value was verified by receiver operating characteristic (ROC) curve and their association with immune infiltration was analyzed by CIBERSORT. Finally, candidate gene's expression was verified in chondrocytes from OA patients and in an in vitro IL-1ß-induced OA model, by RT-PCR. RESULTS: Two ferroptosis-related genes, LPCAT3 and PGD, were identified as OA diagnostic biomarkers and confirmed by ROC diagnostic test. The association of LPCAT3 and PGD with the infiltration of several types of immune cells was identified. The decreased expression of LPCAT3 and PGD was both confirmed in OA chondrocytes and IL-1ß-induced OA condition. CONCLUSIONS: We identified ferroptosis-related genes LPCAT3 and PGD as potential diagnostic biomarkers for OA, which may offer insight into the role of ferroptosis in OA and provides useful information for the diagnosis and treatment of OA.

Lysophospholipid acyltransferases orchestrate the compositional diversity of phospholipids.

Lysophospholipid acyltransferases (LPLATs), in concert with glycerol-3-phosphate acyltransferases (GPATs) and phospholipase A1/2s, orchestrate the compositional diversity of the fatty chains in membrane phospholipids. Fourteen LPLAT enzymes which come from two distinct families, AGPAT and MBOAT, have been identified, and in this mini-review we provide an overview of their roles in de novo and remodeling pathways of membrane phospholipid biosynthesis. Recently new nomenclature for LPLATs has been introduced (LPLATx, where x is a number 1-14), and we also give an overview of key biological functions that have been discovered for LPLAT1-14, revealed primarily through studies of LPLAT-gene-deficient mice as well as by linkages to various human diseases.

Inhibiting Phosphatidylcholine Remodeling in Adipose Tissue Increases Insulin Sensitivity.

Cell membrane phosphatidylcholine (PC) composition is regulated by lysophosphatidylcholine acyltransferase (LPCAT); changes in membrane PC saturation are implicated in metabolic disorders. Here, we identified LPCAT3 as the major isoform of LPCAT in adipose tissue and created adipocyte-specific Lpcat3-knockout mice to study adipose tissue lipid metabolism. Transcriptome sequencing and plasma adipokine profiling were used to investigate how LPCAT3 regulates adipose tissue insulin signaling. LPCAT3 deficiency reduced polyunsaturated PCs in adipocyte plasma membranes, increasing insulin sensitivity. LPCAT3 deficiency influenced membrane lipid rafts, which activated insulin receptors and AKT in adipose tissue, and attenuated diet-induced insulin resistance. Conversely, higher LPCAT3 activity in adipose tissue from ob/ob, db/db, and high-fat diet-fed mice reduced insulin signaling. Adding polyunsaturated PCs to mature human or mouse adipocytes in vitro worsened insulin signaling. We suggest that targeting LPCAT3 in adipose tissue to manipulate membrane phospholipid saturation is a new strategy to treat insulin resistance.