Dysregulation of mitochondrial dynamics modulates malignant cell fate; however, the substantial heterogeneity in mitochondrial dynamics among tumor cells within individual tumor nodules and the resultant functional consequences remain inadequately characterized. In this study, we induced mosaic impairment of mitochondrial fusion in mouse liver under tumorigenic conditions and unexpectedly identified the formation of combined hepatocellular-cholangiocarcinoma (cHC), a monoclonal tumor displaying features of both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). Restoration of the mitochondrial fusion protein MFN1 effectively suppressed cHC development. Analysis of human cHC samples revealed that ICC-like cells exhibit more pronounced mitochondrial fusion impairment compared to HCC-like cells. Mechanistically, increasing impairment of mitochondrial fusion resulted in a dose-dependent elevation of reactive oxygen species (ROS). Low levels of ROS upregulated HNF4α, promoting HCC-like differentiation, whereas high ROS levels activated HES1, facilitating ICC-like differentiation. Collectively, these results demonstrate that heterogeneity in mitochondrial dynamics is a critical determinant of cHC path-ogenesis.

Mitochondria-endoplasmic reticulum contacts (MERCs) play important roles in mitochondrial calcium homeostasis and apoptosis. Although MERC dysregulation has been implicated in the progression of various liver diseases, its role in hepatocellular carcinoma (HCC) remains poorly understood. Here, we report that mitochondria-localized Rab32 is required for maintaining MERC integrity in HCC cells. Mechanistically, Rab32 promotes the mitochondrial localization of protein kinase A (PKA), which facilitates protein tyrosine phosphatase-interacting protein 51 (PTPIP51) phosphorylation and maintains MERC integrity. Rab32 deficiency disrupts MERCs, reduces mitochondrial Ca²⁺ levels, and increases mitochondrial ROS accumulation. MERC restoration with a synthetic mitochondria-ER linker increased apoptosis and inhibited the proliferation of HCC cells. Together, these findings identify mitochondrial Rab32 as an important regulator of MERC integrity and apoptosis in HCC cells, suggesting that MERC stabilization may have potential for HCC treatment.

Intracerebral hemorrhage (ICH) often has a poor prognosis, necessitating the exploration of effective therapeutic targets. Stromal interaction molecule 1 (STIM1) is a crucial regulator of cellular calcium homeostasis, but its specific role in ICH remains unclear. This study finds consistent elevation of STIM1 in neurons after ICH, with increased plasma levels in patients correlating with poor prognosis. Neuronal knockout of STIM1 in mice improves brain tissue damage and neurological injury. Mechanistically, STIM1 exacerbates neuronal injury primarily by promoting ferroptosis. Importantly, in addition to regulating calcium signaling pathways, STIM1 directly regulates iron homeostasis through its interaction with transferrin receptor 1 (TFR1) to promote ferroptosis. Finally, through virtual screening, S-IN-1 is identified as an inhibitor targeting STIM1-TFR1 interaction, protecting against neuronal ferroptosis and brain injury. These findings confirm the molecular function of STIM1 in regulating iron homeostasis, providing valuable insights and promising targets for ICH treatment.

To prevent cell death induced by elevated oxidative stress, cancer cells activate a series of antioxidant defense mechanisms to mitigate cytotoxicity, thereby enhancing the resistance to pro-oxidative therapy. However, the underlying antioxidant mechanisms in cancer cells remain inadequately understood. Through co-immunoprecipitation followed by quantitative mass spectrometry analysis, we for the first time identified that cytoplasmic ALDH1L1 translocates into mitochondria and co-localizes with mitochondrial transcription factor TFAM in cancer cells in a ROS-dependent feedback manner. Mitochondria-translocated ALDH1L1 maintains mitochondrial redox homeostasis by producing NADPH. Moreover, our findings revealed that the ROS-mediated oxidative modification of ALDH1L1 is necessary for its interaction with HSP90β and subsequent translocation into mitochondria via TOM70, where it binds to TFAM to prevent degradation by LONP1. Furthermore, we found that mitochondrial ALDH1L1 antagonized the double-edged role of ROS in cancer cell survival, indicating that disruption of ALDH1L1 expression promoted cancer cell proliferation and autophagy but concurrently diminished cellular capacity to counteract ROS-induced apoptosis. Consistently, ALDH1L1 knockout enhanced the anti-tumor effect of low-dose pro-oxidant Elesclomol, thereby achieving better efficacy and safety of pro-oxidant therapy. Furthermore, our results demonstrated that the combination of Elesclomol with HSP90 inhibitor Ganetespib exhibited synergistic anti-tumor effects. In conclusion, our findings that mitochondria-translocated ALDH1L1 functions as a feedback regulator of redox homeostasis in cancer cells to enhance the resistance to pro-oxidative therapy can provide critical insights into developing effective pro-oxidative therapies against tumors.

Resistance to tyrosine kinase inhibitors (TKIs) poses a significant challenge in the treatment of hepatocellular carcinoma (HCC). Although dysregulation of mitochondrial dynamics has been implicated in the aggressive behaviors of various tumors, the specific role and underlying mechanisms by which this dysregulation contributes to cabozantinib resistance in HCC cells remains insufficiently characterized. By investigating mitochondrial dynamics as central regulators of cabozantinib resistance, this work specifically aims to discover actionable targets for restoring drug sensitivity in treatment-refractory HCC cells. We employed transmission electron microscopy (TEM) and confocal microscopy to analyze mitochondrial morphology in HCC cells resistant to TKIs. Additionally, we utilized an oncogene hydrodynamic injection-induced primary liver cancer mouse model to assess the therapeutic efficacy of combining cabozantinib with other pharmacological agents. Our results demonstrated significant increases in mitochondrial fragmentation, p62 aggregation, and mitophagy in cabozantinib-resistant HCC cells, which correlated with overexpression of c-Myc. Notably, inhibiting mitochondrial fission, p62 aggregation, or autophagy effectively reversed the resistance of HCC cells to cabozantinib. Mechanistically, cabozantinib treatment was shown to induce c-Myc expression, which significantly enhanced mitochondrial fragmentation and p62 aggregation, thereby promoting mitophagy. This mitophagic process selectively eliminated damaged mitochondria, reducing cytochrome C-induced apoptosis in cabozantinib-resistant cells. Ultimately, combining cabozantinib with either the autophagy inhibitor chloroquine or the p62 aggregation inhibitor XRK3F2 resulted in improved anticancer efficacy. In conclusion, c-Myc overexpression facilitates p62 aggregation-mediated mitophagy, leading to cabozantinib resistance in HCC cells. Inhibition of autophagy effectively restores cabozantinib sensitivity in HCC.

Hepatocellular carcinoma (HCC) ranks third in global cancer-related mortality, with limited therapies for advanced stages. Retinol, the alcohol form of vitamin A, has long been associated with liver diseases. Plasma retinol levels have been inversely correlated with the risk and poor prognosis of HCC. In this study, transcriptome data analysis identified retinol metabolism as the seventh KEGG-dysregulated pathway in cirrhosis tissue, ascending to the top position in HCC tissue compared to normal tissue. Specifically, a consistent downregulation of ADH4 (alcohol dehydrogenase 4), the retinol dehydrogenase among human ADHs, was observed, which correlated with poor prognosis in HCC patients. In vivo experiments demonstrated that silencing ADH4 enhances liver fibrosis and the progression of HCC. Mechanistically, ADH4 elevated intracellular levels of RA (retinoic acid), a biologically active derivative of retinol. RA-activated retinoid receptors RARs/RXRs, leading to inhibition of the downstream Wnt/beta-catenin pathway and thereby hindering HCC progression. In contrast, the knockdown of ADH4 in hepatocytes triggers apoptosis. Notably, additional results demonstrated that the combined treatment of RA and cisplatin achieved synergistic antitumor effects in a mouse HCC model. In summary, our research elucidates that ADH4-mediated RA production suppresses HCC growth, providing a theoretical foundation for HCC treatment.

目的 观察溶酶体相关细胞器生物发生复合体-3(biogenesis of lysosome-related organelles complex-3,BLOC-3)亚基Hps1和Hps4对肝癌细胞中Rab32线粒体定位的影响及在肝癌细胞生长中的作用.方法 通过公共数据库GenDoma,String和InBio Discover分析Hps1和Hps4与Rab32的相互作用情况.体外培养人肝癌细胞系SNU-739和Hep-3B,利用脂质体转染方法分别转染Hps1和Hps4相关的siRNAs和质粒,采用免疫荧光和Western blot观察Hps1和Hps4对Rab32线粒体定位的影响,细胞划痕、克隆形成、EdU、MTS及Transwell侵袭实验检测Hps1和Hps4调控Rab32线粒体定位后肝癌细胞迁移、增殖和侵袭的变化情况.通过公共数据库UALCAN中的数据集CPTAC分析Rab32蛋白在肝癌和正常肝脏组织中的表达差异.结果 数据库分析结果显示,Hps1和Hps4均可以与Rab32相互作用;与正常肝脏组织相比,Rab32蛋白在肝癌组织中的表达显著降低(P<0.001).在肝癌细胞系SNU-739中干涉Hps1或Hps4或同时干涉Hps1和Hps4后,Rab32线粒体定位均减少(均P<0.01),细胞线粒体Rab32蛋白表达均降低(均P<0.001),细胞增殖、迁移和侵袭能力增强(均P<0.05);在肝癌细胞系Hep-3B中过表达Hps1和Hps4后,Rab32线粒体定位增多(P<0.01),细胞线粒体Rab32蛋白表达增加(P<0.001),细胞增殖、迁移和侵袭能力均受抑制(均P<0.01),而单独过表达Hps1或Hps4时无显著抑制作用(均P>0.05).结论 BLOC-3亚基Hps1和Hps4均可与Rab32相互作用并增加Rab32线粒体定位,进而抑制肝癌细胞生长.

Mycoplasma is widespread in various hosts and may cause various diseases in animals. Interestingly, the occurrence of mycoplasma infection was observed in many tumor types. However, the mechanism regulating its infection is far from clear. We unexpectedly found that the knockdown of mitochondrial transcription factor A (TFAM) remarkably enhanced mycoplasma infection in hepatocellular carcinoma (HCC) cells. More importantly, we found that mycoplasma infection facilitated by TFAM knockdown significantly promoted HCC cell metastasis. Mycoplasma infection was further found to be positively correlated with poor prognosis in patients with HCC. Mechanistically, the decreased TFAM expression upregulated the transcription factor Sp1 to increase the expression level of Annexin A2 (ANXA2), which was reported to interact with membrane protein of mycoplasma. Moreover, we found that mycoplasma infection enhanced by the TFAM downregulation promoted HCC migration and invasion by activating the nuclear factor-κB signaling pathway. The downregulation of TFAM enhanced mycoplasma infection in HCC cells and promoted HCC cell metastasis. Our study contributes to the understanding of the pathological role of mycoplasma infection and provides supporting evidence that targeting TFAM could be a potential strategy for the treatment of HCC with mycoplasma infection.

线粒体是细胞进行有氧呼吸的主要场所,被誉为细胞的动力工厂.除了供能,线粒体还可作为信号转导中心在调控细胞生理功能及疾病发生等过程发挥重要作用.例如,在线粒体受损情况下,其会将损伤信号传递到细胞核,从而激活适应性的转录以调节细胞的代谢状态.这些由线粒体向细胞核传递的信号被称为"线粒体-细胞核逆行信号".最近的研究揭示,通过改变线粒体代谢物的水平或应激信号,线粒体向细胞核的逆行信号可引起各种表观遗传变化,并与肿瘤的发生、恶性进展和治疗等过程密切相关.鉴于此,本文总结了近期关于线粒体-细胞核逆行信号如何调控表观基因组及调控肿瘤的研究进展,以期为建立肿瘤预防和治疗新策略提供新视角.

Next-generation sequencing (NGS) of mitochondrial DNA (mtDNA) has widespread applications in aging and cancer studies. However, cross-contamination of mtDNA constitutes a major concern. Previous methods for the detection of mtDNA contamination mainly focus on haplogroup-level phylogeny, but neglect haplotype-level differences, leading to limited sensitivity and accuracy. In our study, we present mitoDataclean, a random-forest-based machine learning package for accurate identification of cross-contamination, evaluation of contamination levels and detection of contamination-derived variants in mtDNA NGS data. Comprehensive optimization of mitoDataclean revealed that training simulation with mixtures of small haplogroup distance and low polymorphic difference was critical for optimal modeling. Compared to existing methods, mitoDataclean exhibited significantly improved sensitivity and accuracy for the detection of sample contamination in simulated data. In addition, mitoDataclean achieved area under the curve values of 0.91 and 0.97 for discerning genuine and contamination-derived mtDNA variants in a simulated Western dataset and private sequencing contamination data, respectively, suggesting that this tool may be applicable for different populations and samples with different sources of contamination. Finally, mitoDataclean was further evaluated in several private and public datasets and showed a robust ability for contamination detection. Altogether, our study demonstrates that mitoDataclean may be used for accurate detection of contaminated samples and contamination-derived variants in mtDNA NGS data.

Adv. Sci. 2021, 8, 2002794 DOI: 10.1002/advs.202002794 In the original published article, the previous forward and reverse primers of Cidea in Table S1 (Supporting Information) were reversed. Now these two primers of Cidea have been exchanged. Please find the correct Table S1 below. The authors apologize for any inconvenience caused.

目的 探讨血清磷脂酰肌醇蛋白聚糖3(glypican?3,GPC3)蛋白和外周血GPC3 mRNA及其联合检测在肝细胞癌(hepatocellular carcinoma,HCC)中的诊断价值.方法 采用免疫组化法检测1例肝癌组织标本中GPC3的表达情况.检索PubMed、Web of Science、Embase和Cochrane library数据库中关于GPC3诊断HCC的文献,根据纳入和排除标准筛选文献,对纳入文献进行数据提取和质量评价,采用MetaDisc 1.4软件进行Meta分析,计算合并的诊断评估指标,绘制综合受试者工作特征(summary receiver operating characteristic,SROC)曲线评估其诊断效能.结果 免疫组化结果显示肝癌组织中特异性表达GPC3.进一步纳入19篇文献(其中血清GPC3蛋白16篇,外周血GPC3 mRNA 5篇)评价外周血中GPC3分子对HCC的诊断效能,结果显示,单独血清GPC3蛋白诊断HCC的合并敏感度、特异度、SROC曲线下面积分别为0.62(95％CI:0.60～0.64)、0.67(95％CI:0.65～0.69)、0.75;外周血GPC3 mRNA诊断HCC的合并敏感度、特异度、SROC曲线下面积分别为0.76(95％CI:0.70～0.81)、0.87(95％CI:0.84～0.91)、0.92.血清GPC3蛋白联合甲胎蛋白(α?fetoprotein,AFP)诊断HCC的敏感度、特异度分别为0.82(95％CI:0.80～0.85)、0.81(95％CI:0.79～0.84),联合异常凝血酶原(des?γ?carboxy prothrombin,DCP)诊断的敏感度和特异度分别为0.74(95％CI:0.68～0.79)、0.76(95％CI:0.70～0.81).结论 GPC3分子特异性表达于肝癌组织,相对于血清GPC3蛋白,外周血GPC3 mRNA诊断HCC可能具有更高的效能;单独GPC3蛋白的诊断效能有限,联合AFP或DCP可提高其诊断效能,有助于HCC早期诊断.

Hepatoblastoma is the most common liver cancer in children, and the aggressive subtype often has a poor prognosis and lacks effective targeted therapy. Although aggressive hepatoblastoma (HB) is often accompanied by abnormally high expression of the transcription factor c-Myc, the underlying mechanism remains unclear. In this study, we found that mitochondrial fragmentation was enhanced by c-Myc overexpression in human aggressive HB tissues and was associated with poor prognosis. Then, a mouse model resembling human HB was established via hydrodynamic injection of c-Myc plasmids. We observed that liverspecific knockout of the mitochondrial fusion molecule MFNI or overexpression of mitochondrial fission molecule DRP1 promoted the occurrence of c-Myc-driven liver cancer. In contrast, when MFNI was overexpressed in the liver, tumor formation was delayed. In vitro experiments showed that c-Myc transcriptionally upregulated the expression of DRP1 and decreased MFNI expression through upregulation of miR-373-3p. Moreover, enhanced mitochondrial fragmentation significantly promoted aerobic glycolysis and the proliferation of HB cells by significantly increasing reactive oxygen species (ROS) production and activating the RAC-alpha serine/threonine-protein kinase (AKT)/mammalian target of rapamycin (mTOR) and nuclear factor kappa B (NF-kappa B) pathways. Taken together, our results indicate that c-Myc-mediated mitochondrial fragmentation promotes the malignant transformation and progression of HB by activating ROS-mediated multi-oncogenic signaling.

BackgroundMitochondrial DNA (mtDNA) mutations alter mitochondrial function in oxidative metabolism and play an important role in tumorigenesis. A series of studies have demonstrated that the mtDNA control region (mtCTR), which is essential for mtDNA replication and transcription, represents a mutational hotspot in human tumors. However, a comprehensive pan-cancer evolutionary pattern analysis of mtCTR mutations is urgently needed.MethodsWe generated a comprehensive combined dataset containing 10026 mtDNA somatic mutations from 4664 patients, covering 20 tumor types based on public and private next-generation sequencing data.FindingsOur results demonstrated a significantly higher and much more variable mutation rate in mtCTR than in the coding region across different tumor types. Moreover, our data showed a remarkable distributional bias of tumor somatic mutations between the hypervariable segment (HVS) and non-HVS, with a significantly higher mutation density and average mutation sites in HVS. Importantly, the tumor-specific mutational pattern between mtCTR HVS and non-HVS was identified, which was classified into three evolutionary selection types (relaxed, moderate, and strict constraint types). Analysis of substitution patterns revealed that the prevalence of CH > TH in non-HVS greatly contributed to the mutational selection pattern of mtCTR across different tumor types. Furthermore, we found that the mutational pattern of mtCTR in the four tumor types was clearly associated with mitochondrial biogenesis, mitochondrial oxidative metabolism, and the overall survival of patients.InterpretationOur results suggest that somatic mutations in mtCTR may be shaped by tumor-specific selective pressure and are involved in tumorigenesis.FundingsNational Natural Science Foundation of China [grants 82020108023, 81830070, 81872302], and Autonomous Project of State Key Laboratory of Cancer Biology, China [grants CBSKL2019ZZ06, CBSKL2019ZZ27].

Objective To establish a prognosis model using immune-related genes in hepatocellular carcinoma (HCC) patients, which could provide a theoretical foundation for HCC immunotherapy. Methods Immune-related genes were identified by differential expression analysis, and risk prognosis prediction models were established using univariate, multivariate Cox and Least absolute shrinkage and selection operator (LASSO) regression analysis. The predictive value of the prognostic model was evaluated using the concordance index (C-index), receiver operating characteristic curve (ROC curve), and calibration curve and decision curve. In addition, risk score was used to stratify patients to assess prognostic differences in patients at different risk levels. Results We identified 1403 immune-related genes, mainly involved in biological processes such as immune response, adaptive immune response, and immunoglobulin production, as well as pathways such as cytokine interactions, chemokine signaling pathways and allograft rejection. Univariate Cox analysis found that 53 immune-related genes were associated with prognosis, and eight prognostic immune-related genes cytochrome P450 1A2(CYP1A2), ficolin 3(FCN3), hepatoma derived growth factor-like 1(HDGFL1), lipocalin 2(LCN2), mitochondrially encoded cytochrome C oxidase II pseudogene 12(MTCO2P12), peptidyl arginine deiminase 3(PADI3) and regulator of G protein signaling 16(RGS16) were further screened by LASSO and multivariate Cox regression analysis. Subsequently, a prognostic nomogram based on risk score was established. The ROC curve, calibration curve and decision curve confirmed that the model has good discrimination, accuracy and clinical value. Furthermore, stratified analysis showed that patients with higher risk scores had poorer prognosis. Conclusion We establish a prognostic nomogram model using eight immune-related genes, which can reliably predict the prognosis of HCC patients.

BACKGROUND:Many studies have demonstrated the high efficacy of cell-free nuclear DNA in cancer diagnostics. Compared to nuclear DNA, mitochondrial DNA (mtDNA) exhibits distinct characteristics, including multiple copies per cell and higher mutation frequency. However, the potential applicability of cell-free mtDNA (cf-mtDNA) in plasma and urine remains poorly investigated.METHODS:Here, we comprehensively analyzed the fragmentomic and mutational characteristics of cf-mtDNA in urine and plasma samples from controls and cancer patients using next-generation sequencing.RESULTS:Compared to plasma cf-mtDNA, urine cf-mtDNA exhibited increased copy numbers and wider spread in fragment size distributions. Based on 2 independent animal models, urine cf-mtDNA originated predominantly from local shedding and transrenal excretion. Further analysis indicated an enhanced fragmentation of urine cf-mtDNA in renal cell carcinoma (RCC) and colorectal cancer (CRC) patients. Using the mtDNA sequence of peripheral blood mononuclear cells for reference, the mutant fragments were shorter than wild-type fragments in urine cf-mtDNA. Size selection of short urine cf-mtDNA fragments (<150 bp) significantly enhanced the somatic mutation detection. Our data revealed remarkably different base proportions of fragment ends between urine and plasma cf-mtDNA that also were associated with fragment size. Moreover, both RCC and CRC patients exhibited significantly higher T-end and lower A-end proportions in urine cf-mtDNA than controls. By integrating the fragmentomic and mutational features of urine cf-mtDNA, our nomogram model exhibited a robust efficacy for cancer diagnosis.CONCLUSIONS:Our proof-of-concept findings revealed aberrant fragmentation and mutation profiles of urine cf-mtDNA in cancer patients that have diagnostic potential.

The mechanisms underlying cancer metastasis remain poorly understood. Here, we report that TFAM deficiency rapidly and stably induced spontaneous lung metastasis in mice with liver cancer. Interestingly, unexpected polymerization of nuclear actin was observed in TFAM-knockdown HCC cells when cytoskeleton was examined. Polymerization of nuclear actin is causally linked to the high-metastatic ability of HCC cells by modulating chromatin accessibility and coordinating the expression of genes associated with extracellular matrix remodeling, angiogenesis, and cell migration. Mechanistically, TFAM deficiency blocked the TCA cycle and increased the intracellular malonyl-CoA levels. Malonylation of mDia2, which drives actin assembly, promotes its nuclear translocation. Importantly, inhibition of malonyl-CoA production or nuclear actin polymerization significantly impeded the spread of HCC cells in mice. Moreover, TFAM was significantly downregulated in metastatic HCC tissues and was associated with overall survival and time to tumor recurrence of HCC patients. Taken together, our study connects mitochondria to the metastasis of human cancer via uncovered mitochondria-to-nucleus retrograde signaling, indicating that TFAM may serve as an effective target to block HCC metastasis.

Mitochondria are key regulators in cell proliferation and apoptosis. Alterations in mitochondrial function are closely associated with inflammation and tumorigenesis. This study aimed to investigate whether mitochondrial transcription factor A ( TFAM ), a key regulator of mitochondrial DNA transcription and replication, is involved in the initiation and progression of colitis-associated cancer (CAC). TFAM expression was examined in tissue samples of inflammatory bowel diseases (IBD) and CAC by immunohistochemistry. Intestinal epithelial cell (IEC)-specific TFAM- knockout mice ( TFAM △IEC ) and colorectal cancer (CRC) cells with TFAM knockdown or overexpression were used to evaluate the role of TFAM in colitis and the initiation and progression of CAC. The underlying mechanisms of TFAM were also explored by analyzing mitochondrial respiration function and biogenesis. The expression of TFAM was downregulated in active IBD and negatively associated with the disease activity. The downregulation of TFAM in IECs was induced by interleukin-6 in a signal transducer and activator of transcription 3 (STAT3)/miR-23b-dependent manner. In addition, TFAM knockout impaired IEC turnover to promote dextran sulfate sodium (DSS)-induced colitis in mice. Of note, TFAM knockout increased the susceptibility of mice to azoxymethane/DSS-induced CAC and TFAM overexpression protected mice from intestinal inflammation and colitis-associated tumorigenesis. By contrast, TFAM expression was upregulated in CAC tissues and contributed to cell growth. Furthermore, it was demonstrated that β-catenin induced the upregulation of TFAM through c-Myc in CRC cells. Mechanistically, TFAM promoted the proliferation of both IECs and CRC cells by increasing mitochondrial biogenesis and activity. TFAM plays a dual role in the initiation and progression of CAC, providing a novel understanding of CAC pathogenesis.

Altering the balance between energy intake and expenditure is a major strategy for treating obesity. Nonetheless, despite the progression in antiobesity drugs on appetite suppression, therapies aimed at increasing energy expenditure are limited. Here, knockout ofAKAP1, a signaling hub on outer mitochondrial membrane, renders mice resistant to diet-induced obesity.AKAP1 knockout significantly enhances energy expenditure and thermogenesis in brown adipose tissues (BATs) of obese mice. Restoring AKAP1 expression in BAT clearly reverses the beneficial antiobesity effect in AKAP1-/- mice. Mechanistically, AKAP1 remarkably decreases fatty acid β-oxidation (FAO) by phosphorylating ACSL1 to inhibit its activity in a protein-kinase-A-dependent manner and thus inhibits thermogenesis in brown adipocytes. Importantly, AKAP1 peptide inhibitor effectively alleviates diet-induced obesity and insulin resistance. Altogether, the findings demonstrate that AKAP1 functions as a brake of FAO to promote diet-induced obesity, which may be used as a potential therapeutic target for obesity.