Molecular Metabolism最新文献_第6页

Glucose-1,6-bisphosphate: A new gatekeeper of cerebral mitochondrial pyruvate uptake 1,6-二磷酸葡萄糖：大脑线粒体丙酮酸摄取的新看门人。

IF 7 2区医学 Q1 ENDOCRINOLOGY & METABOLISM

Molecular Metabolism

Pub Date : 2024-08-24 DOI: 10.1016/j.molmet.2024.102018

Motahareh Solina Safari , Priska Woerl , Carolin Garmsiri , Dido Weber , Marcel Kwiatkowski , Madlen Hotze , Louisa Kuenkel , Luisa Lang , Matthias Erlacher , Ellen Gelpi , Johannes A. Hainfellner , Gottfried Baier , Gabriele Baier-Bitterlich , Stephanie zur Nedden

Objective

Glucose-1,6-bisphosphate (G-1,6-BP), a byproduct of glycolysis that is synthesized by phosphoglucomutase 2 like 1 (PGM2L1), is particularly abundant in neurons. G-1,6-BP is sensitive to the glycolytic flux, due to its dependence on 1,3-bisphosphoglycerate as phosphate donor, and the energy state, due to its degradation by inosine monophosphate-activated phosphomannomutase 1. Since the exact role of this metabolite remains unclear, our aim was to elucidate the specific function of G-1,6-BP in the brain.

Methods

The effect of PGM2L1 on neuronal post-ischemic viability was assessed by siRNA-mediated knockdown of PGM2L1 in primary mouse neurons. Acute mouse brain slices were used to correlate the reduction in G-1,6-BP upon ischemia to changes in carbon metabolism by ¹³C₆-glucose tracing. A drug affinity responsive target stability assay was used to test if G-1,6-BP interacts with the mitochondrial pyruvate carrier (MPC) subunits in mouse brain protein extracts. Human embryonic kidney cells expressing a MPC bioluminescence resonance energy transfer sensor were used to analyze how PGM2L1 overexpression affects MPC activity. The effect of G-1,6-BP on mitochondrial pyruvate uptake and oxygen consumption rates was analyzed in isolated mouse brain mitochondria. PGM2L1 and a predicted upstream kinase were overexpressed in a human neuroblastoma cell line and G-1,6-BP levels were measured.

Results

We found that G-1,6-BP in mouse brain slices was quickly degraded upon ischemia and reperfusion. Knockdown of PGM2L1 in mouse neurons reduced post-ischemic viability, indicating that PGM2L1 plays a neuroprotective role. The reduction in G-1,6-BP upon ischemia was not accompanied by alterations in glycolytic rates but we did see a reduced ¹³C₆-glucose incorporation into citrate, suggesting a potential role in mitochondrial pyruvate uptake or metabolism. Indeed, G-1,6-BP interacted with both MPC subunits and overexpression of PGM2L1 increased MPC activity. G-1,6-BP, at concentrations found in the brain, enhanced mitochondrial pyruvate uptake and pyruvate-induced oxygen consumption rates. Overexpression of a predicted upstream kinase inhibited PGM2L1 activity, showing that besides metabolism, also signaling pathways can regulate G-1,6-BP levels.

Conclusions

We provide evidence that G-1,6-BP positively regulates mitochondrial pyruvate uptake and post-ischemic neuronal viability. These compelling data reveal a novel mechanism by which neurons can couple glycolysis-derived pyruvate to the tricarboxylic acid cycle. This process is sensitive to the glycolytic flux, the cell's energetic state, and upstream signaling cascades, offering many regulatory means to fine-tune this critical metabolic step.

目的：葡萄糖-1,6-二磷酸（G-1,6-BP葡萄糖-1,6-二磷酸（G-1,6-BP）是糖酵解的副产物，由磷酸葡萄糖突变酶 2 样 1（PGM2L1）合成，在神经元中特别丰富。由于 G-1,6-BP 依赖 1,3-二磷酸甘油酯作为磷酸盐供体，因此它对糖酵解通量和能量状态都很敏感。由于这种代谢物的确切作用尚不清楚，我们的目的是阐明 G-1,6-BP 在大脑中的具体功能：方法：通过 siRNA 介导的小鼠原代神经元 PGM2L1 基因敲除，评估 PGM2L1 对神经元缺血后活力的影响。通过13C6-葡萄糖描记法将小鼠急性脑片缺血时G-1,6-BP的减少与碳代谢的变化联系起来。利用药物亲和力反应靶标稳定性测定法检测 G-1,6-BP 是否与小鼠脑蛋白提取物中的线粒体丙酮酸载体（MPC）亚基发生相互作用。表达 MPC 生物荧光共振能量转移传感器的人胚肾细胞被用来分析 PGM2L1 的过表达如何影响 MPC 的活性。在分离的小鼠脑线粒体中分析了 G-1,6-BP 对线粒体丙酮酸摄取和耗氧率的影响。在人神经母细胞瘤细胞系中过表达 PGM2L1 和一种预测的上游激酶，并测量 G-1,6-BP 的水平：结果：我们发现小鼠脑片中的G-1,6-BP在缺血和再灌注后迅速降解。小鼠神经元中 PGM2L1 的敲除降低了缺血后的存活率，表明 PGM2L1 起着神经保护作用。缺血时 G-1,6-BP 的减少并不伴随糖酵解率的改变，但我们确实看到 13C6 葡萄糖掺入柠檬酸盐的减少，这表明它可能在线粒体丙酮酸摄取或代谢中发挥作用。事实上，G-1,6-BP 与 MPC 的两个亚基都有相互作用，过量表达 PGM2L1 会增加 MPC 的活性。在大脑中发现的浓度下，G-1,6-BP 可提高线粒体的丙酮酸摄取量和丙酮酸诱导的氧消耗率。预测的上游激酶的过表达抑制了 PGM2L1 的活性，这表明除了新陈代谢外，信号通路也能调节 G-1,6-BP 的水平：我们提供的证据表明，G-1,6-BP 能积极调节线粒体丙酮酸摄取和缺血后神经元的存活能力。这些令人信服的数据揭示了神经元将糖酵解产生的丙酮酸与三羧酸循环耦合的新机制。这一过程对糖酵解通量、细胞能量状态和上游信号级联都很敏感，为微调这一关键代谢步骤提供了多种调节手段。

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引用次数: 0

Renal L-2-hydroxyglutarate dehydrogenase activity promotes hypoxia tolerance and mitochondrial metabolism in Drosophila melanogaster 肾脏 L-2-羟基戊二酸脱氢酶活性促进黑腹果蝇耐缺氧能力和线粒体代谢

IF 7 2区医学 Q1 ENDOCRINOLOGY & METABOLISM

Molecular Metabolism

Pub Date : 2024-08-23 DOI: 10.1016/j.molmet.2024.102013

Nader H. Mahmoudzadeh , Yasaman Heidarian , Jason P. Tourigny , Alexander J. Fitt , Katherine Beebe , Hongde Li , Arthur Luhur , Kasun Buddika , Liam Mungcal , Anirban Kundu , Robert A. Policastro , Garrett J. Brinkley , Gabriel E. Zentner , Travis Nemkov , Robert Pepin , Geetanjali Chawla , Sunil Sudarshan , Aylin R. Rodan , Angelo D'Alessandro , Jason M. Tennessen

Objectives

The mitochondrial enzyme L-2-hydroxyglutarate dehydrogenase (L2HGDH) regulates the abundance of L-2-hydroxyglutarate (L-2HG), a potent signaling metabolite capable of influencing chromatin architecture, mitochondrial metabolism, and cell fate decisions. Loss of L2hgdh activity in humans induces ectopic L-2HG accumulation, resulting in neurodevelopmental defects, altered immune cell function, and enhanced growth of clear cell renal cell carcinomas. To better understand the molecular mechanisms that underlie these disease pathologies, we used the fruit fly Drosophila melanogaster to investigate the endogenous functions of L2hgdh.

Methods

L2hgdh mutant adult male flies were analyzed under normoxic and hypoxic conditions using a combination of semi-targeted metabolomics and RNA-seq. These multi-omic analyses were complemented by tissue-specific genetic studies that examined the effects of L2hgdh mutations on the Drosophila renal system (Malpighian tubules; MTs).

Results

Our studies revealed that while L2hgdh is not essential for growth or viability under standard culture conditions, L2hgdh mutants are hypersensitive to hypoxia and expire during the reoxygenation phase with severe disruptions of mitochondrial metabolism. Moreover, we find that the fly renal system is a key site of L2hgdh activity, as L2hgdh mutants that express a rescuing transgene within the MTs survive hypoxia treatment and exhibit normal levels of mitochondrial metabolites. We also demonstrate that even under normoxic conditions, L2hgdh mutant MTs experience significant metabolic stress and are sensitized to aberrant growth upon Egfr activation.

Conclusions

These findings present a model in which renal L2hgdh activity limits systemic L-2HG accumulation, thus indirectly regulating the balance between glycolytic and mitochondrial metabolism, enabling successful recovery from hypoxia exposure, and ensuring renal tissue integrity.

线粒体酶 L-2-羟基戊二酸脱氢酶（L2HGDH）调节 L-2-羟基戊二酸（L-2HG）的丰度，L-2HG 是一种强效的信号代谢产物，能够影响染色质结构、线粒体代谢和细胞命运决定。人体内 L2hgdh 活性的缺失会诱导 L-2HG 的异位积累，从而导致神经发育缺陷、免疫细胞功能改变以及透明细胞肾细胞癌的生长增强。为了更好地了解这些疾病病理的分子机制，我们利用黑腹果蝇研究了L2hgdh的内源性功能。我们的研究发现，虽然在标准培养条件下，L2hgdh 对生长或存活并不重要，但 L2hgdh 突变体对缺氧不敏感，并在复氧阶段死亡，线粒体代谢受到严重破坏。此外，我们还发现苍蝇肾脏系统（Malpighian tubules; MTs）是 L2hgdh 活性的关键部位，因为在 MTs 中表达拯救转基因的 L2hgdh 突变体能在缺氧处理中存活，并表现出正常水平的线粒体代谢物。我们还证明，即使在正常缺氧条件下，L2hgdh 突变体 MT 也会经受巨大的代谢压力，并在 Egfr 激活后出现异常生长。总之，我们的研究结果提出了一个模型，在该模型中，肾脏 L2hgdh 的活性限制了全身 L-2HG 的积累，从而间接调节了糖酵解和线粒体代谢之间的平衡，使其能够从缺氧暴露中成功恢复，并确保肾脏组织的完整性。

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引用次数: 0

An INSULIN and IAPP dual reporter enables tracking of functional maturation of stem cell-derived insulin producing cells INSULIN 和 IAPP 双报告器可追踪干细胞衍生的胰岛素分泌细胞的功能成熟。

IF 7 2区医学 Q1 ENDOCRINOLOGY & METABOLISM

Molecular Metabolism

Pub Date : 2024-08-23 DOI: 10.1016/j.molmet.2024.102017

Carmen L. Bayly , Xiao-Qing Dai , Cuilan Nian , Paul C. Orban , C. Bruce Verchere , Patrick E. MacDonald , Francis C. Lynn

Objective

Human embryonic stem cell (hESC; SC)-derived pancreatic β cells can be used to study diabetes pathologies and develop cell replacement therapies. Although current differentiation protocols yield SCβ cells with varying degrees of maturation, these cells still differ from deceased donor human β cells in several respects. We sought to develop a reporter cell line that could be used to dynamically track SCβ cell functional maturation.

Methods

To monitor SCβ cell maturation in vitro, we created an IAPP-2A-mScar and INSULIN-2A-EGFP dual fluorescent reporter (INS^2A-EGFP/+;IAPP^{2A-mScarlet/+}) hESC line using CRISPR/Cas9. Pluripotent SC were then differentiated using a 7-stage protocol to islet-like cells. Immunohistochemistry, flow cytometry, qPCR, GSIS and electrophysiology were used to characterise resulting cell populations.

Results

We observed robust expression of EGFP and mScarlet fluorescent proteins in insulin- and IAPP-expressing cells without any compromise to their differentiation. We show that the proportion of insulin-producing cells expressing IAPP increases over a 4-week maturation period, and that a subset of insulin-expressing cells remain IAPP-free. Compared to this IAPP-free population, we show these insulin- and IAPP-expressing cells are less polyhormonal, more glucose-sensitive, and exhibit decreased action potential firing in low (2.8 mM) glucose.

Conclusions

The INS^2A-EGFP/+;IAPP^{2A-mScarlet/+} hESC line provides a useful tool for tracking populations of maturing hESC-derived β cells in vitro. This tool has already been shared with 3 groups and is freely available to all.

目的：人类胚胎干细胞（hESC；SC）衍生的胰腺β细胞可用于研究糖尿病病理和开发细胞替代疗法。尽管目前的分化方案可产生不同成熟度的SCβ细胞，但这些细胞在多个方面仍与已故供体人类β细胞不同。我们试图开发一种可用于动态跟踪SCβ细胞功能成熟的报告细胞系：为了在体外监测SCβ细胞的成熟，我们利用CRISPR/Cas9创建了一个IAPP-2A-mScar和INSULIN-2A-EGFP双荧光报告细胞系（INS2A-EGFP/+;IAPP2A-mScarlet/+）。然后用 7 级方案将多能细胞分化成小岛样细胞。免疫组化、流式细胞术、qPCR、GSIS 和电生理学被用来描述由此产生的细胞群：结果：我们在胰岛素和 IAPP 表达细胞中观察到 EGFP 和 mScarlet 荧光蛋白的强健表达，而它们的分化没有受到任何影响。我们发现，表达 IAPP 的胰岛素分泌细胞的比例在 4 周的成熟期内不断增加，而表达胰岛素的细胞中仍有一部分不表达 IAPP。与不表达 IAPP 的细胞群相比，我们发现这些表达胰岛素和 IAPP 的细胞的多激素性更低，对葡萄糖更敏感，在低葡萄糖（2.8 mM）条件下的动作电位点燃减少：结论：INS2A-EGFP/+;IAPP2A-mScarlet/+ hESC品系为追踪体外成熟的hESC衍生β细胞群提供了有用的工具。该工具已与 3 个研究小组共享，可供所有人免费使用。

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引用次数: 0

Imatinib therapy of chronic myeloid leukemia significantly reduces carnitine cell intake, resulting in adverse events 伊马替尼治疗慢性粒细胞白血病会显著减少肉碱细胞的摄入量，从而导致不良反应。

IF 7 2区医学 Q1 ENDOCRINOLOGY & METABOLISM

Molecular Metabolism

Pub Date : 2024-08-23 DOI: 10.1016/j.molmet.2024.102016

Pavel Burda , Alzbeta Hlavackova , Vendula Polivkova , Nikola Curik , Adam Laznicka , Jitka Krizkova , Jiri Suttnar , Pavel Klener , Katerina Machova Polakova

Objective

A prominent, safe and efficient therapy for patients with chronic myeloid leukemia (CML) is inhibiting oncogenic protein BCR::ABL1 in a targeted manner with imatinib, a tyrosine kinase inhibitor. A substantial part of patients treated with imatinib report skeletomuscular adverse events affecting their quality of life. OCTN2 membrane transporter is involved in imatinib transportation into the cells. At the same time, the crucial physiological role of OCTN2 is cellular uptake of carnitine which is an essential co-factor for the mitochondrial β-oxidation pathway. This work investigates the impact of imatinib treatment on carnitine intake and energy metabolism of muscle cells.

Methods

HTB-153 (human rhabdomyosarcoma) cell line and KCL-22 (CML cell line) were used to study the impact of imatinib treatment on intracellular levels of carnitine and vice versa. The energy metabolism changes in cells treated by imatinib were quantified and compared to changes in cells exposed to highly specific OCTN2 inhibitor vinorelbine. Mouse models were used to test whether in vitro observations are also achieved in vivo in thigh muscle tissue. The analytes of interest were quantified using a Prominence HPLC system coupled with a tandem mass spectrometer.

Results

This work showed that through the carnitine-specific transporter OCTN2, imatinib and carnitine intake competed unequally and intracellular carnitine concentrations were significantly reduced. In contrast, carnitine preincubation did not influence imatinib cell intake or interfere with leukemia cell targeting. Blocking the intracellular supply of carnitine with imatinib significantly reduced the production of most Krebs cycle metabolites and ATP. However, subsequent carnitine supplementation rescued mitochondrial energy production. Due to specific inhibition of OCTN2 activity, the influx of carnitine was blocked and mitochondrial energy metabolism was impaired in muscle cells in vitro and in thigh muscle tissue in a mouse model.

Conclusions

This preclinical experimental study revealed detrimental effect of imatinib on carnitine-mediated energy metabolism of muscle cells providing a possible molecular background of the frequently occurred side effects during imatinib therapy such as fatigue, muscle pain and cramps.

目的：酪氨酸激酶抑制剂伊马替尼（imatinib）以靶向方式抑制致癌蛋白 BCR::ABL1，是治疗慢性髓性白血病（CML）患者的一种显著、安全和高效的疗法。相当一部分接受伊马替尼治疗的患者报告了影响其生活质量的骨骼肌肉不良事件。OCTN2 膜转运体参与了伊马替尼向细胞内的转运。同时，OCTN2 的关键生理作用是细胞摄取肉碱，而肉碱是线粒体 β 氧化途径的重要辅助因子。方法：使用 HTB-153（人横纹肌肉瘤）细胞系和 KCL-22（CML 细胞系）研究伊马替尼治疗对细胞内肉碱水平的影响，反之亦然。对伊马替尼处理的细胞的能量代谢变化进行了量化，并与暴露于高度特异性 OCTN2 抑制剂长春瑞滨的细胞的变化进行了比较。使用小鼠模型测试体外观察结果是否也能在大腿肌肉组织体内实现。相关分析物的定量采用了与串联质谱仪相结合的 Prominence HPLC 系统：这项研究表明，通过肉碱特异性转运体 OCTN2，伊马替尼和肉碱的摄入量竞争不均，细胞内肉碱浓度显著降低。相反，肉碱预孵育不会影响伊马替尼细胞的摄入量，也不会干扰白血病细胞的靶向性。用伊马替尼阻断细胞内左旋肉碱的供应会显著减少大多数克雷布斯循环代谢产物和 ATP 的产生。不过，随后补充的肉碱能挽救线粒体能量的产生。由于特异性抑制了 OCTN2 的活性，肉碱流入受阻，体外肌肉细胞和小鼠模型大腿肌肉组织的线粒体能量代谢受损：这项临床前实验研究揭示了伊马替尼对肉碱介导的肌肉细胞能量代谢的有害影响，为伊马替尼治疗期间经常出现的副作用（如疲劳、肌肉疼痛和痉挛）提供了可能的分子背景。

{"title":"Imatinib therapy of chronic myeloid leukemia significantly reduces carnitine cell intake, resulting in adverse events","authors":"Pavel Burda , Alzbeta Hlavackova , Vendula Polivkova , Nikola Curik , Adam Laznicka , Jitka Krizkova , Jiri Suttnar , Pavel Klener , Katerina Machova Polakova","doi":"10.1016/j.molmet.2024.102016","DOIUrl":"10.1016/j.molmet.2024.102016","url":null,"abstract":"<div><h3>Objective</h3><p>A prominent, safe and efficient therapy for patients with chronic myeloid leukemia (CML) is inhibiting oncogenic protein BCR::ABL1 in a targeted manner with imatinib, a tyrosine kinase inhibitor. A substantial part of patients treated with imatinib report skeletomuscular adverse events affecting their quality of life. OCTN2 membrane transporter is involved in imatinib transportation into the cells. At the same time, the crucial physiological role of OCTN2 is cellular uptake of carnitine which is an essential co-factor for the mitochondrial β-oxidation pathway. This work investigates the impact of imatinib treatment on carnitine intake and energy metabolism of muscle cells.</p></div><div><h3>Methods</h3><p>HTB-153 (human rhabdomyosarcoma) cell line and KCL-22 (CML cell line) were used to study the impact of imatinib treatment on intracellular levels of carnitine and vice versa. The energy metabolism changes in cells treated by imatinib were quantified and compared to changes in cells exposed to highly specific OCTN2 inhibitor vinorelbine. Mouse models were used to test whether <em>in vitro</em> observations are also achieved <em>in vivo</em> in thigh muscle tissue. The analytes of interest were quantified using a Prominence HPLC system coupled with a tandem mass spectrometer.</p></div><div><h3>Results</h3><p>This work showed that through the carnitine-specific transporter OCTN2, imatinib and carnitine intake competed unequally and intracellular carnitine concentrations were significantly reduced. In contrast, carnitine preincubation did not influence imatinib cell intake or interfere with leukemia cell targeting. Blocking the intracellular supply of carnitine with imatinib significantly reduced the production of most Krebs cycle metabolites and ATP. However, subsequent carnitine supplementation rescued mitochondrial energy production. Due to specific inhibition of OCTN2 activity, the influx of carnitine was blocked and mitochondrial energy metabolism was impaired in muscle cells <em>in vitro</em> and in thigh muscle tissue in a mouse model.</p></div><div><h3>Conclusions</h3><p>This preclinical experimental study revealed detrimental effect of imatinib on carnitine-mediated energy metabolism of muscle cells providing a possible molecular background of the frequently occurred side effects during imatinib therapy such as fatigue, muscle pain and cramps.</p></div>","PeriodicalId":18765,"journal":{"name":"Molecular Metabolism","volume":"88 ","pages":"Article 102016"},"PeriodicalIF":7.0,"publicationDate":"2024-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2212877824001479/pdfft?md5=5d06e3c1393685b044f76a4004fd6375&pid=1-s2.0-S2212877824001479-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142056053","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A novel lncRNA GM47544 modulates triglyceride metabolism by inducing ubiquitination-dependent protein degradation of APOC3 新型lncRNA GM47544通过诱导泛素化依赖性蛋白降解APOC3来调节甘油三酯代谢。

IF 7 2区医学 Q1 ENDOCRINOLOGY & METABOLISM

Molecular Metabolism

Pub Date : 2024-08-20 DOI: 10.1016/j.molmet.2024.102011

Qianqian Xiao , Luyun Wang , Jing Wang , Man Wang , Dao Wen Wang , Hu Ding

Objective

Emerging evidence highlights the pivotal roles of long non-coding RNAs (lncRNAs) in lipid metabolism. Apoprotein C3 (ApoC3) is a well-established therapeutic target for hypertriglyceridemia and exhibits a strong association with cardiovascular disease. However, the exact mechanisms via which the lncRNAs control ApoC3 expression remain unclear.

Methods

We identified a novel long noncoding RNA (lncRNA), GM47544, within the ApoA1/C3/A4/A5 gene cluster. Subsequently, the effect of GM47544 on intracellular triglyceride metabolism was analyzed. The diet-induced mouse models of hyperlipidemia and atherosclerosis were established to explore the effect of GM47544 on dyslipidemia and plaque formation in vivo. The molecular mechanism was explored through RNA sequencing, immunoprecipitation, RNA pull-down assay, and RNA immunoprecipitation.

Results

GM47544 was overexpressed under high-fat stimulation. GM47544 effectively improved hepatic steatosis, reduced blood lipid levels, and alleviated atherosclerosis in vitro and in vivo. Mechanistically, GM47544 directly bound to ApoC3 and facilitated the ubiquitination at lysine 79 in ApoC3, thereby facilitating ApoC3 degradation via the ubiquitin-proteasome pathway. Moreover, we identified AP006216.5 as the human GM47544 transcript, which fulfills a comparable function in human hepatocytes.

Conclusions

The identification of GM47544 as a lncRNA modulator of ApoC3 reveals a novel mechanism of post-translational modification, with significant clinical implications for the treatment of hypertriglyceridemia and atherosclerosis.

目的：新的证据凸显了长非编码 RNA（lncRNA）在脂质代谢中的关键作用。载脂蛋白 C3（ApoC3）是治疗高甘油三酯血症的公认靶点，并与心血管疾病密切相关。然而，lncRNA控制载脂蛋白C3表达的确切机制仍不清楚：我们在载脂蛋白A1/C3/A4/A5基因簇中发现了一种新型长非编码RNA（lncRNA）GM47544。随后，我们分析了 GM47544 对细胞内甘油三酯代谢的影响。建立了饮食诱导的高脂血症和动脉粥样硬化小鼠模型，以探讨 GM47544 对体内血脂异常和斑块形成的影响。通过 RNA 测序、免疫沉淀、RNA 下拉实验和 RNA 免疫沉淀等方法探讨其分子机制：结果：GM47544在高脂刺激下过度表达。结果：GM47544 在高脂肪刺激下被过表达，GM47544 在体外和体内均能有效改善肝脂肪变性，降低血脂水平，缓解动脉粥样硬化。从机理上讲，GM47544直接与载脂蛋白C3结合，促进载脂蛋白C3赖氨酸79的泛素化，从而促进载脂蛋白C3通过泛素-蛋白酶体途径降解。此外，我们还鉴定出 AP006216.5 为人类 GM47544 转录本，它在人类肝细胞中发挥着类似的功能：结论：GM47544作为载脂蛋白C3的lncRNA调控因子的发现揭示了一种新的翻译后修饰机制，对治疗高甘油三酯血症和动脉粥样硬化具有重要的临床意义。

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引用次数: 0

Conditional deletion of CEACAM1 in hepatic stellate cells causes their activation 肝星状细胞中 CEACAM1 的条件性缺失会导致其活化。

IF 7 2区医学 Q1 ENDOCRINOLOGY & METABOLISM

Molecular Metabolism

Pub Date : 2024-08-19 DOI: 10.1016/j.molmet.2024.102010

Harrison T. Muturi , Hilda E. Ghadieh , Suman Asalla , Sumona G. Lester , Getachew D. Belew , Sobia Zaidi , Raziyeh Abdolahipour , Abhishek P. Shrestha , Agnes O. Portuphy , Hannah L. Stankus , Raghd Abu Helal , Stefaan Verhulst , Sergio Duarte , Ali Zarrinpar , Leo A. van Grunsven , Scott L. Friedman , Robert F. Schwabe , Terry D. Hinds Jr. , Sivarajan Kumarasamy , Sonia M. Najjar

Objectives

Hepatic CEACAM1 expression declines with advanced hepatic fibrosis stage in patients with metabolic dysfunction-associated steatohepatitis (MASH). Global and hepatocyte-specific deletions of Ceacam1 impair insulin clearance to cause hepatic insulin resistance and steatosis. They also cause hepatic inflammation and fibrosis, a condition characterized by excessive collagen production from activated hepatic stellate cells (HSCs). Given the positive effect of PPARγ on CEACAM1 transcription and on HSCs quiescence, the current studies investigated whether CEACAM1 loss from HSCs causes their activation.

Methods

We examined whether lentiviral shRNA-mediated CEACAM1 donwregulation (KD-LX2) activates cultured human LX2 stellate cells. We also generated LratCre + Cc1^fl/fl mutants with conditional Ceacam1 deletion in HSCs and characterized their MASH phenotype. Media transfer experiments were employed to examine whether media from mutant human and murine HSCs activate their wild-type counterparts.

Results

LratCre + Cc1^fl/fl mutants displayed hepatic inflammation and fibrosis but without insulin resistance or hepatic steatosis. Their HSCs, like KD-LX2 cells, underwent myofibroblastic transformation and their media activated wild-type HSCs. This was inhibited by nicotinic acid treatment which blunted the release of IL-6 and fatty acids, both of which activate the epidermal growth factor receptor (EGFR) tyrosine kinase. Gefitinib inhibition of EGFR and its downstream NF-κB/IL-6/STAT3 inflammatory and MAPK-proliferation pathways also blunted HSCs activation in the absence of CEACAM1.

Conclusions

Loss of CEACAM1 in HSCs provoked their myofibroblastic transformation in the absence of insulin resistance and hepatic steatosis. This response is mediated by autocrine HSCs activation of the EGFR pathway that amplifies inflammation and proliferation.

目的肝脏 CEACAM1 的表达随 MASH 患者肝纤维化晚期而下降。Ceacam1的全局性缺失和肝细胞特异性缺失会影响胰岛素清除，从而导致肝胰岛素抵抗和脂肪变性。它们还会导致肝脏炎症和纤维化，这种情况的特点是活化的肝星状细胞（HSCs）产生过多的胶原蛋白。鉴于 PPARγ 对 CEACAM1 的转录和造血干细胞的静止有积极作用，本研究探讨了造血干细胞中 CEACAM1 的缺失是否会导致其活化：我们研究了慢病毒 shRNA 介导的 CEACAM1 调控（KD-LX2）是否会激活培养的人 LX2 星状细胞。我们还在造血干细胞中产生了条件性Ceacam1缺失的LratCre+Cc1fl/fl突变体，并鉴定了它们的MASH表型。我们采用了培养基转移实验来研究突变体人类和鼠类造血干细胞的培养基是否能激活野生型造血干细胞：结果：LratCre+Cc1fl/fl 突变体表现出肝脏炎症和纤维化，但没有胰岛素抵抗或肝脏脂肪变性。它们的造血干细胞和 KD-LX2 细胞一样，都发生了肌成纤维细胞转化，而且它们的介质激活了野生型造血干细胞。烟酸可抑制IL-6和脂肪酸的释放，而这两种物质都能激活表皮生长因子受体（EGFR）酪氨酸激酶。吉非替尼（Gefitinib）抑制表皮生长因子受体及其下游的NF-κB/IL-6/STAT3炎症和MAPK-增殖通路也会在CEACAM1缺失的情况下抑制造血干细胞的活化：结论：在没有胰岛素抵抗和肝脏脂肪变性的情况下，造血干细胞中 CEACAM1 的缺失会引起其肌成纤维细胞的转化。这种反应是由自分泌的造血干细胞激活表皮生长因子受体通路介导的，该通路会放大炎症和增殖。

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引用次数: 0

Mitochondrial protein deacetylation by SIRT3 in osteoclasts promotes bone resorption with aging in female mice SIRT3 在破骨细胞中的线粒体蛋白去乙酰化作用会随着雌性小鼠的衰老而促进骨吸收。

IF 7 2区医学 Q1 ENDOCRINOLOGY & METABOLISM

Molecular Metabolism

Pub Date : 2024-08-16 DOI: 10.1016/j.molmet.2024.102012

Kimberly K. Richardson , Gareeballah Osman Adam , Wen Ling , Aaron Warren , Adriana Marques-Carvalho , Jeff D. Thostenson , Kimberly Krager , Nukhet Aykin-Burns , Stephanie D. Byrum , Maria Almeida , Ha-Neui Kim

Objectives

The mitochondrial deacetylase sirtuin-3 (SIRT3) is necessary for the increased bone resorption and enhanced function of mitochondria in osteoclasts that occur with advancing age; how SIRT3 drives bone resorption remains elusive.

Methods

To determine the role of SIRT3 in osteoclast mitochondria, we used mice with conditional loss of Sirt3 in osteoclast lineage and mice with germline deletion of either Sirt3 or its known target Pink1.

Results

SIRT3 stimulates mitochondrial quality in osteoclasts in a PINK1-independent manner, promoting mitochondrial activity and osteoclast maturation and function, thereby contributing to bone loss in female but not male mice. Quantitative analyses of global proteomes and acetylomes revealed that deletion of Sirt3 dramatically increased acetylation of osteoclast mitochondrial proteins, particularly ATPase inhibitory factor 1 (ATPIF1), an essential protein for mitophagy. Inhibition of mitophagy via mdivi-1 recapitulated the effect of deletion of Sirt3 or Atpif1 in osteoclast formation and mitochondrial function.

Conclusions

Decreasing mitophagic flux in osteoclasts may be a promising pharmacotherapeutic approach to treat osteoporosis in older adults.

研究目的线粒体去乙酰化酶sirtuin-3（SIRT3）是随着年龄增长而出现的骨吸收增加和破骨细胞线粒体功能增强的必要条件；SIRT3如何驱动骨吸收仍是一个谜：为了确定SIRT3在破骨细胞线粒体中的作用，我们使用了破骨细胞系中Sirt3条件性缺失的小鼠和Sirt3或其已知靶标Pink1种系缺失的小鼠：结果：SIRT3以一种与PINK1无关的方式刺激破骨细胞中线粒体的质量，促进线粒体活性以及破骨细胞的成熟和功能，从而导致雌性小鼠的骨质流失，而非雄性小鼠的骨质流失。全局蛋白质组和乙酰基组的定量分析显示，Sirt3的缺失显著增加了破骨细胞线粒体蛋白的乙酰化，尤其是ATPase抑制因子1（ATPIF1），它是有丝分裂的必需蛋白。通过mdivi-1抑制有丝分裂再现了缺失Sirt3或ATPIF1对破骨细胞形成和线粒体功能的影响：结论：降低破骨细胞的有丝分裂通量可能是治疗老年人骨质疏松症的一种很有前景的药物治疗方法。

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引用次数: 0

HSP60 chaperone deficiency disrupts the mitochondrial matrix proteome and dysregulates cholesterol synthesis HSP60 合子缺乏会破坏线粒体基质蛋白组，并导致胆固醇合成失调。

IF 7 2区医学 Q1 ENDOCRINOLOGY & METABOLISM

Molecular Metabolism

Pub Date : 2024-08-14 DOI: 10.1016/j.molmet.2024.102009

Cagla Cömert , Kasper Kjær-Sørensen , Jakob Hansen , Jasper Carlsen , Jesper Just , Brandon F. Meaney , Elsebet Østergaard , Yonglun Luo , Claus Oxvig , Lisbeth Schmidt-Laursen , Johan Palmfeldt , Paula Fernandez-Guerra , Peter Bross

Objective

Mitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized.

Methods

We studied two model systems for HSP60 deficiency: (1) engineered HEK cells carrying an inducible dominant negative HSP60 mutant protein, (2) zebrafish HSP60 knockout larvae. Both systems were analyzed by RNASeq, proteomics, and targeted metabolomics, and several functional assays relevant for the respective model. In addition, skin fibroblasts from patients with disease-associated HSP60 variants were analyzed by proteomics.

Results

We show that HSP60 deficiency leads to a differentially downregulated mitochondrial matrix proteome, transcriptional activation of stress responses, and dysregulated cholesterol biosynthesis. This leads to lipid accumulation in zebrafish knockout larvae.

Conclusions

Our data provide a compendium of the effects of HSP60 deficiency on the mitochondrial matrix proteome. We show that HSP60 is a master regulator and modulator of mitochondrial functions and metabolic pathways. HSP60 dysfunction also affects cellular metabolism and disrupts the integrated stress response. The effect on cholesterol synthesis explains the effect of HSP60 dysfunction on myelination observed in patients carrying genetic variants of HSP60.

目的：线粒体蛋白稳态对细胞功能至关重要：线粒体蛋白稳态对细胞功能至关重要。分子伴侣 HSP60 对细胞功能至关重要，HSP60 表达失调与癌症和糖尿病有关。据报道，少数携带 HSP60 基因变异的患者表现出神经发育迟缓和脑髓鞘功能减退。HSP60 与 260 多种线粒体蛋白相互作用，但受 HSP60 缺乏症影响的线粒体蛋白和功能特征尚不明确：我们研究了两种 HSP60 缺乏的模型系统：（1）携带诱导性显性阴性 HSP60 突变蛋白的工程 HEK 细胞；（2）斑马鱼 HSP60 基因敲除幼体。这两个系统都通过 RNASeq、蛋白质组学、靶向代谢组学以及与各自模型相关的几种功能测试进行了分析。此外，我们还通过蛋白质组学分析了患有与疾病相关的 HSP60 变体的患者的皮肤成纤维细胞：结果：我们发现，HSP60 缺乏会导致线粒体基质蛋白组的不同程度下调、应激反应的转录激活以及胆固醇生物合成失调。这导致斑马鱼基因敲除幼体中的脂质积累：我们的数据提供了 HSP60 缺乏对线粒体基质蛋白组影响的概要。我们的研究表明，HSP60 是线粒体功能和代谢途径的主调节器和调节器。HSP60 功能障碍也会影响细胞代谢并破坏综合应激反应。对胆固醇合成的影响解释了在携带 HSP60 基因变体的患者身上观察到的 HSP60 功能障碍对髓鞘形成的影响。

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引用次数: 0

Glucose intolerance as a consequence of hematopoietic stem cell dysfunction in offspring of obese mice 肥胖小鼠后代造血干细胞功能障碍导致葡萄糖耐受不良

IF 7 2区医学 Q1 ENDOCRINOLOGY & METABOLISM

Molecular Metabolism

Pub Date : 2024-08-12 DOI: 10.1016/j.molmet.2024.102008

Merve Denizli , James Ropa , Lindsay Beasley , Joydeep Ghosh , Kelli DeVanna , Taylor Spice , Laura S. Haneline , Maegan Capitano , Kok Lim Kua

Objective

Maternal obesity is increasingly common and negatively impacts offspring health. Children of mothers with obesity are at higher risk of developing diseases linked to hematopoietic system abnormalities and metabolism such as type 2 diabetes. Interestingly, disease risks are often dependent on the offspring's sex, suggesting sex-specific reprogramming effect of maternal obesity on offspring hematopoietic stem and progenitor cell (HSPC) function. However, the impact of maternal obesity exposure on offspring HSPC function, and the capability of HSPC to regulate offspring metabolic health is largely understudied. This study aims to test the hypothesis that offspring of obese mice exhibit sex-differences in HSPC function that affect offspring's metabolic health.

Methods

We first assessed bone marrow hematopoietic stem and progenitor cell phenotype using postnatal day 21 (P21) and 8-week-old C57BL/6J mice born to control and diet-induced obese dams. We also sorted HSPC (Lineage-, Sca1+, cKit + cells) from P21 mice for competitive primary and secondary transplant, as well as transcriptomic analysis. Body weight, adiposity, insulin tolerance test and glucose tolerance tests were performed in primary and secondary transplant recipient animals.

Results

We discovered sex-differences in offspring HSPC function in response to maternal obesity exposure, where male offspring of obese dams (MatOb) showed decreased HSPC numbers and engraftment, while female MatOb offspring remained largely unaffected. RNA-seq revealed immune stimulatory pathways in female MatOb offspring. Finally, only recipients of male MatOb offspring HSPC exhibited glucose intolerance.

Conclusions

This study demonstrated the lasting effect of maternal obesity exposure on offspring HSPC function and implicates HSPC in metabolic regulation.

背景/目的：母亲肥胖越来越常见，对后代的健康产生了负面影响。肥胖母亲的子女罹患与造血系统异常和代谢有关的疾病（如 2 型糖尿病）的风险较高。有趣的是，疾病风险往往取决于后代的性别，这表明母体肥胖对后代造血干细胞和祖细胞（HSPC）功能的重编程效应具有性别特异性。然而，母体肥胖对子代造血干细胞功能的影响以及造血干细胞调节子代代谢健康的能力在很大程度上还未得到充分研究。本研究旨在验证肥胖小鼠的后代在HSPC功能上表现出性别差异从而影响后代代谢健康的假设：方法：我们首先使用对照组和饮食诱导肥胖母鼠所生的出生后第21天（P21）和8周大的C57BL/6J小鼠评估骨髓造血干细胞和祖细胞表型。我们还对 P21 小鼠的 HSPC（Lineage-、Sca1+、cKit+ 细胞）进行了分拣，以进行竞争性原代和继代移植以及转录组分析。对初次和二次移植受体动物进行了体重、脂肪、胰岛素耐量试验和葡萄糖耐量试验：结果：我们发现母体肥胖对后代HSPC功能的影响存在性别差异，肥胖母体（MatOb）的雄性后代显示出HSPC数量和移植率下降，而雌性MatOb后代基本不受影响。RNA-seq发现了雌性MatOb后代的免疫刺激通路。最后，只有雄性 MatOb 后代 HSPC 受体表现出葡萄糖不耐受：这项研究证明了母体肥胖对后代 HSPC 功能的持久影响，并将 HSPC 与代谢调节联系起来。

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引用次数: 0

The mitochondrial pyruvate carrier regulates adipose glucose partitioning in female mice 线粒体丙酮酸载体调节雌性小鼠的脂肪葡萄糖分配。

IF 7 2区医学 Q1 ENDOCRINOLOGY & METABOLISM

Molecular Metabolism

Pub Date : 2024-08-11 DOI: 10.1016/j.molmet.2024.102005

Christopher E. Shannon , Terry Bakewell , Marcel J. Fourcaudot , Iriscilla Ayala , Annie A. Smelter , Edgar A. Hinostroza , Giovanna Romero , Mara Asmis , Leandro C. Freitas Lima , Martina Wallace , Luke Norton

Objective

The mitochondrial pyruvate carrier (MPC) occupies a critical node in intermediary metabolism, prompting interest in its utility as a therapeutic target for the treatment of obesity and cardiometabolic disease. Dysregulated nutrient metabolism in adipose tissue is a prominent feature of obesity pathophysiology, yet the functional role of adipose MPC has not been explored. We investigated whether the MPC shapes the adaptation of adipose tissue to dietary stress in female and male mice.

Methods

The impact of pharmacological and genetic disruption of the MPC on mitochondrial pathways of triglyceride assembly (lipogenesis and glyceroneogenesis) was assessed in 3T3L1 adipocytes and murine adipose explants, combined with analyses of adipose MPC expression in metabolically compromised humans. Whole-body and adipose-specific glucose metabolism were subsequently investigated in male and female mice lacking adipocyte MPC1 (Mpc1^AD−/−) and fed either standard chow, high-fat western style, or high-sucrose lipid restricted diets for 24 weeks, using a combination of radiolabeled tracers and GC/MS metabolomics.

Results

Treatment with UK5099 or siMPC1 impaired the synthesis of lipids and glycerol-3-phosphate from pyruvate and blunted triglyceride accumulation in 3T3L1 adipocytes, whilst MPC expression in human adipose tissue was negatively correlated with indices of whole-body and adipose tissue metabolic dysfunction. Mature adipose explants from Mpc1^AD−/− mice were intrinsically incapable of incorporating pyruvate into triglycerides. In vivo, MPC deletion restricted the incorporation of circulating glucose into adipose triglycerides, but only in female mice fed a zero fat diet, and this associated with sex-specific reductions in tricarboxylic acid cycle pool sizes and compensatory transcriptional changes in lipogenic and glycerol metabolism pathways. However, whole-body adiposity and metabolic health were preserved in Mpc1^AD−/− mice regardless of sex, even under conditions of zero dietary fat.

Conclusions

These findings highlight the greater capacity for mitochondrially driven triglyceride assembly in adipose from female versus male mice and expose a reliance upon MPC-gated metabolism for glucose partitioning in female adipose under conditions of dietary lipid restriction.

目的：线粒体丙酮酸载体（MPC）是中间代谢的关键节点：线粒体丙酮酸载体（MPC）在中间代谢中占据着一个关键节点，促使人们将其作为治疗肥胖症和心脏代谢疾病的治疗靶点。脂肪组织营养代谢失调是肥胖症病理生理学的一个突出特征，但脂肪 MPC 的功能作用尚未得到探讨。我们研究了MPC是否会影响雌性和雄性小鼠脂肪组织对饮食压力的适应：方法：我们在 3T3L1 脂肪细胞和小鼠脂肪外植体中评估了药理学和遗传学干扰 MPC 对线粒体甘油三酯组装途径（脂肪生成和甘油酯生成）的影响，并结合分析了代谢受损人类的脂肪 MPC 表达。随后，采用放射性标记示踪剂和 GC/MS 代谢组学相结合的方法，对缺乏脂肪细胞 MPC1（Mpc1AD-/-）的雄性和雌性小鼠的全身和脂肪特异性葡萄糖代谢进行了研究：结果：用UK5099或siMPC1处理会损害丙酮酸合成脂类和甘油-3-磷酸，并减弱3T3L1脂肪细胞中甘油三酯的积累，而人体脂肪组织中MPC的表达与全身和脂肪组织代谢功能障碍指数呈负相关。Mpc1AD-/-小鼠的成熟脂肪外植体本质上无法将丙酮酸转化为甘油三酯。在体内，MPC缺失限制了循环葡萄糖与脂肪甘油三酯的结合，但仅限于以零脂肪饮食喂养的雌性小鼠，这与三羧酸循环池大小的性别特异性减少以及生脂和甘油代谢途径的代偿性转录变化有关。然而，Mpc1AD-/-小鼠无论性别如何，即使在零膳食脂肪的条件下，其全身脂肪含量和代谢健康状况仍能保持不变：这些发现凸显了雌性与雄性小鼠脂肪中线粒体驱动的甘油三酯组装能力更强，并揭示了在饮食脂质限制条件下，雌性脂肪中的葡萄糖分配依赖于线粒体驱动的新陈代谢。

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引用次数: 0