{"title":"比较诱导小鼠 3T3-L1 细胞胰岛素抵抗的方法","authors":"Hend Al-Jaber, Shamma Al-Muraikhy, Aldana Jabr Jabr, Aisha Yousuf, Najeha Anwardeen, Mohamed Elrayess, Layla Al-Mansoori","doi":"10.2174/0115733998263359231211044539","DOIUrl":null,"url":null,"abstract":"<p><p>Cell culture plays a crucial role in addressing fundamental research questions, particularly in studying insulin resistance (IR) mechanisms. Multiple in vitro models are utilized for this purpose, but their technical distinctions and relevance to in vivo conditions remain unclear. This study aims to assess the effectiveness of existing in vitro models in inducing IR and their ability to replicate in vivo IR conditions.</p><p><strong>Background: </strong>Insulin resistance (IR) is a cellular condition linked to metabolic disorders. Despite the utility of cell culture in IR research, questions persist regarding the suitability of various models. This study seeks to evaluate these models' efficiency in inducing IR and their ability to mimic in vivo conditions. Insights gained from this research could enhance our understanding of model strengths and limitations, potentially advancing strategies to combat IR and related disorders.</p><p><strong>Objective: </strong>1- Investigate the technical differences between existing cell culture models used to study molecular mediators of insulin resistance (IR). 2- Compare the effectiveness of present in vitro models in inducing insulin resistance (IR). 3- Assess the relevance of the existing cell culture models in simulating the in vivo conditions and environment that provoke the induction of insulin resistance (IR).</p><p><strong>Methods and material: </strong>In vitro, eight sets of 3T3-L1 cells were cultured until they reached 90% confluence. Subsequently, adipogenic differentiation was induced using a differentiation cocktail (media). These cells were then divided into four groups, with four subjected to normal conditions and the other four to hypoxic conditions. Throughout the differentiation process, each cell group was exposed to specific factors known to induce insulin resistance (IR). These factors included 2.5nM tumor necrosis factor-alpha (TNFα), 20 ng/ml interleukin-6 (IL-6), 10 micromole 4-hydroxynonenal (4HNE), and high insulin (HI) at a concentration of 100nM. To assess cell proliferation, DAPI staining was employed, and the expression of genes associated with various metabolic pathways affected by insulin resistance was investigated using Real-Time PCR. Additionally, insulin signaling was examined using the Bio-plex Pro cell signaling Akt panel.</p><p><strong>Results: </strong>We induced insulin resistance in 3T3-L1 cells using IL-6, TNFα, 4HNE, and high insulin in both hypoxic and normoxic conditions. Hypoxia increased HIF1a gene expression by approximately 30% (P<0.01). TNFα reduced cell proliferation by 10-20%, and chronic TNFα treatment significantly decreased mature adipocytes due to its cytotoxicity. We assessed the impact of insulin resistance (IR) on metabolic pathways, focusing on genes linked to branched-chain amino acid metabolism, detoxification, and chemotaxis. Notably, ALDH6A1 and MCCC1 genes, related to amino acid metabolism, were significantly affected under hypoxic conditions. TNFα treatment notably influenced MCP-1 and MCP-2 genes linked to chemotaxis, with remarkable increases in MCP-1 levels and MCP-2 expression primarily under hypoxia. Detoxification-related genes showed minimal impact, except for a significant increase in MAOA expression under acute hypoxic conditions with TNFα treatment. Additional genes displayed varying effects, warranting further investigation. To investigate insulin signaling's influence in vitro by IRinducing factors, we assessed phospho-protein levels. Our results reveal a significant p-Akt induction with chronic high insulin (10%) and acute TNFα (12%) treatment under hypoxia (both P<0.05). Other insulin resistance-related phospho-proteins (GSK3B, mTOR, PTEN) increased with IL-6, 4HNE, TNFα, and high insulin under hypoxia, while p-IRS1 levels remained unaffected.</p><p><strong>Conclusion: </strong>In summary, different in vitro models using inflammatory, oxidative stress, and high insulin conditions under hypoxic conditions can capture various aspects of in vivo adipose tissue insulin resistance (IR). Among these models, acute TNFα treatment may offer the most robust approach for inducing IR in 3T3-L1 cells.</p>","PeriodicalId":10825,"journal":{"name":"Current diabetes reviews","volume":null,"pages":null},"PeriodicalIF":2.4000,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Comparing Methods for Induction of Insulin Resistance in Mouse 3T3-L1 Cells.\",\"authors\":\"Hend Al-Jaber, Shamma Al-Muraikhy, Aldana Jabr Jabr, Aisha Yousuf, Najeha Anwardeen, Mohamed Elrayess, Layla Al-Mansoori\",\"doi\":\"10.2174/0115733998263359231211044539\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Cell culture plays a crucial role in addressing fundamental research questions, particularly in studying insulin resistance (IR) mechanisms. Multiple in vitro models are utilized for this purpose, but their technical distinctions and relevance to in vivo conditions remain unclear. This study aims to assess the effectiveness of existing in vitro models in inducing IR and their ability to replicate in vivo IR conditions.</p><p><strong>Background: </strong>Insulin resistance (IR) is a cellular condition linked to metabolic disorders. Despite the utility of cell culture in IR research, questions persist regarding the suitability of various models. This study seeks to evaluate these models' efficiency in inducing IR and their ability to mimic in vivo conditions. Insights gained from this research could enhance our understanding of model strengths and limitations, potentially advancing strategies to combat IR and related disorders.</p><p><strong>Objective: </strong>1- Investigate the technical differences between existing cell culture models used to study molecular mediators of insulin resistance (IR). 2- Compare the effectiveness of present in vitro models in inducing insulin resistance (IR). 3- Assess the relevance of the existing cell culture models in simulating the in vivo conditions and environment that provoke the induction of insulin resistance (IR).</p><p><strong>Methods and material: </strong>In vitro, eight sets of 3T3-L1 cells were cultured until they reached 90% confluence. Subsequently, adipogenic differentiation was induced using a differentiation cocktail (media). These cells were then divided into four groups, with four subjected to normal conditions and the other four to hypoxic conditions. Throughout the differentiation process, each cell group was exposed to specific factors known to induce insulin resistance (IR). These factors included 2.5nM tumor necrosis factor-alpha (TNFα), 20 ng/ml interleukin-6 (IL-6), 10 micromole 4-hydroxynonenal (4HNE), and high insulin (HI) at a concentration of 100nM. To assess cell proliferation, DAPI staining was employed, and the expression of genes associated with various metabolic pathways affected by insulin resistance was investigated using Real-Time PCR. Additionally, insulin signaling was examined using the Bio-plex Pro cell signaling Akt panel.</p><p><strong>Results: </strong>We induced insulin resistance in 3T3-L1 cells using IL-6, TNFα, 4HNE, and high insulin in both hypoxic and normoxic conditions. Hypoxia increased HIF1a gene expression by approximately 30% (P<0.01). TNFα reduced cell proliferation by 10-20%, and chronic TNFα treatment significantly decreased mature adipocytes due to its cytotoxicity. We assessed the impact of insulin resistance (IR) on metabolic pathways, focusing on genes linked to branched-chain amino acid metabolism, detoxification, and chemotaxis. Notably, ALDH6A1 and MCCC1 genes, related to amino acid metabolism, were significantly affected under hypoxic conditions. TNFα treatment notably influenced MCP-1 and MCP-2 genes linked to chemotaxis, with remarkable increases in MCP-1 levels and MCP-2 expression primarily under hypoxia. Detoxification-related genes showed minimal impact, except for a significant increase in MAOA expression under acute hypoxic conditions with TNFα treatment. Additional genes displayed varying effects, warranting further investigation. To investigate insulin signaling's influence in vitro by IRinducing factors, we assessed phospho-protein levels. Our results reveal a significant p-Akt induction with chronic high insulin (10%) and acute TNFα (12%) treatment under hypoxia (both P<0.05). Other insulin resistance-related phospho-proteins (GSK3B, mTOR, PTEN) increased with IL-6, 4HNE, TNFα, and high insulin under hypoxia, while p-IRS1 levels remained unaffected.</p><p><strong>Conclusion: </strong>In summary, different in vitro models using inflammatory, oxidative stress, and high insulin conditions under hypoxic conditions can capture various aspects of in vivo adipose tissue insulin resistance (IR). Among these models, acute TNFα treatment may offer the most robust approach for inducing IR in 3T3-L1 cells.</p>\",\"PeriodicalId\":10825,\"journal\":{\"name\":\"Current diabetes reviews\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.4000,\"publicationDate\":\"2024-01-09\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Current diabetes reviews\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.2174/0115733998263359231211044539\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"ENDOCRINOLOGY & METABOLISM\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current diabetes reviews","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.2174/0115733998263359231211044539","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"ENDOCRINOLOGY & METABOLISM","Score":null,"Total":0}
引用次数: 0
摘要
细胞培养在解决基础研究问题,尤其是研究胰岛素抵抗(IR)机制方面发挥着至关重要的作用。为此,人们使用了多种体外模型,但它们的技术区别以及与体内条件的相关性仍不清楚。本研究旨在评估现有体外模型在诱导 IR 方面的有效性及其复制体内 IR 条件的能力:背景:胰岛素抵抗(IR)是一种与代谢紊乱有关的细胞病症。背景:胰岛素抵抗(IR)是一种与代谢紊乱有关的细胞病症。尽管细胞培养在 IR 研究中很有用,但有关各种模型是否适用的问题仍然存在。本研究旨在评估这些模型诱导 IR 的效率及其模拟体内条件的能力。目标:1- 调查用于研究胰岛素抵抗(IR)分子介质的现有细胞培养模型之间的技术差异。 2- 比较现有体外模型在诱导胰岛素抵抗(IR)方面的有效性。3- 评估现有细胞培养模型在模拟诱导胰岛素抵抗(IR)的体内条件和环境方面的相关性:在体外培养八组 3T3-L1 细胞,直到它们达到 90% 的融合度。随后,使用分化鸡尾酒(培养基)诱导脂肪分化。然后将这些细胞分成四组,其中四组在正常条件下培养,另外四组在缺氧条件下培养。在整个分化过程中,每组细胞都暴露在已知会诱导胰岛素抵抗(IR)的特定因子中。这些因子包括 2.5nM 肿瘤坏死因子-α(TNFα)、20 纳克/毫升白细胞介素-6(IL-6)、10 微摩尔 4-羟基壬烯醛(4HNE)和浓度为 100nM 的高胰岛素(HI)。为了评估细胞增殖情况,采用了 DAPI 染色法,并使用实时 PCR 技术研究了受胰岛素抵抗影响的各种代谢途径相关基因的表达情况。此外,还使用 Bio-plex Pro 细胞信号 Akt 面板检测了胰岛素信号转导:我们使用 IL-6、TNFα、4HNE 和高胰岛素在缺氧和常氧条件下诱导 3T3-L1 细胞产生胰岛素抵抗。缺氧使 HIF1a 基因表达增加了约 30%(PConclusion:总之,在缺氧条件下使用炎症、氧化应激和高胰岛素条件的不同体外模型可以捕捉到体内脂肪组织胰岛素抵抗(IR)的各个方面。在这些模型中,急性 TNFα 处理可能是诱导 3T3-L1 细胞产生 IR 的最有效方法。
Comparing Methods for Induction of Insulin Resistance in Mouse 3T3-L1 Cells.
Cell culture plays a crucial role in addressing fundamental research questions, particularly in studying insulin resistance (IR) mechanisms. Multiple in vitro models are utilized for this purpose, but their technical distinctions and relevance to in vivo conditions remain unclear. This study aims to assess the effectiveness of existing in vitro models in inducing IR and their ability to replicate in vivo IR conditions.
Background: Insulin resistance (IR) is a cellular condition linked to metabolic disorders. Despite the utility of cell culture in IR research, questions persist regarding the suitability of various models. This study seeks to evaluate these models' efficiency in inducing IR and their ability to mimic in vivo conditions. Insights gained from this research could enhance our understanding of model strengths and limitations, potentially advancing strategies to combat IR and related disorders.
Objective: 1- Investigate the technical differences between existing cell culture models used to study molecular mediators of insulin resistance (IR). 2- Compare the effectiveness of present in vitro models in inducing insulin resistance (IR). 3- Assess the relevance of the existing cell culture models in simulating the in vivo conditions and environment that provoke the induction of insulin resistance (IR).
Methods and material: In vitro, eight sets of 3T3-L1 cells were cultured until they reached 90% confluence. Subsequently, adipogenic differentiation was induced using a differentiation cocktail (media). These cells were then divided into four groups, with four subjected to normal conditions and the other four to hypoxic conditions. Throughout the differentiation process, each cell group was exposed to specific factors known to induce insulin resistance (IR). These factors included 2.5nM tumor necrosis factor-alpha (TNFα), 20 ng/ml interleukin-6 (IL-6), 10 micromole 4-hydroxynonenal (4HNE), and high insulin (HI) at a concentration of 100nM. To assess cell proliferation, DAPI staining was employed, and the expression of genes associated with various metabolic pathways affected by insulin resistance was investigated using Real-Time PCR. Additionally, insulin signaling was examined using the Bio-plex Pro cell signaling Akt panel.
Results: We induced insulin resistance in 3T3-L1 cells using IL-6, TNFα, 4HNE, and high insulin in both hypoxic and normoxic conditions. Hypoxia increased HIF1a gene expression by approximately 30% (P<0.01). TNFα reduced cell proliferation by 10-20%, and chronic TNFα treatment significantly decreased mature adipocytes due to its cytotoxicity. We assessed the impact of insulin resistance (IR) on metabolic pathways, focusing on genes linked to branched-chain amino acid metabolism, detoxification, and chemotaxis. Notably, ALDH6A1 and MCCC1 genes, related to amino acid metabolism, were significantly affected under hypoxic conditions. TNFα treatment notably influenced MCP-1 and MCP-2 genes linked to chemotaxis, with remarkable increases in MCP-1 levels and MCP-2 expression primarily under hypoxia. Detoxification-related genes showed minimal impact, except for a significant increase in MAOA expression under acute hypoxic conditions with TNFα treatment. Additional genes displayed varying effects, warranting further investigation. To investigate insulin signaling's influence in vitro by IRinducing factors, we assessed phospho-protein levels. Our results reveal a significant p-Akt induction with chronic high insulin (10%) and acute TNFα (12%) treatment under hypoxia (both P<0.05). Other insulin resistance-related phospho-proteins (GSK3B, mTOR, PTEN) increased with IL-6, 4HNE, TNFα, and high insulin under hypoxia, while p-IRS1 levels remained unaffected.
Conclusion: In summary, different in vitro models using inflammatory, oxidative stress, and high insulin conditions under hypoxic conditions can capture various aspects of in vivo adipose tissue insulin resistance (IR). Among these models, acute TNFα treatment may offer the most robust approach for inducing IR in 3T3-L1 cells.
期刊介绍:
Current Diabetes Reviews publishes frontier reviews on all the latest advances on diabetes and its related areas e.g. pharmacology, pathogenesis, complications, epidemiology, clinical care, and therapy. The journal"s aim is to publish the highest quality review articles dedicated to clinical research in the field. The journal is essential reading for all researchers and clinicians who are involved in the field of diabetes.