Department of Chemistry, University of California, Davis, California 95616, United States.
Department of Biochemistry, University of California, Davis, California 95616, United States.
Anal Chem. 2023 Aug 29;95(34):12884-12892. doi: 10.1021/acs.analchem.3c02230. Epub 2023 Aug 16.
Glycogen is a highly branched biomacromolecule that functions as a glucose buffer. It is involved in multiple diseases such as glycogen storage disorders, diabetes, and even liver cancer, where the imbalance between biosynthetic and catabolic enzymes results in structural alterations and abnormal accumulation of glycogen that can be toxic to cells. Accurate and sensitive glycogen quantification and structural determination are prerequisites for understanding the phenotypes and biological functions of glycogen under these conditions. In this research, we furthered cell glycogen characterization by presenting a highly sensitive method to measure the glycogen content and degree of branching. The method employed a novel fructose density gradient as an alternative to the traditional sucrose gradient to fractionate glycogen from cell mixtures using ultracentrifugation. Fructose was used to avoid the large glucose background, allowing the method to be highly quantitative. The glycogen content was determined by quantifying 1-phenyl-3-methyl-5-pyrazolone (PMP)-derivatized glucose residues obtained from acid-hydrolyzed glycogen using ultra-high-performance liquid chromatography/triple quadrupole mass spectrometry (UHPLC/QqQ-MS). The degree of branching was determined through linkage analysis where the glycogen underwent permethylation, hydrolysis, PMP derivatization, and UHPLC/QqQ-MS analysis. The new approach was used to study the effect of insulin on the glycogen phenotypes of human hepatocellular carcinoma (Hep G2) cells. We observed that cells produced greater amounts of glycogen with less branching under increasing insulin levels before reaching the cell's insulin-resistant state, where the trend reversed and the cells produced less but higher-branched glycogen. The advantage of this method lies in its high sensitivity in characterizing both the glycogen level and the structure of biological samples.
糖原是一种高度分支的生物大分子，作为葡萄糖缓冲剂发挥作用。它与多种疾病有关，如糖原贮积症、糖尿病，甚至肝癌，其中合成和分解酶之间的失衡导致糖原结构改变和异常积累，从而对细胞产生毒性。准确和敏感的糖原定量和结构测定是理解这些条件下糖原表型和生物学功能的前提。在这项研究中，我们通过提出一种高度敏感的方法来测量糖原含量和分支程度，进一步研究了细胞糖原的特征。该方法使用一种新颖的果糖密度梯度代替传统的蔗糖梯度，通过超速离心从细胞混合物中分离糖原。使用果糖可避免大量葡萄糖背景，使该方法具有高度定量性。通过定量分析从酸水解糖原中获得的 1-苯基-3-甲基-5-吡唑啉酮（PMP）衍生的葡萄糖残基，来确定糖原含量，使用超高效液相色谱/三重四极杆质谱（UHPLC/QqQ-MS）。通过连接分析确定分支程度，其中糖原经历了全甲基化、水解、PMP 衍生化和 UHPLC/QqQ-MS 分析。新方法用于研究胰岛素对人肝癌（Hep G2）细胞糖原表型的影响。我们观察到，在达到细胞胰岛素抵抗状态之前，随着胰岛素水平的升高，细胞产生的糖原量增加，分支较少，但当达到胰岛素抵抗状态时，趋势发生逆转，细胞产生的糖原量减少，但分支较多。该方法的优点在于其在表征生物样品的糖原水平和结构方面具有高度的灵敏度。
