Fructose is an abundant source of carbon and energy for cells to use for metabolism, but only certain cell types use fructose to proliferate. Tumor cells that acquire the ability to metabolize fructose have a fitness advantage over their neighboring cells, but the proteins that mediate fructose metabolism in this context are unknown. Here, we investigated the determinants of fructose-mediated cell proliferation. Live cell imaging and crystal violet assays were used to characterize the ability of several cell lines (RKO, H508, HepG2, Huh7, HEK293T (293T), A172, U118-MG, U87, MCF-7, MDA-MB-468, PC3, DLD1 HCT116, and 22RV1) to proliferate in fructose (i.e., the fructolytic ability). Fructose metabolism gene expression was determined by RT-qPCR and western blot for each cell line. A positive selection approach was used to “train” non-fructolytic PC3 cells to utilize fructose for proliferation. RNA-seq was performed on parental and trained PC3 cells to find key transcripts associated with fructolytic ability. A CRISPR-cas9 plasmid containing KHK-specific sgRNA was transfected in 293T cells to generate KHK-/- cells. Lentiviral transduction was used to overexpress empty vector, KHK, or GLUT5 in cells. Metabolic profiling was done with seahorse metabolic flux analysis as well as LC/MS metabolomics. Cell Titer Glo was used to determine cell sensitivity to 2-deoxyglucose in media containing either fructose or glucose. We found that neither the tissue of origin nor expression level of any single gene related to fructose catabolism determine the fructolytic ability. However, cells cultured chronically in fructose can develop fructolytic ability. SLC2A5, encoding the fructose transporter, GLUT5, was specifically upregulated in these cells. Overexpression of GLUT5 in non-fructolytic cells enabled growth in fructose-containing media across cells of different origins. GLUT5 permitted fructose to flux through glycolysis using hexokinase (HK) and not ketohexokinase (KHK). We show that GLUT5 is a robust and generalizable driver of fructose-dependent cell proliferation. This indicates that fructose uptake is the limiting factor for fructose-mediated cell proliferation. We further demonstrate that cellular proliferation with fructose is independent of KHK.

中文翻译：

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GLUT5 (SLC2A5) 使果糖介导的增殖独立于酮己糖激酶

果糖是细胞用于新陈代谢的丰富的碳和能量来源，但只有某些细胞类型使用果糖进行增殖。获得代谢果糖能力的肿瘤细胞比其邻近细胞具有适应性优势，但在这种情况下介导果糖代谢的蛋白质是未知的。在这里，我们研究了果糖介导的细胞增殖的决定因素。活细胞成像和结晶紫测定用于表征几种细胞系（RKO、H508、HepG2、Huh7、HEK293T (293T)、A172、U118-MG、U87、MCF-7、MDA-MB-468、PC3 、DLD1、HCT116 和 22RV1）在果糖中增殖（即果糖分解能力）。通过 RT-qPCR 和蛋白质印迹确定每个细胞系的果糖代谢基因表达。正选择方法用于“训练”非果糖分解 PC3 细胞以利用果糖进行增殖。RNA-seq 在亲本和训练的 PC3 细胞上进行，以找到与果糖分解能力相关的关键转录本。将含有 KHK 特异性 sgRNA 的 CRISPR-cas9 质粒转染到 293T 细胞中以产生 KHK-/- 细胞。慢病毒转导用于在细胞中过表达空载体、KHK 或 GLUT5。使用海马代谢通量分析以及 LC/MS 代谢组学进行代谢分析。Cell Titer Glo 用于确定细胞对含有果糖或葡萄糖的培养基中的 2-脱氧葡萄糖的敏感性。我们发现，与果糖分解代谢相关的任何单个基因的起源组织和表达水平都不能决定果糖分解能力。然而，在果糖中长期培养的细胞可以发展果糖分解能力。编码果糖转运蛋白 GLUT5 的 SLC2A5 在这些细胞中被特异性上调。GLUT5 在非果糖分解细胞中的过表达使不同来源的细胞能够在含果糖的培养基中生长。GLUT5 允许果糖使用己糖激酶 (HK) 而不是酮己糖激酶 (KHK) 通过糖酵解流动。我们表明 GLUT5 是果糖依赖性细胞增殖的强大且可推广的驱动因素。这表明果糖摄取是果糖介导的细胞增殖的限制因素。我们进一步证明果糖的细胞增殖与 KHK 无关。GLUT5 在非果糖分解细胞中的过表达使不同来源的细胞能够在含果糖的培养基中生长。GLUT5 允许果糖使用己糖激酶 (HK) 而不是酮己糖激酶 (KHK) 通过糖酵解流动。我们表明 GLUT5 是果糖依赖性细胞增殖的强大且可推广的驱动因素。这表明果糖摄取是果糖介导的细胞增殖的限制因素。我们进一步证明果糖的细胞增殖与 KHK 无关。GLUT5 在非果糖分解细胞中的过表达使不同来源的细胞能够在含果糖的培养基中生长。GLUT5 允许果糖使用己糖激酶 (HK) 而不是酮己糖激酶 (KHK) 通过糖酵解流动。我们表明 GLUT5 是果糖依赖性细胞增殖的强大且可推广的驱动因素。这表明果糖摄取是果糖介导的细胞增殖的限制因素。我们进一步证明果糖的细胞增殖与 KHK 无关。这表明果糖摄取是果糖介导的细胞增殖的限制因素。我们进一步证明果糖的细胞增殖与 KHK 无关。这表明果糖摄取是果糖介导的细胞增殖的限制因素。我们进一步证明果糖的细胞增殖与 KHK 无关。