Department of Functional Genomics, Yamagata University School of Medicine, Japan; Research Center for Molecular Genetics, Institute for Promotion of Medical Science Research, Yamagata University Faculty of Medicine, Japan.
Department of Functional Genomics, Yamagata University School of Medicine, Japan; Research Center for Molecular Genetics, Institute for Promotion of Medical Science Research, Yamagata University Faculty of Medicine, Japan; Department of Orthopedics, Yamagata University School of Medicine, Japan.
Arch Biochem Biophys. 2021 Jan 15;697:108721. doi: 10.1016/j.abb.2020.108721. Epub 2020 Dec 8.
5-Aminolevulinic acid (ALA) is the rate-limiting intermediate in heme biosynthesis in vertebrate species; a reaction catalyzed by the mitochondrial ALA synthase 1 (ALAS1) enzyme. Previously we reported that knockdown of the ubiquitously expressed ALAS1 gene in mice disrupts normal glucose metabolism, attenuates mitochondrial function and results in a prediabetic like phenotype when animals pass 20-weeks of age (Saitoh et al., 2018). Contrary to our expectations, the cytosolic and mitochondrial heme content of ALAS1 heterozygous (A1+/-) mice were similar to WT animals. Therefore, we speculated that regulatory "free heme" may be reduced in an age dependent manner in A1+/- mice, but not total heme. Here, we examine free and total heme from the skeletal muscle and liver of WT and A1+/- mice using a modified acetone extraction method and examine the effects of aging on free heme by comparing the amounts at 8-12 weeks and 30-36 weeks of age, in addition to the mRNA abundance of ALAS1. We found an age-dependent reduction in free heme in the skeletal muscle and liver of A1+/- mice, while WT mice showed only a slight decrease in the liver. Total heme levels showed no significant difference between young and aged WT and A1+/- mice. ALAS1 mRNA levels showed an age-dependent reduction similar to that of free heme levels, indicating that ALAS1 mRNA expression levels are a major determinant for free heme levels. The free heme pools in skeletal muscle tissue were almost 2-fold larger than that of liver tissue, suggesting that the heme pool varies across different tissue types. The expression of heme oxygenase 1 (HO-1) mRNA, which is expressed proportionally to the amount of free heme, were similar to those of free heme levels. Taken together, this study demonstrates that the free heme pool differs across tissues, and that an age-dependent reduction in free heme levels is accelerated in mice heterozygous for ALAS1, which could account for the prediabetic phenotype and mitochondrial abnormality observed in these animals.
5-氨基酮戊酸（ALA）是脊椎动物血红素生物合成的限速中间产物；这一反应由线粒体 ALA 合酶 1（ALAS1）酶催化。此前，我们报道过，在小鼠中敲低广泛表达的 ALAS1 基因会破坏正常的葡萄糖代谢，减弱线粒体功能，并导致动物在 20 周龄时出现类似糖尿病前期的表型（Saitoh 等人，2018 年）。与我们的预期相反，ALAS1 杂合子（A1+/-）小鼠的细胞质和线粒体血红素含量与 WT 动物相似。因此，我们推测，A1+/- 小鼠中的调节“游离血红素”可能随年龄呈依赖性降低，但总血红素没有。在这里，我们使用改良的丙酮提取方法从 WT 和 A1+/- 小鼠的骨骼肌和肝脏中检测游离和总血红素，并通过比较 8-12 周和 30-36 周龄时的量来检查衰老对游离血红素的影响，此外还检测了 ALAS1 的 mRNA 丰度。我们发现 A1+/- 小鼠的骨骼肌和肝脏中的游离血红素随年龄呈依赖性降低，而 WT 小鼠的肝脏中仅略有下降。WT 和 A1+/- 小鼠的年轻和年老组之间的总血红素水平没有显著差异。ALAS1 mRNA 水平的降低与游离血红素水平相似，表明 ALAS1 mRNA 表达水平是游离血红素水平的主要决定因素。骨骼肌组织中的游离血红素池几乎是肝脏组织的两倍，这表明血红素池在不同组织类型之间存在差异。血红素加氧酶 1（HO-1）mRNA 的表达与游离血红素的量成比例，与游离血红素水平相似。总之，这项研究表明，游离血红素池在不同组织中存在差异，并且 ALAS1 杂合子小鼠中的游离血红素水平随年龄的降低呈加速趋势，这可能解释了这些动物中观察到的糖尿病前期表型和线粒体异常。
