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-氨基乙酰丙酸（ALA）是脊椎动物血红素生物合成中的限速中间体。线粒体ALA合酶1（ALAS1）酶催化的反应。之前我们曾报道过敲除小鼠中普遍表达的ALAS1基因会破坏正常的葡萄糖代谢，削弱线粒体功能并在动物超过20周龄时导致糖尿病样表型（Saitoh等人，2018）。与我们的预期相反，ALAS1杂合（A1 +/-）小鼠的胞质和线粒体血红素含量与野生型动物相似。因此，我们推测在A1 +/-小鼠中，调节性“游离血红素”可能会以年龄依赖性方式降低，但总血红素不会降低。

在这里，我们使用改良的丙酮提取方法检查了WT和A1 +/-小鼠骨骼肌和肝脏的游离血红素和总血红素，并通过比较8-12周和30-36周的量对衰老对游离血红素的影响周龄，以及ALAS1的mRNA丰度。我们发现年龄依赖性的A1 +/-小鼠骨骼肌和肝脏中游离血红素减少，而WT小鼠肝脏中仅显示轻度减少。总血红素水平在年轻和老年的WT和A1 +/-小鼠之间没有显着差异。ALAS1 mRNA水平显示出与游离血红素水平相似的年龄依赖性降低，表明ALAS1mRNA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库几乎是肝组织中的2倍，这表明血红素库在不同的组织类型中有所不同。的表达血红素加氧酶1（HO-1 ）的mRNA，这是成比例地表达以游离血红素的量，的那些相似的游离血红素水平。总而言之，这项研究表明游离血红素库在不同组织之间存在差异，并且ALAS1杂合小鼠的血红素血红蛋白水平随年龄增长而降低，这可能是这些动物中观察到的糖尿病前期表型和线粒体异常的原因。