Heterozygous disruption of ALAS1 in mice causes an accelerated age-dependent reduction in free heme, but not total heme, in skeletal muscle and liver

Koen Van Wijk,Takeru Akabane,Tomohiro Kimura,Shinichi Saitoh,Satoshi Okano,Vincent P Kelly,Michiaki Takagi,Ken Kodama,Kiwamu Takahashi,Tohru Tanaka,Motowo Nakajima,Osamu Nakajima

doi:10.1016/j.abb.2020.108721

Koen Van Wijk, Takeru Akabane + Show 10 more

Open Access

https://doi.org/10.1016/j.abb.2020.108721

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Abstract

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.

Highlights

Heme serves as an important regulatory component for hemopro teins such as those involved in translation and transcription [1], and is an essential component of complex II, III, and IV of the mitochondrial electron transport chain (ETC)
In mice that were 8–12 weeks of age the reduction in ALA synthase 1 (ALAS1) mRNA expression did not differ significantly be tween A1+/- mice and WT mice. This suggests that the levels of ALAS1 mRNA in skeletal muscle of A1+/- mice decreased with age, beyond a minimum threshold level which could possibly be a causative factor for the aberrant glucose metabolism and mitochondrial function observed in aged A1+/- mice
We found an age-dependent reduction in the levels of free rather than total heme, both in the skeletal muscle and the liver of A1+/- mice, while wildtype mice showed only a slight decrease

Summary

Introduction

Heme serves as an important regulatory component for hemopro teins such as those involved in translation and transcription [1], and is an essential component of complex II, III, and IV of the mitochondrial electron transport chain (ETC). Regulatory heme, known as “free heme”, is a form of heme that has yet to be incorporated into protein or that is nonspecifically asso ciated with protein or the membrane This mobile form of heme can act as a signal molecule [3] with the ability to influence transcription, translation and enzyme activity; vide infra. 5-Aminolevulinate synthase 1 ALAS1 heterozygous 5-Aminolevulinic acid Bovine serum albumin Wild-type which are combined in the mitochondria to form 5-aminolevulinic acid (ALA) by the enzyme 5-aminolevulinate synthase (ALAS). This is the rate-limiting step in heme synthesis. Our previous studies on ALAS gene knockout mice showed that the ubiquitously expressed isozyme ALAS1 has an indispensable function for early embryogenesis [4], whereas the erythroid specific isozyme ALAS2 is crucial for heme production and iron metabolism in erythroid cells [5,6]

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