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

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.

Crosstalk between Metabolic Disorders and Immune Cells.

Metabolic syndrome results from multiple risk factors that arise from insulin resistance induced by abnormal fat deposition. Chronic inflammation owing to obesity primarily results from the recruitment of pro-inflammatory M1 macrophages into the adipose tissue stroma, as the adipocytes within become hypertrophied. During obesity-induced inflammation in adipose tissue, pro-inflammatory cytokines are produced by macrophages and recruit further pro-inflammatory immune cells into the adipose tissue to boost the immune response. Here, we provide an overview of the biology of macrophages in adipose tissue and the relationship between other immune cells, such as CD4+ T cells, natural killer cells, and innate lymphoid cells, and obesity and type 2 diabetes. Finally, we discuss the link between the human pathology and immune response and metabolism and further highlight potential therapeutic targets for the treatment of metabolic disorders.

Heme Biosynthesis is Crucial for Cell Survival and Mitochondrial OXPHOS after X Irradiation.

Heme is an essential component of the hemoproteins involved in the mitochondrial electron transport chain (ETC). Cancer cells have been reported to display high heme levels and increased activity of heme-containing proteins. Consistently, inhibition of heme biosynthesis by the ALAD inhibitor succinylacetone (SA) has been shown to reduce tumor cell survival. These observations indicate that heme biosynthesis is essential for cancer cell proliferation. X irradiation has been shown to increase mitochondrial mass, membrane potential, oxygen consumption, reactive oxygen species (ROS) production, and ATP synthesis. This finding suggests that radiation activates mitochondrial oxidative phosphorylation (OXPHOS). However, although heme is an essential component of the mitochondrial ETC, whether radiation influences heme biosynthesis remains unclear. In this study, we evaluated heme biosynthesis activity after X irradiation and examined the effects of heme biosynthesis inhibition by SA on cellular radiosensitivity and mitochondrial OXPHOS function. We demonstrated that X irradiation significantly increased ALAS1 mRNA levels and cellular heme content. Inhibition of heme biosynthesis by SA significantly decreased cellular heme content and sensitized cancer cells to radiation. We also showed that SA reduced cellular ATP levels, mitochondrial membrane potential, and mitochondrial ROS production, suggesting mitochondrial OXPHOS dysfunction. SA decreased the expression of mitochondrial heme-related proteins COX2 and cytochrome c but did not influence COX1 and VDAC expression. These results indicate that inhibition of heme biosynthesis decreased mitochondrial ETC protein expression and OXPHOS activity, which triggered cellular ATP depletion and radiosensitization after X irradiation. In summary, heme biosynthesis is upregulated by X irradiation and is essential for mitochondrial OXPHOS and cell survival.

Inheritance of the Golgi Apparatus and Cytokinesis Are Controlled by Degradation of GBF1.

Although much is known about how chromosome segregation is coupled to cell division, how intracellular organelles partition during mitotic division is poorly understood. We report that the phosphorylation-dependent degradation of the ARFGEF GBF1 regulates organelle trafficking during cell division. We show that, in mitosis, GBF1 is phosphorylated on Ser292 and Ser297 by casein kinase-2 allowing recognition by the F-box protein ßTrCP. GBF1 interaction with ßTrCP recruits GBF1 to the SCFßTrCP ubiquitin ligase complex, triggering its degradation. Phosphorylation and degradation of GBF1 occur along microtubules at the intercellular bridge of telophase cells and are required for Golgi membrane positioning and postmitotic Golgi reformation. Indeed, expression of a non-degradable GBF1 mutant inhibits the transport of the Golgi cluster adjacent to the midbody toward the Golgi twin positioned next to the centrosome and results in defective Golgi reassembly and cytokinesis failure. These findings define a mechanism that controls postmitotic Golgi reassembly and inheritance.