Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis.

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Abcb10 regulates murine hematopoietic stem cell potential and erythroid differentiation.

Erythropoiesis in the adult bone marrow relies on mitochondrial membrane transporters to facilitate heme and hemoglobin production. Erythrocytes in the bone marrow are produced although the differentiation of erythroid progenitor cells that originate from hematopoietic stem cells (HSCs). Whether and how mitochondria transporters potentiate HSCs and affect their differentiation toward erythroid lineage remains unclear. Here, we show that the ATP-binding cassette (ABC) transporter 10 (Abcb10), located on the inner mitochondrial membrane, is essential for HSC maintenance and erythroid-lineage differentiation. Induced deletion of Abcb10 in adult mice significantly increased erythroid progenitor cell and decreased HSC number within the bone marrow (BM). Functionally, Abcb10-deficient HSCs exhibited significant decreases in stem cell potential but with a skew toward erythroid-lineage differentiation. Mechanistically, deletion of Abcb10 rendered HSCs with excess mitochondrial iron accumulation and oxidative stress yet without alteration in mitochondrial bioenergetic function. However, impaired hematopoiesis could not be rescued through the in vivo administration of a mitochondrial iron chelator or antioxidant to Abcb10-deficient mice. Abcb10-mediated mitochondrial iron transfer is thus pivotal for the regulation of physiologic HSC potential and erythroid-lineage differentiation.

Imeglimin improves systemic metabolism by targeting brown adipose tissue and gut microbiota in obese model mice.

Imeglimin is a recently developed anti-diabetic drug that could concurrently promote insulin secretion and insulin sensitivity, while its mechanisms of action are not fully understood. Here we show that imeglimin administration could protect mice from high fat diet-induced weight gain with enhanced energy expenditure and attenuated whitening of brown adipose tissue. Imeglimin administration led to significant alteration of gut microbiota, which included an increase of Akkermansia genus, with attenuation of obesity-associated gut pathologies. Ablation of microbiota by antibiotic treatment partially abrogated the insulin sensitizing effects of imeglimin, while not affecting its actions on body weight gain or brown adipose tissue. Collectively, our results characterize imeglimin as a potential agent promoting energy expenditure and gut integrity, providing new insights into its mechanisms of action.

Distinct roles of the preparatory and payoff phases of glycolysis in hematopoietic stem cells.

In physiological conditions, most adult hematopoietic stem cells (HSCs) maintain a quiescent state. Glycolysis is a metabolic process that can be divided into preparatory and payoff phases. Although the payoff phase maintains HSC function and properties, the role of the preparatory phase remains unknown. In this study, we aimed to investigate whether the preparatory or payoff phases of glycolysis were required for maintenance of quiescent and proliferative HSCs. We used glucose-6-phosphate isomerase (Gpi1) as a representative gene for the preparatory phase and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as a representative gene for the payoff phase of glycolysis. First, we identified that stem cell function and survival were impaired in Gapdh-edited proliferative HSCs. Contrastingly, cell survival was maintained in quiescent Gapdh- and Gpi1-edited HSCs. Gapdh- and Gpi1-defective quiescent HSCs maintained adenosine-triphosphate (ATP) levels by increasing mitochondrial oxidative phosphorylation (OXPHOS), whereas ATP levels were decreased in Gapdh-edited proliferative HSCs. Interestingly, Gpi1-edited proliferative HSCs maintained ATP levels independent of increased OXPHOS. Oxythiamine, a transketolase inhibitor, impaired proliferation of Gpi1-edited HSCs, suggesting that the nonoxidative pentose phosphate pathway (PPP) is an alternative means to maintain glycolytic flux in Gpi1-defective HSCs. Our findings suggest that OXPHOS compensated for glycolytic deficiencies in quiescent HSCs, and that in proliferative HSCs, nonoxidative PPP compensated for defects in the preparatory phase of glycolysis but not for defects in the payoff phase. These findings provide new insights into regulation of HSC metabolism, which could have implications for development of novel therapies for hematologic disorders.

Mesenchymal loss of p53 alters stem cell capacity and models human soft tissue sarcoma traits.

Soft tissue sarcomas (STSs) are a heterogeneous group of tumors that originate from mesenchymal cells. p53 is frequently mutated in human STS. In this study, we found that the loss of p53 in mesenchymal stem cells (MSCs) mainly causes adult undifferentiated soft tissue sarcoma (USTS). MSCs lacking p53 show changes in stem cell properties, including differentiation, cell cycle progression, and metabolism. The transcriptomic changes and genetic mutations in murine p53-deficient USTS mimic those seen in human STS. Furthermore, single-cell RNA sequencing revealed that MSCs undergo transcriptomic alterations with aging-a risk factor for certain types of USTS-and that p53 signaling decreases simultaneously. Moreover, we found that human STS can be transcriptomically classified into six clusters with different prognoses, different from the current histopathological classification. This study paves the way for understanding MSC-mediated tumorigenesis and provides an efficient mouse model for sarcoma studies.

A culture platform to study quiescent hematopoietic stem cells following genome editing.

Other than genetically engineered mice, few reliable platforms are available for the study of hematopoietic stem cell (HSC) quiescence. Here we present a platform to analyze HSC cell cycle quiescence by combining culture conditions that maintain quiescence with a CRISPR-Cas9 genome editing system optimized for HSCs. We demonstrate that preculture of HSCs enhances editing efficiency by facilitating nuclear transport of ribonucleoprotein complexes. For post-editing culture, mouse and human HSCs edited based on non-homologous end joining and cultured under low-cytokine, low-oxygen, and high-albumin conditions retain their phenotypes and quiescence better than those cultured under the proliferative conditions. Using this approach, HSCs regain quiescence even after editing by homology-directed repair. Our results show that low-cytokine culture conditions for gene-edited HSCs are a useful approach for investigating HSC quiescence ex vivo.

SLC15A4 mediates M1-prone metabolic shifts in macrophages and guards immune cells from metabolic stress.

The amino acid and oligopeptide transporter Solute carrier family 15 member A4 (SLC15A4), which resides in lysosomes and is preferentially expressed in immune cells, plays critical roles in the pathogenesis of lupus and colitis in murine models. Toll-like receptor (TLR)7/9- and nucleotide-binding oligomerization domain-containing protein 1 (NOD1)-mediated inflammatory responses require SLC15A4 function for regulating the mechanistic target of rapamycin complex 1 (mTORC1) or transporting L-Ala-γ-D-Glu-meso-diaminopimelic acid, IL-12: interleukin-12 (Tri-DAP), respectively. Here, we further investigated the mechanism of how SLC15A4 directs inflammatory responses. Proximity-dependent biotin identification revealed glycolysis as highly enriched gene ontology terms. Fluxome analyses in macrophages indicated that SLC15A4 loss causes insufficient biotransformation of pyruvate to the tricarboxylic acid cycle, while increasing glutaminolysis to the cycle. Furthermore, SLC15A4 was required for M1-prone metabolic change and inflammatory IL-12 cytokine productions after TLR9 stimulation. SLC15A4 could be in close proximity to AMP-activated protein kinase (AMPK) and mTOR, and SLC15A4 deficiency impaired TLR-mediated AMPK activation. Interestingly, SLC15A4-intact but not SLC15A4-deficient macrophages became resistant to fluctuations in environmental nutrient levels by limiting the use of the glutamine source; thus, SLC15A4 was critical for macrophage's respiratory homeostasis. Our findings reveal a mechanism of metabolic regulation in which an amino acid transporter acts as a gatekeeper that protects immune cells' ability to acquire an M1-prone metabolic phenotype in inflammatory tissues by mitigating metabolic stress.

Obesity accelerates hair thinning by stem cell-centric converging mechanisms.

Obesity is a worldwide epidemic that predisposes individuals to many age-associated diseases, but its exact effects on organ dysfunction are largely unknown1. Hair follicles-mini-epithelial organs that grow hair-are miniaturized by ageing to cause hair loss through the depletion of hair follicle stem cells (HFSCs)2. Here we report that obesity-induced stress, such as that induced by a high-fat diet (HFD), targets HFSCs to accelerate hair thinning. Chronological gene expression analysis revealed that HFD feeding for four consecutive days in young mice directed activated HFSCs towards epidermal keratinization by generating excess reactive oxygen species, but did not reduce the pool of HFSCs. Integrative analysis using stem cell fate tracing, epigenetics and reverse genetics showed that further feeding with an HFD subsequently induced lipid droplets and NF-κB activation within HFSCs via autocrine and/or paracrine IL-1R signalling. These integrated factors converge on the marked inhibition of Sonic hedgehog (SHH) signal transduction in HFSCs, thereby further depleting lipid-laden HFSCs through their aberrant differentiation and inducing hair follicle miniaturization and eventual hair loss. Conversely, transgenic or pharmacological activation of SHH rescued HFD-induced hair loss. These data collectively demonstrate that stem cell inflammatory signals induced by obesity robustly represses organ regeneration signals to accelerate the miniaturization of mini-organs, and suggests the importance of daily prevention of organ dysfunction.

p38α plays differential roles in hematopoietic stem cell activity dependent on aging contexts.

Hematopoietic stem cells (HSCs) and their progeny sustain lifetime hematopoiesis. Aging alters HSC function, number, and composition and increases risk of hematological malignancies, but how these changes occur in HSCs remains unclear. Signaling via p38 mitogen-activated kinase (p38MAPK) has been proposed as a candidate mechanism underlying induction of HSC aging. Here, using genetic models of both chronological and premature aging, we describe a multimodal role for p38α, the major p38MAPK isozyme in hematopoiesis, in HSC aging. We report that p38α regulates differentiation bias and sustains transplantation capacity of HSCs in the early phase of chronological aging. However, p38α decreased HSC transplantation capacity in the late progression phase of chronological aging. Furthermore, codeletion of p38α in mice deficient in ataxia-telangiectasia mutated, a model of premature aging, exacerbated aging-related HSC phenotypes seen in ataxia-telangiectasia mutated single-mutant mice. Overall, these studies provide new insight into multiple functions of p38MAPK, which both promotes and suppresses HSC aging context dependently.

ATP turnover and glucose dependency in hematopoietic stem/progenitor cells are increased by proliferation and differentiation.

Hematopoietic stem cells (HSCs) are quiescent cells in the bone marrow niche and are relatively dependent on glycolytic ATP production. On the other hand, differentiated cells, including hematopoietic progenitor cells (HPCs), preferentially generate ATP via oxidative phosphorylation. However, it is unclear how cellular differentiation and the cell cycle status affect nutritional requirements and ATP production in HSCs and HPCs. Using a newly developed culture system, we demonstrated that survival of HPCs was strongly dependent on glucose, whereas quiescent HSCs survived for a certain duration without glucose. Among HPCs, granulocyte/monocyte progenitors (GMPs) were particularly dependent on glucose during proliferation. By monitoring the ATP concentration in live cells, we demonstrated that the ATP level was maintained for a short duration without glucose in HSCs, possibly due to their metabolic flexibility. In addition, HSCs exhibited low ATP turnover, whereas HPCs including GMPs demonstrated high ATP turnover and required efficient ATP production from glucose. These findings show that ATP turnover and nutritional requirements differ between HSCs and HPCs according to the cell cycle and differentiation status.