TCA cycle turning is dispensable In Vivo and its loss promotes stem cell function during regeneration

Abstract In 1937, Krebs discovered that nutrients are oxidized by the conversion of 6-carbon citrate to 4-carbon oxaloacetate, followed by the synthesis of citrate from oxaloacetate and nutrient-derived acetyl-CoA through citrate synthase (CS). Nearly a century of work has enshrined the Krebs cycle (or tricarboxylic acid, TCA cycle) in textbooks as an integral part of oxidative energy production. The cycle is also thought to play critical roles in biosynthesis. However, the essentiality of a full turning cycle in vivo has never been tested. The bone marrow produces ~300 billion new cells daily, far more than any other tissue. These cells are derived from self-renewing hematopoietic stem cells (HSC). To test if this highly proliferating system relies on an intact TCA cycle, we created a Cs conditional knockout mouse for the first time and deleted Cs in adult hematopoiesis with Mx1Cre.HSPC survival and proliferation do not require an intact TCA cycle. Cs deletion blocked the development of some differentiated cell types, including T cells and erythroid lineages, but to our surprise, it did not reduce the number or frequency of bone marrow hematopoietic stem cells (HSCs) and most progenitors. Genotyping of single HSC-derived colonies, western blot, and metabolic tracing experiments confirmed efficient deletion. Cs deletion increased the frequency and number of spleen HSCs, MPPs, and HPCs, and of bone marrow and spleen common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs). Despite the prevalent idea that the TCA cycle supplies anabolic intermediates, Cs deletion did not impair the proliferation of HSCs and increased the proliferation of myeloid progenitors. HSPCs without a TCA cycle have increased respiration, nutrient consumption, and biosynthesis. The classical metabolic function of the TCA cycle is to extract energy stored in nutrients by linking nutrient catabolism to respiration. Surprisingly, Cs deletion increased respiration in HSPCs, suggesting the TCA cycle is not required for their respiration. We used in vivo stem cell metabolomics and stable isotope tracing methods we developed to show that hematopoietic cells adapted to TCA cycle loss by reprogramming their metabolism, markedly increasing nutrient consumption and biosynthesis. HSPCs without a TCA cycle outcompete HSPCs with a TCA cycle during regeneration HSCs regenerate the hematopoietic system after bone marrow transplantation. We hypothesized that the increased ability of CsΔ/Δ HSPCs to consume nutrients and divert them to anabolism as compared to wild-type HSPCs would increase their regenerative ability. Competitive transplant experiments showed that disruption of citrate cycling increased HSC regeneration, myeloid progenitor proliferation, and myelopoiesis in vivo. HSCs without a TCA cycle outcompeted wild-type HSCs during regeneration. These effects were cell-autonomous and were not phenocopied by genetic ablation of cytosolic citrate use. We made several unanticipated discoveries that change the way we understand metabolism in vivo, in stem cells and more generally: 1) Unlike textbook depictions, a TCA cycle is not required for respiration or cell survival in vivo; 2) The TCA cycle does not support biosynthesis but on the contrary limits nutrient uptake and biosynthesis; 3) Loss of TCA cycling increases HSC self-renewal, and HSCs with reduced cycle activity outcompete wild type HSCs during regeneration.