Logic and mechanisms of metabolite signalling

Abstract

Metabolites have emerged as central regulators of biological function, but understanding mechanisms of metabolite regulation has proven challenging. In 2021 we have seen discoveries in the field of metabolite signalling motivated by a combination of scientific intuition and the elegant deployment of new technologies.

Metabolites are typically considered in catabolic and anabolic roles. A major theme emerging over the past decade is a growing appreciation that many metabolic intermediates have key regulatory functions. This mode of biological adaptation is based on the idea that cells must respond quickly and locally to metabolic perturbations to facilitate appropriate biological outputs. We face two common challenges when discovering and contextualizing modes of metabolite regulation in cells, tissues and organisms. The first challenge can be formulated in the question: “why this metabolite?”. Understanding the logic of why a particular metabolite responds in a certain way to specific inputs can provide unifying models for metabolic regulation and will generate testable hypotheses. The second challenge is technical in nature. Understanding how a metabolite can modify biological processes is inherently difficult to determine and is not readily informed by the systematic techniques that have dominated late 20th and early 21st century cell biology. As such, over the past decade, discoveries in this field have been characterized by a combination of scientific intuition and the elegant deployment of new technologies. Herein, I discuss five such examples from 2021 that exemplify the exciting new directions and research themes in the field of metabolite signalling.

The Vander Heiden group revisited one of the longest standing ‘why’ questions in cell metabolism, seeking to better understand why lactate production as a consequence of aerobic glycolysis coincides so faithfully with the proliferative state in mammalian cells¹. Longstanding, but not entirely satisfying explanations, focused on the role of ATP generated by glycolysis and its contributions to biomass generation². In the study by Luengo et al., the authors used a combination of elegant approaches to uncouple the various energetic outputs of aerobic glycolysis¹. In doing so, they provided strong evidence for a generalizable model for elevated lactate production that is based on its critical role in regenerating NAD⁺, which is a limiting co-factor for numerous biosynthetic reactions required to build a new cell. Conceptualizing lactate production as a driver of NAD⁺ regeneration provides a powerful framework and generates testable hypotheses that link aerobic glycolysis to maintenance of NAD⁺/NADH homeostasis. These principles could prove extremely important in light of the wide range of biological processes that are directly regulated by the cytosolic NAD⁺/NADH couple.

The role of exogenous nutrients in regulating organismal adaptation is a major theme in the field of metabolite signalling. In this context, Goncalves and colleagues explored a different ‘why’ question in their study published in September 2021³. The question centered on the links between fructose consumption and incidence of metabolic diseases and related cancers, and was based on both clinical and pre-clinical data^3,4. Intriguingly, mice that were fed calorie-matched high-fat diets had increased obesity when the diet was supplemented with high levels of fructose, despite identical caloric consumption to the control group, suggesting that fructose somehow alters energy balance³. The authors hypothesized that fructose might remodel intestinal properties and nutrient uptake, which could provide an explanation for the obesogenic role of this metabolite. Remarkably, they show that dietary fructose increased the surface area of intestinal villi to drive enhanced nutrient absorption in mice. Most interestingly, metabolite profiling of fructose-treated villi suggested an effect on pyruvate kinase M2 isoform (PKM2)-dependent pyruvate production. This led the authors to hypothesize that fructose 1-phosphate (F1P), a product of fructose metabolism that is structurally similar to an endogenous regulator of PKM2 (fructose 1,6-bisphosphate), could inhibit PKM2. This model turned out to be correct, and the authors demonstrated that the interaction between F1P and PKM2 was required for fructose-dependent transcriptional remodelling of villi to increase surface area and nutrient absorption.

When considering how cells respond to metabolites of exogenous origin, fundamental insights can also be gleaned from the study of model organisms, as exemplified by a study published in February 2021 from the Pierce lab⁵. The amoeba Dictyostelium discoideum exists in unicellular form under nutrient replete conditions, but aggregates into a multicellular organism upon nutrient limitation. The Pierce group exploited this phenomenon to understand how nutrient status could regulate this fundamental adaptive process. In doing so, they discovered that partitioning of the amino acid cysteine has a central role in this cell fate decision. Upon nutrient limitation, D. discoideum exhibit elevated levels of mitochondrial reactive oxygen species (ROS), which shunts cysteine towards production of the antioxidant thiol glutathione, a major reducing factor in the cell. The authors demonstrated that this process limited availability of cysteine, which was both necessary and sufficient to deplete cells of iron–sulfur containing proteins and inhibit protein synthesis rates. As such, cysteine sequestration into the glutathione pool facilitated the differentiated multicellular switch in D. discoideum. This remarkable mechanism could provide general frameworks to understand how cellular fate could be regulated by cellular thiols and redox biochemistry in multicellular organisms.

The above study highlights the central role of glutathione and its critical biological activities in mitochondria and the cytosol in cellular redox biology. However, how glutathione pools are partitioned within the cell has remained a major mystery, until the November 2021 discovery of the Birsoy group⁶. In this study, the authors applied acute manipulations of cellular glutathione status, combined with an elegant combination of proteomics and differential metabolite profiling to address this longstanding mystery in human cells. In doing so, they discovered an essential role for the orphan mitochondrial transporter SLC25A39 in mitochondrial glutathione uptake. Using an analogous approach, the critical role of this transporter in mitochondrial GSH uptake was confirmed in a pre-print paper from the Shen group⁷. The discovery of SLC25A39 as being essential for mitochondrial glutathione uptake allowed the Birsoy team to assess, for the first time, specific roles of mitochondrial glutathione in cell biology and physiology⁶. Among the most striking discoveries is the essentiality of mitochondrial glutathione for erythropoiesis, which underscores a core physiological role for mitochondrial glutathione in iron–sulfur containing protein assembly. Moreover, they demonstrated in human cells that glutathione availability and redox status regulates mitochondrial import of glutathione via SLC25A39, thereby providing a mechanistic basis for the regulation of glutathione partitioning through cellular redox status.

Intuition regarding the behaviour of metabolites in particular biological contexts often leads to the discovery of mechanisms of metabolic regulation. A converse approach that is often taken in other areas of cell biology is to apply hypotheses-free systematic manipulations to identify causal interactions, for example, between particular proteins. Understanding systematic interactions between metabolites and proteins is far more challenging than understanding protein–protein interactions, as standard ‘big data’ technologies cannot be deployed in this realm of biology. For example, direct determination of causal metabolite-driven regulation is hampered by the fact that interactions between metabolites and proteins are typically low affinity and therefore not amenable to standard screening methods. In findings from a pre-print paper, the Rutter lab has reported the development of a technology that has the potential to be transformative in this respect⁸. The platform, termed MIDAS (Mass spectrometry Integrated with equilibrium Dialysis for the discovery of Allostery Systematically) enables the systematic discovery of metabolite–protein interactions. Based on the principle of equilibrium dialysis, a protein is separated from a defined pool of metabolites by a semi-permeable membrane that only allows passage of small molecules. By allowing the system to reach equilibrium, metabolites that interact with the protein of interest will concentrate in the protein-containing chamber. This differential concentration can be determined by mass spectrometry, enabling the identification of metabolite–protein interactions. The authors exemplify this approach by systematically identifying hundreds of novel metabolite interactions with 33 metabolic enzymes. They then use this information as a basis to discover functional and physiologically relevant models of metabolite regulation over enzyme function. MIDAS not only provides a rich new resource of metabolic regulation, but lays the groundwork for greatly expanding our understanding of how metabolites can remodel cellular function.

Together, the studies cited here illustrate an emerging appreciation for the ubiquitous importance of metabolite regulation in biology. Moreover, this work exemplifies the powerful approaches and principles that can be applied to understand the mechanisms and logic behind how organisms use metabolites to control cell function and fate.

Key advances.

• In mammalian cells, lactate production as a consequence of aerobic glycolysis drives NAD⁺ regeneration, which might influence a wide range of biological processes that are directly regulated by the cytosolic NAD⁺/NADH couple¹.

• In mice, dietary fructose increased the surface area of intestinal villi via the interaction of fructose 1-phosphate and pyruvate kinase M2 isoform, which drove enhanced nutrient absorption, thereby demonstrating the potential of exogenous nutrients to regulate organismal adaptation³.

• Work in the model amoeba Dictyostelium discoideum showed that cysteine sequestration into the glutathione pool facilitated the differentiated multicellular switch, providing a framework for how cellular fate might be regulated by cellular thiols and redox biochemistry⁵.

• In human cells, glutathione availability and redox status regulates mitochondrial import of glutathione via SLC25A39, providing a mechanistic basis for the regulation of glutathione partitioning through cellular redox status⁶.

• A pre-print paper reports a promising new approach using mass spectrometry and the principle of equilibrium dialysis (MIDAS), which will enable the systematic discovery of metabolite–protein interactions⁸.