Introduction to Glucose Metabolism
Many think of glucose as something diabetics have to test each day using a blood monitor, or a quick energy boost for someone exercising intensely. However, the simple sugar glucose, a monosaccharide, fuels most of the cells in our bodies. Disaccharides that contain glucose (e.g., sucrose is comprised of glucose and fructose) and glucose polymers (e.g., starch and glycogen) are carbohydrates that are consumed by organisms from bacteria to humans to produce energy. These carbohydrates are broken down into component monosaccharides like glucose and lactose. The process of glycolysis generates the energy currency of cells, ATP, as well as precursor molecules for nucleotides, lipids and amino acids. Because glucose is the cell fuel source, the uptake of glucose and its subsequent metabolism is increased by cells that divide rapidly like cancer cells. The more energy and precursor molecules the cancer cell can create for itself, the more rapidly the tumor can grow.
Because glucose metabolism is central to cellular functioning, changes that decrease glucose uptake or increase glycolysis have a widespread effect on on both the cells and organism. How does a simple sugar molecule create such broad effects on health? For example, diabetes results from the inability to store glucose because of a lack of insulin, a hormone that draws glucose from the blood and stores it as glycogen in the liver, muscles and adipose tissue. High levels of sugar in the blood negatively affect the body over the long term, damaging blood vessels and eyesight, making the kidneys work harder to excrete the excess sugar and increasing the risk of stroke and coronary artery disease. Because cancer cells have such a high metabolic demand for glucose, many of the mutations in cancers affect pathways that regulate glucose uptake and glucose breakdown, allowing the cancer cells to survive and grow, crowding out nearby normal cells.
Glucose metabolism is altered by processes other than mutations or an reduced production of a hormone. Throughout its life cycle, a cell will vary its requirements for glucose. For example, the cells that comprise our innate immune response are typically in a quiescent or steady state. However, when these immune cells encounter an foreign invader, they become activated and increase their demand for glucose. To respond to a potential pathogen, the activated cells need glucose to fuel cell proliferation and the production of cytokines, chemicals that activate other immune cells and initiate an inflammatory response. The typical signs of inflammation are red inflamed area that may be painful to the touch, such as a cut that becomes infected. Most inflammation resolves when the infection is eliminated, leaving behind whole skin in the instance of a cut, and the activated immune cells become quiescent again.
An Interesting Observation about Glucose Metabolism in M2 Macrophages
Glucose uptake, immunity and metabolism are cellular pathways that are intertwined such that understanding how glucose is utilized in macrophages illuminates gene induction and regulation in activated macrophages. In a recently published eLife article, Covarrubias et al. studied how activation of murine bone marrow-derived macrophages (BMDMs) by interleukin-4 (IL-4), a signaling cytokine, altered glucose metabolism in the cells and regulated a subset of genes involved in macrophage activation. Activated macrophages are categorized as either M1 (promotes inflammation) or M2 (suppresses the immune response) with each type stimulated by different molecules. BMDMs were stimulated with IL-4 then the mass spectrometry profile of metabolic proteins was compared to unstimulated BMDMs. Most of the activated metabolic pathways matched the known M2 macrophage profile with the exception of one pathway normally seen in M1 macrophages: Upregulated glycolysis. This unexpected metabolic pathway became the focus of further study by the authors.
Glucose uptake in BMDMs increased over time when stimulated by IL-4, and this increased uptake was eliminated in the presence of an Akt inhibitor. Akt is a kinase involved in glucose uptake and metabolism and implicated in M2 activation. When a glycolysis inhibitor and IL-4 were both added to the BMDMs, expression of some genes associated with the M2 macrophage were reduced. These results suggest that Akt regulates increased glucose metabolism, which contributes to inducing M2 gene expression.
Uncovering the Role of Akt in Macrophage Activation
To further examine the role Akt may play in activating M2 phenotype, BMDMs were pretreated with one of two structurally distinct Akt inhibitors and stimulated with IL-4, and six genes that are hallmarks of M2 activation were then amplified in qPCR. Akt inhibition decreased expression of three of the six M2 genes. Probing how Akt could affect only a subset of M2 activation genes involved testing its role in histone acetylation. When BMDMs were treated with IL-4, H3 and H4 histone acetylation increased globally. When an Akt inhibitor was added to the IL-4 treatment of BMDMs, the overall level of acetylation was reduced. Closer examination of M2 gene promoters showed that histone acetylation increased at the M2 promoters and the acetylation seemed to correlate to the degree of gene activation. Interestingly, when an Akt inhibitor was added and the M2 promoters examined, the activation was only seen in the three M2 genes the authors classified as Akt dependent while the Akt-independent genes had no change in histone acetylation. Because histone acetylation means DNA is more accessible for expression, researchers tested if RNA polymerase II was recruited to the M2 promoters. The M2 genes that depended on Akt activation and whose histones were acetylated, did have RNA polymerase II associated with them in the presence of Akt.
Seeking to understand how Akt controls histone acetylation, Covarrubias et al. turned to the substrate for acetylation, acetyl coenzyme A (AcCoA). They found that IL-4-stimulated BMDMs did increase levels of AcCoA in the cells. ATP-citrate lyase (Acly) is a known regulator of AcCoA and also known to be phosphorylated and activated by Akt. The M2 macrophages stimulated by IL-4 did have Acly phosphorylated by Akt. To confirm the connection to AcCoA levels, Akt or Acly inhibitors blocked an increase in AcCoA for IL-4-stimulated macrophages.
Understanding How Acly Influenced Macrophage Activation
Now that researchers had uncovered a connection between Akt and Acly that controlled M2 macrophage activation, they wanted to understand the role of Acly in M2 macrophages. Mirroring the results with Akt, two different Acly inhibitors reduced activation and histone acetylation of Akt-dependent M2 macrophage genes in IL-4 stimulated BMDMs but not for genes that were Akt independent. In addition, there was a decrease of RNA polymerase II recruited to the Akt-dependent M2 gene promoters in the presence of an Acyl inhibitor. Because Acyl and Akt could increase the levels of AcCoA, but only induce histone acetylation of a subset of M2 macrophage genes, Covarrubias et al. tested how inhibiting p300, a histone acetyltransferase regulated by the levels of AcCoA, acted on the M2 macrophage genes. Results showed that p300 inhibitor reduced the induction of Akt-dependent M2 genes, but not the Akt-independent M2 genes.
The Role of mTORC1 in Macrophage Activation
mTORC1, also known as mammalian target of rapamycin complex 1, is found downstream of Akt in the Akt-mTORC1 signaling pathway and controls protein synthesis. To test if mTORC1 had a role in M2 macrophage activation, BMDMs missing regulatory-associated protein of mTOR (Raptor), a key component of the mTORC1 complex, were stimulated with IL-4 and the levels of M2 macrophage genes examined. Matching the results from Akt and Acly, only the Akt-dependent M2 gene expression was reduced; there was no change in the Akt-independent genes. Furthermore, both Acly protein levels and Acly phosphorylation were reduced in BMDMs deficient in mTORC1. BMDMs with constitutively active mTORC1 had increased levels of Acly that was reduced when treated with the mTORC1 inhibitor rapamycin. These results suggest that mTORC1 regulates the level of Acly expression and phosphorylation in M2 macrophages.
Because mTORC1 is known to be regulated by amino acid availability, Covarrubias et al. manipulated the levels of amino acids in IL-4-stimulated BMDMs. In amino acid-deficient medium or low amino acid medium, M2 macrophages showed lower mTORC1 activity. Akt phosphorylation was higher in cells with normal levels of amino acids compared to those in deficient or low amino acid medium. Acly protein levels and phosphorylation as well as AcCoA levels were dependent on the amount of amino acids available to the cells and increased with more amino acid availability. In addition, lower levels of amino acids reduced M2 gene induction for Akt-dependent genes, but not the Akt independed M2 genes. These results suggest having sufficient level of nutrients is important for induction of the Akt- and Acly-dependent M2 genes.
The End Result of the Akt-Acly Pathway on M2 Macrophage Activation
A transcriptional profile of IL-4-stimulated BMDMs treated with or without Akt or Acly inhibitors upregulated or downregulated 750 genes; most genes affected by the Akt inhibitor also affected by the Acly inhibitor while only a subset of genes sensitive to Akt inhibition were affected by Acly inhibition. This suggests that in M2 macrophages, Akt acts on Acly for M2 activation. The genes affected by Akt and Acly were enriched for cell cycle, DNA replication and cell proliferation pathways as well as chemokines and eosinophil associated ribonucleases (Ear) family. The Ear family was noted by researchers because these genes are induced during inflammation, not typically part of the M2 macrophage profile.
Covarrubias et al. has demonstrated that the metabolic context of M2 macrophages activates the Akt-mTORC1 to regulate a specific subset of M2 genes. Because of the connection between resources available to the macrophage and the cells’s ability to carry out its immune functions, responding appropriately to the nutrient environment affects how a macrophage invests in its energy-intensive response to a pathogen. Finding that M2 macrophages increased their glucose uptake and glycolysis has revealed that metabolism plays a role in macrophage activation through the Akt-mTORC1 pathway. Who knew a simple glucose molecule could change our understanding of immune regulation?
Covarrubias, A.J. et al. (2016) Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation. eLife 5, e11612. doi: 10.7554/eLife.11612