Carbohydrate metabolism
Recent literature has revealed that macrophage differentiation and polarization strongly influences macrophage metabolism 153. Also the reciprocal relationship, i.e.
that metabolism determines differentiation state may be true 154. When combined,
these observations imply that macrophage metabolic state may be just as hetero- Figure 5. The CK/PCr system for intracellular ATP transfer and temporal and spatial energy buffering.
Creatine is transported across the cell membrane via the creatine transporter (CRT). Cytosolic CK (CK- c) regulates intracellular PCr/Cr and ATP/ADP ratios. CK coupled to glycolytic enzymes (CK-g) accepts ATP produced by glycolysis, while ATP from oxidative phosphorylation (OXPHOS) is accepted by mito- chondrial CK (mtCK), which - in turn - is coupled to the adenine nucleotide translocator (ANT). CK may also be associated with specific sites of ATP consumption (CK-a), for example ATP-gated ion pumps. The CK/PCr circuit, therefore, connects sites of ATP production (glycolysis or OXPHOS) with subcellular sites of ATP utilization (ATPases) without the need for ATP or ADP to diffuse back and forth. (Adapted from Wallimann et al. 141)
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geneous and complex as their polarization phenotype. During the early studies on macrophage metabolism, this was not (fully) acknowledged and general conclusions were drawn from experiments performed on macrophages with different origins and activation/polarization status. A significant number of studies have been per- formed using thioglycollate elicited mouse peritoneal macrophages (TEPMs) which appear - at least partially - polarized towards a M2 phenotype 155, 156. These TEPMs
consume three times more oxygen than resident peritoneal macrophages 157. None-
theless, carbohydrate metabolism in all macrophages is fundamentally glycolytic 158.
Monocytes acquire a glycolytic metabolism as they differentiate into macrophages
in vitro 159, although a concomitant increase in aerobic metabolism (mitochondrial
mass and cytochrome oxidase activity) has also been described under aerobic con- ditions 160, 161. Upon polarization, macrophage metabolism is altered to shape the
new activation state and to meet the specific requirements for performing effec- tor functions such as phagocytosis, cytokine secretion, ROS production, and ECM synthesis 153. After polarization, the largest amount (95%) of all glucose consumed
by macrophages remains converted into lactate 158 instead of being oxidized via
the TCA-cycle and OXPHOS, even under aerobic conditions. In this regard, macro- phage metabolism closely resembles that of cancer cells where this phenomenon is termed the “Warburg phenotype”.
Glucose is imported by macrophages via different GLUT isoforms. The expres- sion of GLUT isoforms seems to be dependent on species, origin (primary cells or cell lines), and differentiation and polarization state. THP-1 cells (human monocytic cell line) express GLUT1,3, and 4 and upon differentiation into macrophages up- regulate GLUT3 and 5 expression 162. In primary human blood monocytes GLUT1 is
expressed and upregulated during differentiation into macrophages 159, while RAW
264.7 cells (murine macrophage cell line) mainly express GLUT3 163. Stimulation
with LPS (driving M1 polarization) causes an upregulation of GLUT isoform expres- sion, leading to elevated influx of glucose 164, 165. This is accompanied by an increase
in glycolysis gene expression and glycolytic flux to lactate 154, 158. The molecular basis
of this upregulation in glycolysis involves a switch in the expression of liver-type phosphofructokinase (L-type PFK2) in resting TEPMs to the more active ubiquitous PFK2 (uPFK2) in M1 TEPMs 158. This causes a nine times rise in PFK2 activity and five
times increase in fructose-2,6-bisphosphate levels. In contrast, IL-4/IL-13 stimula- tion, which drives M2 polarization, causes no alteration in PFK2 expression and has only mild metabolic effects.
The increased rate of gene transcription and synthesis of macromolecules dur- ing macrophage activation increases the bioenergetic demand but also the need for anabolic intermediates. Accordingly, the activity of the pentose phosphate pathway (PPP) is increased in LPS-stimulated M1 macrophages and a substantial portion of
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glucose-derived carbon is routed into the PPP 154. Increased PPP activity switches
these cells to a reductive mode with elevated levels of reduced glutathione (GSH) and NADH. Concomitantly, the oxygen consumption rate and expression of OXPHOS genes are downregulated in classically activated macrophages 154, 158. In contrast,
IL-4 stimulated M2 cells exhibit an upregulation in TCA-cycle genes 158 and mito-
chondrial biogenesis 166 and adapt a more oxidative mode with lower GSH levels 167
and increased superoxide production 168.
Lipid metabolism
Although glucose is the main energy producing substrate in macrophages, these cells are also able to derive cellular energy from fatty acid (FA) oxidation. They are equipped with intracellular triglyceride lipase (TGL) 169, 170 but also produce and se-
crete lipoprotein lipase (LPL) 171-174. These enzymes release FAs through the hydro-
lysis of intracellular triacylglycerol (TG) stores or through the hydrolysis of exog- enously supplied lipoproteins, respectively. LPL activity and fatty acid uptake and oxidation are inhibited in M1 and upregulated in M2 macrophages 166, 175, 176. TEPMs,
which are partially M2 polarized, relatedly express a substantial amount of adipose TGL (ATGL). In the absence of ATGL (by double knockout), TEPMs accumulate TG- rich droplets, reducing the availability of FAs for β-oxidation, and have decreased ATP levels even while glucose is sufficiently available 177. Under conditions where
glucose is limiting, LPL-catalyzed hydrolysis of exogenously supplied lipoproteins have been shown to provide energy for (M2 polarized) macrophages during pe- riods of intense metabolic activity (e.g. phagocytosis) 178. However, the presence
of FAs does not suppress normal glucose or glutamine utilization when this is suf- ficiently available 179. Macrophages probably first esterify and convert internalized
FAs into TGs before hydrolysis by ATGL and transport into the mitochondria for β-oxidation 177. The largest fraction of oxidized FAs is incorporated into phospholip-
ids and TAGs both in TEPMs and unstimulated peritoneal macrophages 179, 180. Apart
from supplying energy, lipid metabolism, therefore, also regulate membrane fluid- ity which is a determining factor in macrophage adhesion and phagocytosis 153, 181.
Since the saturation status of fatty acids determines membrane fluidity, saturated and unsaturated FAs differentially affect phagocytosis in macrophages: saturated FAs, like palmitic acid, inhibit phagocytosis, while unsaturated FAs, like oleic acid, stimulate phagocytosis 180, 181.
Amino acid metabolism
Macrophages, and immune cells in general, consume relatively large amounts of glutamine 182. Polarization state does not seem to affect glutamine utilization or
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increase in glutamine consumption has been observed in LPS stimulated mouse peritoneal macrophages (resident and Bacillus Calmette–Guerin (BCG) activated) later, after 24 and/or 48 hours 183. As mentioned in 3.2 above, glutamine can be
converted to lactate in a reaction pathway that involves the production of NADPH by malic enzyme. Increased NADPH-production may be beneficial to LPS-stimulated (M1) macrophages, since NADPH is required for the production of ROS via NADPH oxidase (NOX) during the oxidative burst, the production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS), and as co-factor in anabolic reactions (e.g. cy- tokine production, and DNA and RNA synthesis) 184. The production of NO involves
the conversion of another amino acid, arginine, into citrulline and NO by iNOS. Glu- tamine itself may also contribute to NO production by being converted into arginine first. However, this has only been shown for macrophages cultured in the absence of extracellular arginine 183.
Although arginine, therefore, plays a role in M1 activation, its metabolism through arginase 1 is upregulated in mouse M2 macrophages and not in M1 cells 153.
Alternatively activated (M2) mouse macrophages metabolize L-Arg into ornithine and urea thereby contributing to polyamine production for collagen synthesis, cell proliferation, and tissue remodeling 185. It is not clear whether arginine metabolism
is also differentially regulated in human M2 macrophages.
Another amino acid with a notable role in immunometabolism is tryptophan. This essential amino acid is the starting substrate for de novo biosynthesis of NAD+
(see 3.3 above). The first enzyme of this pathway, IDO, is downregulated by IL-4 but upregulated upon IFNγ or LPS stimulation in monocytes and macrophages 186, 187,
resulting in increased tryptophan utilization. NAD+ has been implicated in the regu-
lation of inflammatory cytokine production in macrophages and in the modulation of immune function 133, 135, 136, 188 in addition to its role as metabolic cofactor. De novo
NAD+-synthesis from tryptophan may, therefore, have a role in the immune func-
tion of M1 macrophages. The above examples illustrate that also aspects of amino acid metabolism and polarization state of macrophages are clearly - and probably reciprocally - coupled.