Download Prolonged Triglyceride Storage in Macrophages

Prolonged Triglyceride Storage in
Macrophages: pH o Trumps pO2 and TLR4
Mingfang Lu, Terry Kho and Robert S. Munford
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References
J Immunol 2014; 193:1392-1397; Prepublished online 27
June 2014;
doi: 10.4049/jimmunol.1400886
http://www.jimmunol.org/content/193/3/1392
The Journal of Immunology
Prolonged Triglyceride Storage in Macrophages: pHo Trumps
pO2 and TLR4
Mingfang Lu,1 Terry Kho, and Robert S. Munford
C
ontaining mainly cholesteryl esters and triglycerides,
cytosolic lipid droplets (also called lipid bodies) produce
the foamy appearance often seen in macrophages residing in inflammatory lesions such as granulomas, xanthogranulomatous
kidneys, and atherosclerotic plaques. Although cholesteryl esters typically contribute a larger fraction of the stored lipid, triacylglycerol
(TAG) may comprise a substantial component (1), provide a critical
energy source for phagocytosis (2), and be used by intracellular
pathogens as a source of fatty acids (FAs) (3). The common stimuli
known to promote TAG storage in macrophages include low oxygen
tension (pO2) (3–5) and TLR agonists such as bacterial LPS (6),
bacterial lipopeptides, or polyinosinic:polycytidylic acid (7). Hypoxiainduced triglyceride synthesis has been attributed to increases in lipid
droplet proteins, FA synthesis, and TAG synthesis from glucose (8, 9),
whereas changes in key enzymes (acyl-CoA synthetase long 1 [ACSL1], diacylglycerol acyltransferase-2 [DGAT-2], and adipose triglyceride lipase [ATGL]) have been proposed to promote prolonged TAG
retention in response to TLR ligands (10).
As noted by Mackenzie et al. in 1961 (11), another stimulus to lipid
accumulation is low extracellular pH (pHo) (12, 13). Because both low
pO2 and many inflammatory stimuli induce cells to release small
carboxylic acids, tissues that are hypoxic and/or contain microbial
agonists are often acidic (4, 14, 15). Measurements in human patients
found that pH was often ,6.5 in abscesses (16), which typically are
both anaerobic and microbe laden. In other studies, the median pH of
pus, infected peritoneal fluid, or drainage fluid was 6.75, and the
median pO2 was 28 mm Hg (14). In this study, we used a load-chase
Antibacterial Host Defense Unit, Laboratory of Clinical Infectious Diseases, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
MD 20892
1
Current address: Department of Immunology, Shanghai Medical College, Fudan
University, Shanghai, China.
Received for publication April 7, 2014. Accepted for publication May 27, 2014.
This work was supported by the Division of Intramural Research, National Institute
of Allergy and Infectious Diseases, National Institutes of Health.
strategy to study how extracellular acidity (pHo) influences the effects
of ambient pO2 and LPS stimulation on the retention of TAG by
cultured peritoneal macrophages. We found that low pHo strongly
favors TAG retention in both low and high oxygen environments, and
in the presence and absence of LPS. Macrophages that adapted to
a low pHo environment decreased catabolism of both glucose and FAs,
whereas they increased FA uptake and incorporation into TAG, promoting TAG retention throughout a 72-h chase period.
Materials and Methods
Reagents
Oleic and palmitic acids were from NuChek. [1-14C]-palmitate and
[9,10-3H]oleate were from Moravek, and 2-deoxy-3H-glucose was from
PerkinElmer. Buffers, media, and other reagents were from Sigma-Aldrich.
Macrophage cultures
The animal protocol (LCID 11E) was approved by the National Institute of
Allergy and Infectious Diseases Institutional Animal Care and Use Committee. Harvesting and culture of JAX C57BL/6 peritoneal macrophages
were as described previously (10). Thioglycollate-elicited peritoneal
macrophages were harvested 5 d after injecting 1.0 ml 3% thioglycollate
i.p.; they were allowed to adhere to plastic wells for 3–6 h, washed, and
incubated overnight in DMEM that contained 0.5% FBS (Hyclone),
5.5 mM glucose, 50 mM palmitic acid, 100 mM oleic acid, and 1 mCi/ml
radiolabeled oleate (Fig. 1A, FA load). The cells were then washed and
reincubated (Fig. 1A, chase) in medium that contained half the original
concentrations of nonradioactive FAs and no bicarbonate. The chase medium was buffered by adding 25 mM Mops, Hepes, or Tris to achieve
starting pHo of 6.95–7.1, 7.3–7.5, or 7.6–7.7, respectively, and cells were
then cultured either in a humidified incubator in 21% O2 or in a sealed,
humidified chamber that contained a mixture of 4% O2 and 96% N2. The
cells were harvested after a chase period of 48 or 72 h, and the final pHo
was measured using a Mettler Seven Compact S220 pH/ion reader. In
experiments to study the effect of lactate production on LPS-induced TAG
retention, cells were loaded with [3H]oleate and nonradioactive FA as
described earlier, washed, and cultured in a CO2 incubator in cDMEM that
contained 44 mM NaHCO3, 5% FBS (Hyclone), 20 mM Tris, pH 7.5, 50
mM oleate, 25 mm palmitate, and either 5.5 mM glucose or 4 mM glutamine (17, 18). LPS (2.5–5.0 ng/ml) was added to the chase medium as
indicated.
Address correspondence and reprint requests to Dr. Robert S. Munford, 9000 Rockville Pike, Bethesda, MD 20892-3206. E-mail address: [email protected]
Lipid analysis
Abbreviations used in this article: ACSL-1, acyl-CoA synthetase long 1; ATGL,
adipose triglyceride lipase; DGAT-2, diacylglycerol acyltransferase-2; FA, fatty acid;
pHo, extracellular pH; pO2, oxygen tension; TAG, triacylglycerol.
Preparation of albumin-bound FAs was as described previously (10). The
amount of the radiolabel that remained in TAG ([3H]TAG retention) (10)
was determined by extracting the cellular lipids into isopropanol, thin-
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400886
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Lipid-laden macrophages contribute to pathologies as diverse as atherosclerosis and tuberculosis. Three common stimuli are
known to promote macrophage lipid storage: low tissue oxygen tension (pO2), low extracellular pH (pHo), and exposure to
agonists such as bacterial LPS. Noting that cells responding to low pO2 or agonistic bacterial molecules often decrease pHo by
secreting lactic and other carboxylic acids, we studied how pHo influences the stimulation of triacylglycerol (TAG) storage by low
pO2 and LPS. We found that TAG retention after incubation for 48–72 h was inversely related to pHo when primary macrophages
were cultured in 21% oxygen, 4% oxygen, or with LPS at either oxygen concentration. Maintaining pHo at ∼7.4 was sufficient to
prevent the increase in prolonged TAG storage induced by either low pO2 or LPS. The strong influence of pHo on TAG retention
may explain why lipid-laden macrophages are found in some tissue environments and not in others. It is also possible that other
long-term cellular changes currently attributed to low pO2 or bacterial agonists may be promoted, at least in part, by the decrease
in pHo that these stimuli induce. The Journal of Immunology, 2014, 193: 1392–1397.
The Journal of Immunology
layer chromatography on silica gel G to isolate TAG, and b-scintillation
counting of TAG-containing silica (10). The recovered [3H]TAG correlated
strongly (r2 = 0.90) with measurements of TAG mass in the same cells
(10). To estimate the lipolytic rate in intact cells, we loaded macrophages
with [3H]oleate, incubated them in buffered media for 48 h, then washed
and reincubated them for 48 h in the presence of 10 mM triacsin C (ENZO
Life Sciences) before measuring the extent to which [3H]TAG was depleted (10, 19). Triacsin C inhibits macrophage acyl-CoA synthetases (20,
21), blocking FA thioesterification, and thereby reducing the reincorporation of released FA into TAG. TAG depletion is then attributable to lipolysis (19).
Assays
Results
TAG retention increases at low pHo
We studied TAG retention by loading cells with 3H-FA and following the retention of labeled TAG over 48 or 72 h (10) (Fig. 1A).
We first noticed that retention of radiolabeled TAG increased
when the chase medium was buffered at low pHo. The relationship
between pHo and log10[3H dpm in TAG] after a 48- or 72-h chase
in 21% O2 was approximately linear from pHo ∼6.7 to pHo ∼7.6,
with or without supplemental acids (each at 5 mM) to lower pHo
(Fig. 1B). For each decrease of 0.1 U pHo between 7.4 and 7.0,
TAG retention increased ∼25%.
Low pHo increases FA uptake and FA incorporation into TAG
while decreasing lipolysis
Providing supplementary palmitate and oleate during the chase
greatly increased the amount of cellular TAG mass at the 72-h time
point (Fig. 1C). We therefore studied the effect of pHo on TAG
synthesis from exogenous FA. As expected (11–13), FA uptake
(Fig. 1D) and FA incorporation into TAG (Fig. 1E) were enhanced
at low pHo. In addition, [14C]palmitate added to the medium
during the 72-h chase was incorporated into TAG with the same
relationship to pHo that [3H]TAG was retained from the 3H-oleate
load (Fig. 1F). Incubation at low pHo was also associated with
decreased lipolytic activity in cell lysates (Fig. 1G) and there was
less loss of [3H]TAG at low pHo when FA thioesterification was
inhibited with 10 mM triacsin C to provide a measure of the lipolytic rate (10, 19) (Fig. 1H). These results suggest that macrophages that adapt to low pHo increase TAG storage mainly by
increasing the uptake and incorporation of FA into TAG; a decrease in lipolysis may also contribute.
pHo can trump pO2
Because cells living at low pO2 lower their pHo by secreting lactate
(anaerobic glycolysis), a role for pHo in hypoxia-induced TAG
retention seemed plausible. We cultured cells in 4% O2 (pO2 ∼28
mm Hg), a level measured in tissues such as lymph nodes and
spleen (22, 23). As expected, cells cultured in 4% O2 retained
more of the FA load in TAG than did cells cultured in 21% O2
(pO2 ∼150 mm Hg), but again TAG retention was strongly related
to the final pHo (Fig. 2A, 2B). TAG retention was greatly reduced
by preventing the decline in pHo that occurred as cells adapted to
living at a moderate level of hypoxia, suggesting that pHo may
play a more important role in promoting TAG retention than does
oxygen at this concentration.
pHo-dependent changes in cell metabolism
Although glycolysis increases when macrophages become hypoxic, we found that glucose uptake decreased when cells were
cultured for 72 h at acidic pHo (Fig. 3A), as did both glucose
consumption (Fig. 3B) and the amount of lactate that remained in
the culture medium at the end of the chase period (Fig. 3C). The
relationship between glucose consumption and the medium lactate
level after a 72-h chase was similar in both 4 and 21% O2 (Fig. 3D),
and in the presence and absence of supplemental FA (Fig. 3E),
suggesting that neither low pO2 nor providing FA as an alternative
energy source influenced the effect of pHo on glucose catabolism.
FA b-oxidation also decreased at low pHo (Fig. 3F). Both glucose
and FA catabolism thus decreased as the cells adapted to low pHo,
whereas FA uptake and storage increased. Although a role for
extracellular L-lactic acid (or its uptake and utilization) (24) in
promoting TAG retention at low pH o seemed likely, providing
supplemental L-lactic acid during the chase did not increase TAG
retention more than would be predicted by its effect on pHo
(Fig. 1B).
Extracellular acidity may have dramatic effects on cells, altering
their mobility, secretory ability, and other properties (25–27). The
adherent cells studied in this article were impermeable to trypan
blue after the 72-h chase, yet we found 10% less DNA in wells
with final pHo ,7.0 than in wells cultured at final pHo 7.3–7.5
(210 6 3% [SD], 3 experiments, each with n = 4/condition), and
there was a commensurate decrease in cell protein (212 6 11%, 6
experiments with n = 4/condition). Cellular ATP levels were also
lower in cells carried at low pHo (215 6 5% relative to HEPEScultured cells, 3 experiments with n = 4, p , 0.05; Fig. 3G, 3Ia),
yet the AMP/ATP ratio was higher (Fig. 3H), suggesting that the
low ATP levels were produced, at least in part, by greater ATP
utilization. Switching cells from low pHo (MOPS buffer) to pHo
7.4 (HEPES buffer) after the 72-h chase period allowed restoration
of cellular ATP to control levels 72 h later (total 144 h of chase;
Fig. 3I). These results are consistent with prior reports that macrophages cultured at low pHo expend ATP to defend intracellular
pH, enabling recovery if pHo increases (26, 28).
pHo versus TLR4
LPS, a potent TLR4 agonist, induces acute macrophage FA uptake
and TAG synthesis (6). We recently reported that LPS stimulated
TAG storage for as long as 72–96 h in media buffered with 44 mM
NaHCO3, 5% CO2 (initial pHo 7.7) (10). LPS also stimulated
glucose consumption (data not shown) and lactate accumulation
(Fig. 4A) during the chase period. When we noted the effect of
pHo on prolonged TAG storage, we stimulated macrophages with
LPS while controlling pHo. We again found that TAG storage was
maintained above control (unstimulated) levels for 72 h, but now
the importance of final pHo became apparent in cells cultured in
either 4% O2 or 21% O2 (Fig. 4B–D). Low pHo did not account for
the acute effects of LPS on TAG storage, however, because LPSinduced TAG retention was evident 24 h after the stimulus was
added, before sufficient lactate had accumulated to lower pHo
(10), and because macrophages stimulated with LPS in media that
contained glutamine instead of glucose (17) retained TAG after
chasing for 24 or 72 h in the absence of lactate accumulation
(Fig. 4E) and without a low final pHo. In macrophages exposed to
LPS and cultured for prolonged periods, the accumulation of extracellular lactate induced a decline in pHo that likely extended
and enhanced the acute stimulatory effects of LPS on TAG storage
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Quantitative PCR was performed as previously described (10). Glucose
and L-lactate were measured using kits from Sigma and Eton Biosciences,
respectively. Lipolytic activity (cleavage of [3H]oleate from [3H]triolein
[Amersham]) was measured as described previously (10) in lysates of cells
cultured without FA loading. Cell protein was measured by dissolving the
isopropanol-insoluble residue from each culture well in 200 ml 1N NaOH
before assay using the Pierce BCA kit (Thermo Scientific). DNA was
measured using the Invitrogen NF cell proliferation assay kit (C35007).
For ATP and AMP analysis, cells were lysed in buffer from the Promega
AMP kit before assay using the ATP Determination Kit from Molecular
Probes and the AMP kit from Promega. Tests of significance and linear
regression analysis were performed using GraphPad Prism software.
1393
1394
LOW pH PROLONGS TRIGLYCERIDE STORAGE IN MACROPHAGES
(Fig. 5A, 5B). Decreasing pHo may also have prevented the switch
from glycolysis to FA catabolism that was found by Liu et al. (29)
in a human macrophage cell line that was stimulated with LPS for
shorter periods.
As expected (30), LPS stimulation boosted glucose uptake
(Fig. 3A), glucose consumption (Fig. 4F), lactate accumulation in
the medium (Fig. 4A), and ATP stores (Fig. 3G). As was also
FIGURE 2. pHo trumps pO2. (A) Cells cultured in
4% O2 after the FA load retain more [3H]TAG than do
cells cultured in 21% O2. H, Hepes, initial pH 7.3; M,
Mops, initial pH 6.95; T, Tris, initial pH 7.5. (Inset) The
same data plotted to show the relationship between final
pHo and log10 (cellular [ 3H]TAG). (B) pHo influences
TAG retention when cells are cultured in either low or
high O2. Added to the data for 21% O2 shown in Fig. 1B
(closed circles) are data for cells cultured in 4% O2
(open boxes) from seven experiments (total n = 70) in
which cells were chased at both 4 and 21% O2. Linear
regression lines: solid = 21% O2, dashed = 4% O2. Four
percent O2 regression: r2 = 0.76, n = 70. *p , 0.05,
**p , 0.01.
found in cultures without LPS, however, glucose consumption
(Fig. 4F) and lactate accumulation (data not shown) decreased
when the cells were cultured at low pHo. LPS also induced
increases in mRNA abundance for genes associated previously
with FA uptake and TAG synthesis (ACSL-1 and DGAT-2) (31)
whereas decreasing ATGL, the enzyme that carries out the first
step in TAG lipolysis (32) (Fig. 4G). At low pHo the LPS-induced
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FIGURE 1. pHo influences TAG storage. (A) Experimental approach: load-chase. The retention of [3H]TAG is a measure of net TAG synthesis and
degradation during the chase (10). (B) Relationship between final pHo and cellular [3H]TAG for cells chased 72 h in Tris, Hepes, or Mops buffers, with or
without the addition of 5 mM acetic acid (HAc), b-hydroxybutyrate (BHB), L-lactic acid, or sodium L-lactate to Hepes-buffered medium. Compiled from 12
independent experiments; the values from each experiment were normalized so that log10[3H]TAG dpm at pHo 7.25 was ∼3.7. Linear regression was
performed on the values obtained without added acid (blue circles). (C) Providing supplemental FA increases TAG retention at low pHo. Macrophages were
loaded with FA for 18 h, washed, and chased for 72 h in buffered media in the presence (+ FA) or absence (2 FA) of 25 mM palmitate and 50 mM oleate.
Cellular TAG mass was much greater at 72 h if FAs were added to the medium during the chase. (D) FA uptake is pHo dependent. Cells were incubated
overnight in Hepes buffer, pH 7.4, before their ability to take up [3H]oleate (in 20 mM nonradiolabeled oleate) in 5 h was measured at different pHo. n = 3;
symbols show mean 6 1 SD. Representative of two independent experiments. (E) Incorporation of FA into TAG was also pHo dependent. After overnight
culture in buffered media, cells were provided [3H]oleate and [14C]palmitate (in 20 mM oleate and 20 mM palmitate) in media at different pHo for 5 h before
lipid analysis. Results are expressed as nanomoles FA in TAG per milligram cell protein. Representative of two independent experiments. (F) Cells were
loaded overnight with FA containing [3H]oleate, washed, and incubated for 72 h in media that contained [14C]palmitate (with 25 mM nonradiolabeled
palmitate and 50 mM nonradiolabeled oleate). The amounts of [3H]TAG (acquired during load) and [14C]TAG (acquired during chase) were both higher at
low pHo. Representative of three independent experiments. (G) Cellular lipolytic activity decreased at low pHo. [3H]oleate was released from [3H]triolein by
lysates of cells that had been cultured in media at different pHo for 72 h. A similar trend was observed at 24 and 96 h. (H) Lipolysis decreased at low pHo.
Cells loaded with [3H]oleate were incubated with or without 10 mM triacsin C (Tc) for 48 h before measuring the percentage of the pretreatment [3H]TAG
that was lost from the cells. Tc blocks thioesterification so that TAG loss may be attributed to lipolysis (19). More TAG was lost in the presence of Tc at
high pHo. Data combined from two independent experiments, each with n = 3. **p , 0.01. H, Hepes; M, Mops; T, Tris.
The Journal of Immunology
1395
changes in mRNA abundance were significantly smaller (Fig. 4G),
yet TAG retention increased, suggesting that low pHo had a greater
influence on TAG storage than did the observed changes in mRNA
or protein abundance. None of these changes in mRNA abundance
was noted in cells cultured without LPS. Cells cultured in 4%
oxygen had increased levels of mRNAs for HIF-1a target genes
(GLUT1, LDHa, VEGF), but these changes did not differ substantially in cells cultured at different pHo (Fig. 4H–J).
Discussion
The retention of TAG stores for 48–72 h in vitro was closely related
to the final pHo in both low and high oxygen environments, and in
the presence and absence of a potent microbial stimulus. Maintaining pHo at ∼7.3–7.5 prevented the increase in TAG storage
induced by either low pO2 or LPS.
Although low pHo influenced both TAG synthesis and lipolysis,
its major effect was to increase the uptake and incorporation of FA
into TAG, an activity first described by Spector (12). Low pHo
may promote FA uptake by decreasing the affinity of albumin for
FA (12, 33) or by allowing greater protonation of the carboxyl
moiety, increasing transmembrane diffusion or transport (34). It
seems likely that many of the other effects of pHo on cell metabolism are mediated by changes in intracellular pH that alter
protein function (26–28). In fibroblasts and a glioma cell line, for
example, decreased lactate production at low pHo was attributed to
a decrement in phosphofructokinase activity (35). pHo has also
been reported to dominate pO2 in the regulation of glucose utilization by cultured chondrocytes (36).
Peritoneal macrophages elicited by thioglycollate have been
used for many of the foundational studies of macrophage metabolism
(17, 18, 24, 37, 38), including the recent analysis of macrophage
lipids by the Lipid MAPS consortium (39). By harvesting 5 d after
thioglycollate injection and studying only adherent cells, we sought
to reduce contamination by other cells, particularly neutrophils and
eosinophils, and to obtain the “small peritoneal macrophages” described by Ghosn et al. (40). Although we know no evidence that
the FA and glucose metabolism of thioglycollate-elicited peritoneal
macrophage differs substantially from that of other primary macrophages (as distinct from RAW 264.7 cells and possibly other
macrophage cell lines) (10, 39), it will be important to confirm
our results in other macrophage types.
Our findings suggest that local acidity can contribute prominently to the development of “lipid-laden” macrophages, even in
an in vivo environment that is hypoxic and/or contains microbial
agonists (such as an infected tissue, tuberculous granuloma, abscess, or atherosclerotic plaque) (4, 41) (Fig. 5). In addition to
promoting FA uptake and incorporation into TAG (but not cholesteryl esters) (12), low pHo increases macrophage uptake of
lipoproteins (42) and likely contributes to the acidic environment
that facilitates lipoprotein processing and cholesterol uptake
within surface-connected compartments (43). M1 or classically
activated macrophages may be particularly likely to become lipid
laden, because they carry out aerobic glycolysis and secrete much
of the lactate they produce (30). In contrast, macrophages in
uninflamed tissues, even ones with lower levels of pO2, such as the
spleen and lymph nodes (23), rarely appear “lipid laden.” Our
findings suggest that an acidic environment may be needed to
sustain long-term TAG storage in macrophages.
Low pHo promoted TAG retention, yet glucose uptake, lactate
production, lipolysis, and FA b-oxidation all decreased, in keeping
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FIGURE 3. Cell metabolism produces and responds to low pHo. (A) Glucose uptake decreased in acidic media. 2-[3H]deoxyglucose (3H-DOG) uptake
during a 1-h incubation period was measured after 72-h chase at different pHo. n = 4 per condition. Similar results were found in two additional
experiments. (B) Glucose consumption decreased as pHo decreased. 72-h chase. (C) L-lactate levels in the culture medium at the end of the chase were also
lower at low pHo. (D) Lactate accumulation decreased with decreasing glucose consumption when cells were cultured in either 4 or 21% O2. Similar results
to those shown in (B)–(D) were obtained in four additional experiments, each with n = 3 or 4 at each pHo and % O2. (E) Glucose consumption and lactate
accumulation were not influenced by providing supplemental FA during the chase. Representative of two experiments. (F) b-Oxidation, producing 3H2O
from [3H]oleate, decreased when FA-loaded cells were incubated at low pHo. A similar relationship between pHo and 3H2O release was observed in three
additional experiments. (G–I) After the 72-h chase period, ATP levels were lower (G) and the AMP/ATP ratio was higher (H) in cells cultured low pHo. (I)
Restoring physiological pHo (25 mM Hepes buffer, pH 7.3–7.5) allowed cellular ATP levels to increase. After 72-h chase at initial pH, FA-loaded cells were
washed and reincubated either in media containing the same buffer (a) or Hepes buffer, pH 7.4 (b), for an additional 72 h before ATP was measured. (G–I)
Data were combined from three or four independent experiments, each with n = 3 or 4. *p , 0.05, **p , 0.01, ***p , 0.001.
1396
LOW pH PROLONGS TRIGLYCERIDE STORAGE IN MACROPHAGES
with the general decrease in cell metabolism noted by Taylor (25)
and others. Glucose and FA catabolism did not cease, however,
and the cells produced sufficient ATP to maintain a high level of
FIGURE 5. Summary diagrams. (A) Experimental
time course. After loading overnight with FA, macrophages were exposed to TLR agonists (LPS) and/or
low pO2. In cells exposed to LPS, TAG retention was
associated with enzyme changes that either promote
FA uptake and FA incorporation/retention in TAG or
decrease lipolysis. Both hypoxia and LPS stimulated
glycolysis, producing extracellular acidosis that also
promoted TAG retention while decreasing glucose and
FA catabolism. (B) Proposed in vivo scenario. Blood
monocytes enter an infected tissue and encounter TLR
agonists that acutely induce FA uptake, TAG synthesis,
and lactic acid production. As pHo decreases, lipoprotein and FA uptake both increase, as does TAG and
cholesterol storage in lipid droplets. Cells become
more “lipid laden” and their metabolism slows. Low
tissue pO2 also promotes TAG retention as anaerobic
glycolysis decreases pHo; the enzyme changes noted
with TLR stimulation are not induced by low pHo.
viability and allow recovery when pHo was restored to 7.4. These
changes are similar to those described in human tissues during the
chronic phase of severe sepsis (44), raising the possibility that
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FIGURE 4. LPS versus pHo. (A) Lactate accumulated in the media of cells cultured in 44 mM NaHCO3, 5% CO2, 21% O2 for 72 h. Supplemental FAs were
present during the chase (see Materials and Methods). Representative of three experiments with similar results. (B) LPS (2.5 ng/ml) increased [3H]TAG retention in cells cultured in either Mops or Hepes buffers for 72 h. (Inset) log10[3H]TAG versus pHo. (C) LPS-induced TAG retention after 72-h chase was pHo
dependent. Data for cells treated with 2.5 ng/ml LPS for 72 h in 21% O2 (red boxes) are shown with results for cells incubated without LPS (blue circles; see
Fig. 1B). Data from four independent experiments. Linear regression lines: blue = no LPS; red = + LPS. (D) pHo also influences the impact of LPS on TAG
retention at low pO2. Data shown in (B) for 21% O2 + LPS are reproduced (red symbols and regression line) and data are added (green triangles and regression
line) from 3 experiments in which cells were chased for 48–72 h at 4% O2 with LPS (2.5 ng/ml). (E) LPS-stimulated TAG retention in the absence of glucose
and without increased lactate production. Adding LPS (2.5 ng/ml) increased retention of [3H]TAG for 72 h (bottom) whether or not L-lactate was produced
(top). n = 4. LPS also stimulated TAG retention at 24 h and in two other experiments when glutamine was used instead of glucose (not shown). (F) LPS
stimulation of glucose consumption was pHo dependent. Measurements after 72-h chase. (G) mRNA abundance in cells cultured 48 h at different pHo or with
LPS (M+LPS, H+LPS). LPS-induced increases in mRNA abundance for ACSL-1 and DGAT-2 and decreases in ATGL (10); these changes were smaller at pHo
6.9 (M+LPS) than at pH 7.4 (H+LPS). Data (n = 3) are expressed as fold-change relative to the value obtained using HEPES buffer. Bars show mean + 1 SE.
Similar results in two independent experiments. (H–J) mRNA abundance for hypoxia-responsive genes: VEGF, LDHa, and GLUT1. LPS induced increases in
each of these mRNAs in both 21 and 4% oxygen; each increase was significantly higher in cells chased in 4% O2 (for each comparison, p , 0.01, unpaired t test.)
Representative of two (VEGF, LDHa) or three (GLUT1) independent experiments, each with n = 2 or 3. *p , 0.05, **p , 0.01. H, Hepes; M, Mops; T, Tris.
The Journal of Immunology
local or systemic acidosis may contribute to the hibernation-like
phenomena that are reported to occur in critically ill individuals.
Spurred by observing that metabolic pathways play important
roles in macrophage and lymphocyte function, the new field of
“immunometabolism” promises to revise many ideas about immune
cell biology. It is now appreciated that glycolysis, for example, is
important not only for producing nucleotides, glycerolipids, and
ATP, but also for regulating IFN-g production (45) and inflammasome activation (46). The results presented in this study raise
the possibility that longer-term changes in cellular function may
also be influenced, at least in part, by the decline in local pHo that
results from the secretion of lactic acid, or other acids, by the
same or nearby cells (27).
Disclosures
The authors have no financial conflicts of interest.
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