Abstract
Genetic variants annotated to the hedgehog interacting protein (HHIP)
are robustly associated with chronic obstructive pulmonary disease
(COPD). Hhip haploinsufficiency in mice leads to increased
susceptibility towards the development of emphysema following exposure
to chronic cigarette smoke (CS). To explore the molecular pathways
which contribute to increased susceptibility, we performed metabolomic
profiling using high performance liquid chromatography tandem mass
spectroscopy (LC/MS-MS) on plasma, urine, and lung tissue of Hhip ^+/−
heterozygotes and wild type (Hhip ^+/+) C57/BL6 mice exposed to either
room-air or CS for six months. Univariate comparisons between groups
were made with a combined fold change ≥2 and Student’s t-test
p-value < 0.05 to denote significance; associations with mean alveolar
chord length (MACL), a quantitative measure of emphysema, and
gene-by-environment interactions were examined using empiric
Bayes-mediated linear models. Decreased urinary excretion of cotinine
despite comparable plasma levels was observed in Hhip ^+/−
heterozygotes; a strong gene-by-smoking association was also observed.
Correlations between MACL and markers of oxidative stress such as
urinary methionine sulfoxide were observed in Hhip ^+/− but not in Hhip
^+/+ mice. Metabolite set enrichment analyses suggest reduced
antioxidant capacity and alterations in macronutrient metabolism
contribute to increased susceptibility to chronic CS-induced oxidative
stress in Hhip haploinsufficiency states.
Introduction
Chronic obstructive pulmonary disease (COPD), the third leading cause
of death in the United States^[44]1, is a complex disease influenced by
both genetic and environmental risk factors. Recent genome-wide
association studies (GWAS) have consistently identified a COPD
susceptibility locus in an intergenic region on chromosome 4q31^[45]2.
Using chromosomal conformation capture studies, our group demonstrated
that this GWAS region interacts with the hedgehog interacting protein
(HHIP) promoter through a functional genetic variant within a distal
enhancer which alters binding to the SP3 transcription factor^[46]3.
Subsequent studies of human lung epithelial cells exposed to cigarette
smoke in vitro suggested that, in addition to its established roles in
morphogenesis and embryonic development through the hedgehog
pathway^[47]4, HHIP may alter extracellular matrix and cell growth
pathways^[48]5. Recently, we also demonstrated a role for HHIP in the
development of spontaneous, age-related emphysema in murine models of
Hhip haploinsufficiency^[49]6.
To explore the interaction between HHIP and exposure to chronic
cigarette smoke (CS), the leading environmental risk factor for COPD,
we studied a murine model of HHIP haploinsufficiency generated on a
C57/BL6 background. While homozygous Hhip ^−/− mice die shortly after
birth due to defects in lung branching morphogenesis, Hhip ^+/−
heterozygotes are viable, have normal lung development, and exhibit an
approximately 33% reduction in the expression of Hhip, a level
comparable to that observed in human COPD lung tissue samples harboring
HHIP GWAS risk variants^[50]3. Hhip ^+/− heterozygote mice demonstrate
an increased susceptibility towards the development of both functional
and histological emphysema when exposed to chronic cigarette
smoke^[51]3, [52]7; network analysis of lung gene expression data
demonstrated an enrichment of lymphocyte activation pathways in Hhip
^+/− mice relative to similarly exposed wild type mice^[53]7. To date,
investigations into the metabolic perturbations which may contribute to
the increased susceptibility towards the development of emphysema in
Hhip ^+/− heterozygotes have not been performed.
Metabolomic profiling is a relatively novel “-omics” platform where the
comprehensive small molecule composition of a biological material is
assessed. As such, metabolomics represents a more proximal and
integrative snapshot of the environmental and genetic risk factors
which likely contribute to a disease phenotype. Unlike the genetic
profile which remains largely invariant within a given individual,
multiple “metabolomes” representing different biological materials,
physiological states, and exposure conditions can exist in a single
organism. To further explore the impact of HHIP haploinsufficiency on
the development of COPD, metabolomic profiling using an untargeted
liquid chromatography-tandem mass spectroscopy (LC-MS/MS) platform was
performed on the plasma, urine, and lung tissue from Hhip ^+/−
heterozygote and Hhip ^+/+ wild type mice exposed to either room air or
6 months of cigarette smoke (2 × 2 experimental design).
Results
Five mice were analyzed from each group in this 2 × 2 experimental
design: room air-exposed Hhip ^+/+, CS-exposed Hhip ^+/+, room
air-exposed Hhip ^+/−, and CS-exposed Hhip ^+/−. Although profiling was
performed using an untargeted platform, because we wished to perform
downstream pathway and enrichment analyses, we limited our analyses to
identified compounds. In total, 319 compounds were identified in
plasma, 197 in urine, and 323 in lung. Fatty acids were not detected in
urine samples and account for the decreased number of metabolites
reported in urine. The overlap between metabolites in each dataset by
sample type are shown qualitatively in Supplementary Figure [54]S1.
Baseline differences in metabolites by Hhip genotype
The spectral peak intensity of each metabolite, which is proportional
to concentration, was analyzed following log transformation and Pareto
scaling. Metabolites demonstrating a minimum ≥2 fold change and a
Students t-test p-value (unadjusted) <0.05 between groups were
considered significant. Differences in metabolism by Hhip genotype
(Hhip ^+/− heterozygotes versus Hhip ^+/+ wild type) under room air
conditions are shown in Table [55]1. Both C6 and C8 carnitine are
significantly reduced in plasma samples from Hhip ^+/− mice relative to
Hhip ^+/+ mice. No metabolite met both the fold change (≥2) and
statistical threshold for significance in lung tissue.
Table 1.
Metabolites with differential concentrations by Hhip genotype (Hhip^+/−
versus Hhip^+/+) in room air-exposed mice.
Sample Type/Metabolite Log2 (Fold change)* P-value†
Plasma
C6 carnitine −2.58 0.02
C8 carnitine −1.41 0.02
Urine
2-deoxyadenosine −1.17 0.04
Adenine 1.5 0.01
Histidine 1.14 0.01
Pyroglutamic acid −1.3 0.01
[56]Open in a new tab
*Negative values indicate lower concentration in Hhip^+/− heterozygotes
(minimum 2x fold change).
^†Student’s t-test. No metabolites met the thresholds for significance
in lung tissue.
Impact of chronic cigarette smoke exposure on metabolite levels
Exposure to cigarette smoke increased the number of metabolites which
exist at different concentrations relative to baseline (room-air)
conditions in both Hhip ^+/+ (Table [57]2) and Hhip ^+/− mice
(Table [58]3). As a proof of concept, increased levels of cotinine, an
established metabolite of nicotine and biomarker of cigarette smoke
exposure, were consistently identified in both the plasma and urine of
CS-exposed mice relative to room air-exposed mice. Interestingly, while
a strong inverse correlation between urinary and plasma cotinine levels
was noted among Hhip ^+/+ wild type mice exposed to chronic CS, this
relationship was not observed in Hhip ^+/− heterozygotes (Fig. [59]1)
and may suggest genotype-dependent differences in cotinine metabolism.
Table 2.
Metabolites with differential concentrations following exposure to
chronic cigarette smoke exposure in wild-type (Hhip^+/+) mice.
Sample Type/Metabolite Log2 (Fold change)* P-value†
Plasma
Cotinine 5.2 2.48 × 10^−3
Glutathione (oxidized) −1.01 0.03
Urine
1-methylhistamine −1.07 2.91 × 10^−3
5-aminolevulinic acid 2.42 0.02
Adenine 1.46 7.34 × 10^−3
Creatine −2.54 7.52 × 10^−4‡
Cotinine 4.61 7.69 × 10^−4‡
Guanine 1.53 2.38 × 10^−6‡
N-carbamoyl-beta-alanine −1.04 0.02
Nicotinate 4.36 0.02
Oxalate 1.17 0.05
Pantothenate −1.78 0.04
Xanthine 1.34 6.16 × 10^−3
[60]Open in a new tab
*Negative values indicate lower concentration in wild type Hhip^+/+
mice exposed to chronic cigarette smoke (minimum 2x fold change).
^†Student’s t-test p-value.
^‡Denotes significance at a false discovery rate (FDR) < 0.05.
No metabolites met the thresholds for significance in lung tissue.
Table 3.
Metabolites with differential concentrations following exposure to
chronic cigarette smoke exposure in (Hhip^+/−) heterozygote mice.
Sample Type/Metabolite Log2 (Fold change)* P-value^†
Plasma
4-hydroxybenzaldehyde 1.57 0.03
AMP 2.33 0.02
Cotinine 3.95 1.55 × 10^−3
Cytidine 1.39 0.02
Gentisate −1.44 0.01
Glucose −1.06 0.04
Glutamate 1.29 0.01
GMP 1.85 0.02
Threonine 1.11 0.04
UMP 2.53 0.03
Urine
ADP 1.13 0.03
Alpha-ketoglutarate −1.55 0.02
AMP 1.52 0.02
Argininosuccinate −1.72 1.05 × 10^–3
C3-DC-CH3 carnitine 1.38 0.04
Carnosine −1.73 0.04
Cotinine 2.56 0.02
Glutamate 1.02 0.03
Guanine 1.13 9.67 × 10^−3
Histidine −1.34 3.95 × 10^−3
Lactate −1.22 0.01
Malate −1.24 0.03
Pantothenate −1.41 0.02
Putrescine −1.75 0.03
Succinate −1.05 5.83 × 10^−3
XMP 1.91 0.02
Lung
Adenylosuccinate 1.41 4.98 × 10^−3
[61]Open in a new tab
*Negative values indicate lower concentration in Hhip^+/− mice exposed
to chronic cigarette smoke (minimum 2x fold change).
^†Student’s t-test.
Figure 1.
Figure 1
[62]Open in a new tab
Urinary and plasma cotinine levels by genotype. Urinary cotinine
(normalized for creatinine concentration) relative to plasma cotinine
in Hhip ^+/− heterozygotes (left panel) and Hhip ^+/+ wild type mice
(right panel) subjected to chronic cigarette smoke. In Hhip ^+/+ wild
type mice, a strong inverse correlation between urine and plasma
cotinine levels exist (Pearson rho = −0.89, p-value = 0.04) whereas in
Hhip ^+/− heterozygotes, no correlation was found (Pearson rho = 0.12,
p-value = 0.88). The best fit line is plotted in blue while the 95%
confidence interval is plotted in dark gray.
When we compared Hhip ^+/+ wild type to Hhip ^+/− heterozygotes exposed
chronic CS, a more modest number of metabolites were identified as
existing in different concentrations by genotype (Table [63]4).
Significantly lower concentrations of cotinine were observed in the
urine of Hhip ^+/− relative to Hhip ^+/+ mice. Again, no metabolites
met both the ≥2 fold change and statistical threshold for significance
in lung tissue.
Table 4.
Metabolites with differential concentrations by Hhip genotype (Hhip^+/−
versus Hhip^+/+) in cigarette smoke-exposed mice.
Sample Type/Metabolite Log2 (Fold change)* P-value†
Plasma
C30:1 phosphatidylcholine −1.51 0.04
Pantothenate 1.16 3.6 × 10^−3
Sorbitol 1.39 0.05
Urine
Alpha-hydroxybutyrate −1.22 0.04
C5 carnitine −1.82 0.03
Cotinine −1.89 0.03
Creatine 1.17 0.03
[64]Open in a new tab
*Negative values indicate lower concentration in Hhip^+/− heterozygotes
(minimum 2x fold change).
†Student’s t-test.
No metabolites met the thresholds for significance in lung tissue.
Because of the relative paucity of metabolites which met our stringent
criteria for significance in lung tissue in the majority of our
analyses, we performed secondary exploratory analyses using a less
stringent threshold of 1.5x fold change (while keeping the statistical
threshold of p < 0.05 unchanged) to denote significance. Comparisons
between Hhip ^+/+ and Hhip ^+/− mice exposed to room air (Supplementary
Table [65]S1), differences by smoke exposure in Hhip ^+/+
(Supplementary Table [66]S2) and Hhip ^+/− (Supplementary Table [67]S3)
mice, and differences by genotype when both groups were exposed to
chronic CS (Supplementary Table [68]S4) were examined. Wild-type Hhip
^+/+ demonstrate increases in 2 phosphatidylcholine compounds (C30:1 PC
and C32:2 PC) in lung upon exposure to chronic CS; these increases are
not observed in Hhip ^+/− heterozygotes; the differences in C30:1 PC
and C32:2 PC are significant when comparing Hhip ^+/+ and Hhip ^+/−
groups that have both been exposed to chronic CS. Additionally, reduced
levels of gentisate were observed in the plasma and lung tissue of Hhip
^+/− exposed to chronic CS.
Gene-by-smoking analyses
To explore gene-by-environment interactions, we constructed empiric
Bayes’ mediated linear models with raw metabolite peak intensities as
the dependent variable; dichotomized variables for the main effects and
a multiplicative interaction term were modeled as the independent
variables as shown below:
[MATH: [Metabolitepeakintensity]~[Genotype]+[Smoking]+[Genotype:Smoking
] :MATH]
Metabolites with an association p-value < 0.05 for the interaction term
were considered significant. Metabolites with significant
gene-by-environment interactions are listed in Table [69]5. Four of the
six metabolites identified in plasma were also identified in urine; the
two non-overlapping metabolites in plasma are lipid derivatives which
would not be expected to be present in urine. Using permutation testing
to determine the null distribution for the expected overlap between
plasma and urine metabolites, an overlap of ≥4 metabolites is
significantly greater than would be expected by chance
(p[permutation] = 0.002). Only fructose/glucose/galactose were
identified in both the urine and lung analyses while C58:12
triacylglycerol overlapped between the plasma and lung analyses.
Table 5.
Metabolites with significant (p < 0.05) gene-by-environment
interactions.
Sample Type/Metabolite
Plasma
Aconitate
Adipate
C22:6 Lysophosphatidylcholine
C58:12 Triacylglycerol
Isocitrate
Thymine
Urine
2-deoxyadenosine Fructose/glucose/galactose
2-hydroxyglutarate Hydroxyphenylpyruvate
3-hydroxybenzoate Inositol
3-methyladipate Isocitrate
4-hydroxybenzaldehyde Isoleucine^‡
5-adenosylhomocysteine N-carbamoyl-beta-alanine
Aconitate^‡ Phosphocholine
Adenosine Pyroglutamic acid
Adipate Salicylurate
Alpha-glycerophosphate Symmetric dimethylarginine
Alpha-glycerophosphocholine Suberate
Asparagine Taurocholate^‡
Asymmetric dimethylarginine Thymine
cAMP Tryptophan
chenodeoxycholate Tyrosine
creatinine Uracil
cotinine Valine
cytosine Xanthosine
Lung
2-hydroxyglutarate
2-phosphoglycerate
3-phosphoglycerate
Alpha-hydroxybutyrate
Betaine
Butyrobetaine
C14:0 sphingomyelin
C22:0 sphingomyelin
C32:2 phosphatidylcholine
C58:12 triacylglycerol
Fructose/glucose/galactose
Lactose
Malondialdehyde
Sucrose
[70]Open in a new tab
^‡Denotes significance at a false discovery rate (FDR) < 0.05.
Association with histological emphysema severity
The mean alveolar chord length (MACL), a quantitative histological
measurement of the average vertical and horizontal distances between
the alveolar walls, was used as a surrogate for emphysema severity and
was determined according to previously published protocols^[71]8.
Empiric Bayes-mediated linear models were constructed for each
experimental condition using the raw metabolite peak values and mean
MACL as the dependent and independent variables, respectively;
metabolites with an association p-value < 0.05 were considered
significant.
Results are shown in Supplementary Tables [72]S5 (plasma), [73]S6
(urine) and [74]S7 (lung). A strong association between two lipids,
C56:10 and C58:10 triacylglycerol (TAG), and MACL was noted in the
plasma of Hhip ^+/+ mice exposed to room air; these associations were
no longer present following exposure to chronic CS nor was it present
in Hhip ^+/− heterozygotes under either experimental condition
(Fig. [75]2, panels A and B, respectively). Urinary thiamine and
methionine sulfoxide levels (normalized to creatinine) were
significantly associated with MACL in Hhip ^+/− heterozygotes exposed
to chronic CS (Figs [76]3 and [77]4, respectively). No correlation was
observed in Hhip ^+/− mice exposed to room air or in Hhip ^+/+ wild
type mice exposed to either experimental condition.
Figure 2.
Figure 2
[78]Open in a new tab
Association between plasma C56:10 (panel A) and C58:10 (panel B)
triacylglycerol (TAG) and lung mean alveolar chord length (MACL) by
experimental condition. A strong correlation between C56:10 TAG (Panel
A, Pearson’s rho = 0.97, p-value = 7.22 × 10^–3) and C58:10 TAG (Panel
B, Pearson’s rho = 0.97, p-value = 7.14 × 10^–3) and MACL is observed
Hhip ^+/+ wild type exposed to room air which is not observed in Hhip
^+/+ wild type exposed to chronic cigarette smoke or in Hhip ^+/−
heterozygotes exposed to either experimental condition. The best fit
line is plotted in blue while the 95% confidence interval is plotted in
dark gray.
Figure 3.
Figure 3
[79]Open in a new tab
Association between urinary thiamine/creatinine ratio and lung mean
alveolar chord length (MACL). A significant association between urinary
thiamine/creatinine was noted in Hhip ^+/− heterozygotes exposed to
chronic cigarette smoke (CS) (lower right panel, Pearson’s rho = 0.99,
p-value = 1.67 × 10^–3). No association was observed in Hhip ^+/− mice
exposed to room air or in Hhip ^+/+ wild type mice in either
experimental condition. The best fit line is plotted in blue while the
95% confidence interval is plotted in dark gray (except Hhip ^+/+, room
air, where wide 95%CI exceeds panel borders).
Figure 4.
Figure 4
[80]Open in a new tab
Association between urinary methionine sulfoxide/creatinine ratio and
lung mean alveolar chord length (MACL). A significant association
between urinary methionine sulfoxide/creatinine was noted in Hhip ^+/−
heterozygotes exposed to chronic cigarette smoke (CS) (lower right
panel, Pearson’s rho = 0.99, p-value = 3.94 × 10^–3). No association
was observed in Hhip ^+/− mice exposed to room air or in Hhip ^+/+ wild
type mice in either experimental condition. The best fit line is
plotted in blue while the 95% confidence interval is plotted in dark
gray.
Metabolite set enrichment analysis (MSEA) and pathway analyses
Metabolites significant in the (a) univariate analyses by both fold
change and p-value testing, (b) gene-by-smoking analysis, and (c) MACL
analyses above were mapped to the appropriate unique identifier in the
Human Metabolome Database (HMDB ID); these identifiers were used as
input for MSEA and pathway analyses as implemented in MetaboAnalyst
3.0^[81]9; a false discovery rate (FDR) ≤ 0.05 was considered
significant.
Of the metabolite groups identified in the univariate comparisons,
metabolites from the analysis of Hhip ^+/− heterozygotes mice exposed
to chronic CS versus room air were enriched for several processes. An
enrichment of plasma metabolites annotated to RNA transcription was
observed, while metabolites annotated to the urea cycle, ammonia
recycling, and the citric acid cycle were enriched in the urine
(Fig. [82]5, panels a and b, respectively). Metabolite groups from
other univariate comparisons did not demonstrate significant
enrichments on either MSEA or pathway analyses.
Figure 5.
Figure 5
[83]Open in a new tab
Metabolite set enrichment analysis based on differentially expressed
metabolites identified in plasma (a) and urine (b) from Hhip ^+/−
heterozygotes exposed to chronic cigarette smoke relative to mice
exposed to room air. Metabolite sets significant at an FDR < 0.05 are
denoted with a blue star.
When we performed a similar analysis on metabolites identified in the
gene-by-smoking analysis, a trend towards enrichment in metabolites
annotated to protein synthesis (FDR = 0.07) was identified in the
analysis of urine. As a subgroup analysis, we performed pathway
enrichment analysis using the 4 overlapping metabolites identified in
both plasma and urine gene-by-environment analyses and noted a
significant enrichment of metabolites annotated to (1) glyoxylate and
dicarboxylate metabolism and (2) the citrate cycle pathways
(Supplementary Figure [84]S2). Pathway analysis of metabolite groups
associated with MACL in the urine of Hhip^+/+ exposed to room air
demonstrated a trend towards enrichment of metabolites annotated to
glycerophospholipid metabolism (FDR = 0.054).
Validation of selected metabolites
To confirm the results of our mass spectroscopy-based metabolomic
profiling data, we performed ELISA-based assays for cotinine and
creatine on samples collected from a largely non-overlapping cohort of
mice from the same experimental conditions. Results of the quantitative
urinary cotinine assay are shown in Supplementary Figure [85]S3.
Significantly higher amounts of cotinine were detected in the urine of
both Hhip ^+/+ and Hhip ^+/− mice exposed to chronic CS relative to
those exposed to room air; these findings are consistent with the mass
spectroscopy-based metabolomics data. However, no significant
difference in urinary cotinine was noted between Hhip ^+/+ and Hhip
^+/− when both were exposed to chronic CS; this may be due to limited
sample size or differential sensitivity between the two assays. Serum
cotinine levels are illustrated in Supplementary Figure [86]S4.
Surprisingly, while there was a trend towards higher cotinine levels in
both Hhip ^+/+ and Hhip ^+/− mice exposed to chronic CS relative to
mice exposed to room air, the differences did not meet the thresholds
for statistical significance in either group. We also validated the
effects of chronic CS on creatine, an organic acid involved in energy
supply which is largely stored in skeletal muscle cells. Decreased
levels of urinary creatine were observed in Hhip ^+/+, but not in Hhip
^+/−, mice after chronic CS exposure (Supplementary Figure [87]S5).
There were, however, no differences in urinary creatine levels by
genotype when we compared Hhip ^+/+ to Hhip ^+/− mice after both were
exposed to chronic CS. While most of samples assayed in the validation
cohort were from an independent group of mice, one mouse overlapped
with the original cohort profiled using the untargeted
mass-spectroscopy based metabolomic profiling. Exclusion of the single
overlapping mouse did not change the significance of the results
reported in each of the comparisons above.
Discussion
The hedgehog family of proteins is classified as growth factors and
morphogens which mediate an expansive number of processes during
embryogenesis and development^[88]10. Hedgehog interacting protein
(HHIP) is a highly conserved, vertebrate-specific protein which is both
induced by and serves as a negative regulator of hedgehog
signaling^[89]11. HHIP has an established role in branching
morphogenesis of the lung during embryonic development^[90]4; based on
subsequent RNA interference studies in a human airway epithelial cell
line, HHIP was also implicated in lung extracellular matrix and cell
growth pathways^[91]5. In addition to associations with adult
height^[92]12 and several malignancies^[93]13, variants annotated to
the HHIP locus have been robustly associated with lung function and the
development of COPD^[94]2, [95]14. To date, the mechanisms which
underlie the association between HHIP and late-onset (adult) complex
diseases have not been fully characterized. A murine model of Hhip
haploinsufficiency which closely mimics the impact of genetic variants
identified by genome-wide association studies on the gene expression of
HHIP in the lung demonstrates increased susceptibility towards the
development of emphysema upon exposure to chronic cigarette
smoke^[96]7; using this model, we explored both baseline differences in
metabolism as well as differences induced by exposure to a significant
environmental challenge (i.e., chronic cigarette smoke exposure).
While complete loss of Hhip function results in perinatal lethality,
haploinsufficiency at the Hhip locus does not appear to significantly
alter the viability or lung morphology relative to wild-type mice^[97]7
under normal conditions. Likewise, baseline differences in metabolism
between Hhip ^+/− and Hhip ^+/+ mice also appear to be modest.
Decreased levels of C6 and C8 carnitine were detected in the plasma of
Hhip ^+/− heterozygotes; both compounds belong to the broader category
of acyl carnitines, a group of metabolites involved in fatty acid
oxidation. Interestingly, increased plasma levels of C6 and C8
carnitine are characteristic of medium chain acyl-CoA dehydrogenase
deficiency (MCAD; OMIM 201450), an autosomal recessive disorder
characterized by an intolerance to fasting^[98]15, [99]16. Although the
functional impact of decreased levels of plasma C6 and C8 carnitine is
not known, decreased levels of L-carnitine in lung tissues were
recently reported in a porcine pancreatic elastase murine model of
emphysema^[100]17.
Exposure to cigarette smoke appears to elicit a number of changes to
metabolite concentrations in the plasma and urine of both Hhip ^+/+
wild type and Hhip ^+/− heterozygotes. Decreased urinary excretion of
pantothenate, an essential nutrient also known as vitamin B5 which is
involved in the synthesis of coenzyme A (CoA) and the metabolism of
almost all macronutrients (proteins, carbohydrates, and fats), was
observed in both Hhip ^+/+ and Hhip ^+/− mice exposed to cigarette
smoke. In both instances, a corresponding increase in plasma levels was
not observed. Thus, whether cigarette smoke exposure alters the
synthesis or absorption of the nutrient from the gastrointestinal tract
or the localization, turnover, or sequestration of pantothenate in
different tissues remains unknown. Interestingly, increased levels of
pantothenate have been reported in a human alveolar lung tissue cell
line exposed to mainstream cigarette smoke in vitro ^[101]18; this was
not observed in our data which was ascertained on whole lung tissue
samples from an in vivo exposure model.
Another metabolite consistently altered in both Hhip ^+/+ and Hhip ^+/−
mice exposed to chronic CS was guanine, a purine derivative which is
one of five bases integral to DNA and RNA. Increased urinary guanine in
CS-exposed mice was one of the strongest associations in our study,
remaining robust even after application of a conservative Bonferroni
adjustment for multiple testing. Interestingly, several derivatives of
guanine, such as 8-hydroxydeoxyguanosine and 7-methylguanine, have been
associated with cigarette smoking and may reflect levels of oxidative
damage and DNA methylation, respectively^[102]19–[103]21; these guanine
derivatives were not assessed directly in our study and represent
potential avenues for future investigations.
Cotinine, a major pharmacologically-active metabolite of nicotine, is
an established biomarker of both active and passive cigarette smoke
exposure^[104]22 and was significantly increased in the urine and
plasma of CS-exposed Hhip ^+/+ and Hhip ^+/− mice. The finding of
increased levels of cotinine and a relative depletion of glutathione in
the plasma of Hhip ^+/+ wild type mice exposed to chronic cigarette
smoke is also consistent with known hepatic xenobiotic detoxification
pathways^[105]23 and serves as a proof-of-concept finding.
Interestingly, when we compared Hhip ^+/+ and Hhip ^+/− mice which had
both been exposed to chronic CS, Hhip ^+/− heterozygotes had
significantly lower urinary cotinine relative to Hhip ^+/+ wild type
mice. The decreased urinary excretion of cotinine in Hhip ^+/−
heterozygotes occurs despite similar plasma cotinine levels in both
strains and does not appear to be due to differential renal function as
assessed by plasma creatinine levels (p-value = 0.86). Previous studies
have demonstrated a strong correlation between plasma and urinary
levels of cotinine in both humans and mice ^[106]22, [107]23; a strong
inverse correlation between plasma and urinary cotinine levels was
noted for Hhip ^+/+ wild type mice exposed to cigarette smoke
(Pearson’s rho = −0.89, p-value = 0.04), but no correlation was noted
in Hhip ^+/− heterozygotes (Pearson’s rho = 0.12, p-value = 0.88)
(Fig. [108]1). These results are consistent with the finding of a
strong gene-by-smoking effect for cotinine identified in our earlier
analysis (Table [109]5). Whether cotinine is differentially metabolized
into alternative or downstream metabolites, such as
trans-3-hydroxy-cotinine or cotinine-N-oxide by Hhip ^+/− heterozygotes
remains unknown as the majority of nicotine and cotinine derivatives
remain unannotated in our dataset.
We were able to support some of the mass spectroscopy-based results
reported above using ELISA and colorimetric assays in a largely
non-overlapping cohort of mice from the same experimental conditions.
Urinary cotinine excretion was increased among both Hhip ^+/+ and Hhip
^+/− mice exposed to chronic CS relative to mice exposed to room air,
however no difference in cotinine excretion was found when we compared
Hhip ^+/+ and Hhip ^+/− mice who had both been exposed to chronic CS.
However, due to limited sample availability, urinary creatinine levels
could not be assayed, thus these values were not normalized to urinary
creatinine excretion. Interestingly, while a trend towards increased
plasma cotinine was observed in both Hhip ^+/+ and Hhip ^+/− mice
exposed to chronic CS, these differences did not reach statistical
significance in either group despite known and controlled exposure to
cigarette smoke. Whether this is due to the rapid clearance of
cigarette smoke metabolites in plasma or due to limitations of the
assay employed remains undetermined. We similarly support the mass
spectroscopy-based findings of reduced in urinary creatine observed in
Hhip ^+/+ mice exposed to chronic CS as well as the lack of decrease in
urinary creatine excretion in Hhip ^+/− heterozygotes. There was,
again, no statistically significant difference by genotype when
comparing the two groups of mice exposed to chronic CS, but these
values were similarly not adjusted for urinary creatinine excretion.
The mechanisms which contribute to the propensity of Hhip ^+/−
heterozygotes towards developing more severe histological and
functional emphysema when exposed to chronic CS or during aging
relative to their wild type counterparts are incompletely understood
but are likely mediated through an increased sensitivity towards
oxidative stress^[110]6. Given the “standardized” exposure to oxidative
stress in our experiment, we hypothesize that this increased
sensitivity may be due to a) reduced antioxidant capacity and b)
changes in metabolism which perpetuate oxidative damage. Findings which
support the hypothesis of reduced antioxidant capacity in Hhip ^+/−
heterozygotes include reduced levels of gentisate in plasma and lung
(Table [111]3 and Supplementary Table [112]S3) following exposure to CS
and the strong association between histological emphysema and urinary
excretion of methionine sulfoxide, a biological marker of oxidative
stress and aging. Gentisate, an intermediate in both salicylic acid and
benzoate metabolism, has been shown to have antioxidant and
free-radical scavenging properties in vitro ^[113]24; depletion in
plasma and lung is thus biologically plausible. Methionine residues in
protein complexes also demonstrate similar scavenging properties and
play a significant role as endogenous antioxidants; exposure to
reactive oxygen species leads to the formation of methionine sulfoxide
and reflects the oxidative burden experienced by the organism^[114]25.
Regeneration of methionine through methionine sulfoxide reductases
allows the cell/organism to retain the protein’s structural integrity
as well antioxidant capacity. Whether Hhip directly impacts methionine
sulfoxide reductase function is a potentially intriguing area for
future studies.
Changes in metabolism in Hhip ^+/− heterozygotes which may perpetuate
oxidative damage are based upon evidence of differential macronutrient
utilization which supports increased dependence on glucose and
carbohydrate metabolism and reduced reliance upon fatty acid
metabolism. In metabolite set enrichment analysis, the citric acid
cycle, malate-aspartate shuttle, and gluconeogenesis were significantly
enriched (Fig. [115]5). Thiamine, which was strongly correlated with
histological emphysema in Hhip ^+/− heterozygotes (Fig. [116]3), is
also an essential micronutrient integral to glucose metabolism^[117]26.
This increased reliance upon carbohydrate metabolism, which has been
shown to cause increased oxidative stress relative to fatty acid
metabolism^[118]27, has similarly been demonstrated in human COPD
subjects^[119]28, [120]29.
The net result of increased oxidative burden from both of the
mechanisms above is disrupted proteostasis, with increased degradation
of structural proteins due to damage and shunting of amino acids into
pathways to serve as substrates for the citric acid cycle to meet
essential energy requirements. Increased protein turnover and
degradation is supported by the finding of enrichment for metabolites
annotated to ammonia recycling, the urea cycle, and histidine
metabolism in Hhip ^+/− mice (Fig. [121]5), with similar disruptions in
metabolism described in human COPD patients^[122]30. Examining specific
disrupted pathways and potential therapeutic strategies represent
future directions of investigation.
A unique strength of our data involves the simultaneous profiling of
several sample types at once, an approach which allowed us to leverage
established physiology and biochemistry in the interpretation of our
findings. In addition to expanding our insight into the specific
metabolic pathways which may be involved in HHIP-mediated
susceptibility towards developing COPD, we made several general
observations. First, we noted a relative lack of significant
metabolites on univariate analyses of lung tissue despite the fact that
this tissue was directly exposed to cigarette smoke. Previous studies
utilizing alternative pulmonary-derived samples, such as expectorated
sputum, bronchoalveolar lavage fluid, or exhaled breath condensate,
have reported differences in metabolism between smokers, non-smokers,
and COPD subjects^[123]31, [124]32. We speculate that the relative
paucity of significant findings in lung tissue may be due to tissue and
cell-type heterogeneity as profiling was performed on lung homogenates
as opposed to largely acellular biofluids such as plasma and urine. We
additionally addressed this in our exploratory secondary analyses using
a less stringent fold-change threshold for all metabolites and identify
additional potentially interesting metabolites altered by either
genotype or cigarette smoke exposure. A second observation was that in
both the univariate and gene-by-environment analyses, the number of
significantly associated metabolites was consistently highest in urine.
Increased sensitivity to differences in metabolite concentrations in
urine relative to plasma has been previously reported for
tobacco-related metabolites^[125]33, [126]34; this was validated in our
ELISA cotinine assay which found greater sensitivity in detecting
differences by known exposure status in urine relative to plasma. Our
metabolomic profiling analyses suggest that, in addition to increased
sensitivity, urinary metabolomic profiling may offer complementary
information on metabolic processes relative to plasma metabolomic
profiling alone. Given the abundance and relative ease of collection,
as well as expanding resources cataloging compounds and reference
values^[127]35, urinary metabolomic profiling may be a promising avenue
for large-scale metabolomic investigations in the future.
Our exploratory analyses using metabolomic profiling of a murine model
of HHIP haploinsufficiency have generated a hypothesis that HHIP could
play a role in the development of COPD through differential handling of
environmental toxins and increased sensitivity towards oxidative
damage; further studies will be required to assess this hypothesis. We
assert that contemporaneous profiling of plasma and urine offers
complementary information on metabolic processes affected by
environmental exposures. We acknowledge limited power to detect small
to moderate differences in metabolites due to the small number of
animals in each experimental condition but contend that the use of
genetically identical mice and a highly controlled exposure model, as
well as validation of selected metabolites using independent
technologies, contribute to the rigor of the experiment and the
reported findings. We acknowledge that the majority of metabolites
identified in our analyses would not remain significant after
correction for multiple testing and that a certain proportion of our
associations may represent false positive findings. However, we assert
that the inclusion of a fold-change threshold, as well as the
identification of biologically plausible changes in metabolites
supports the validity our findings. Regarding our gene-by-environment
analysis, we acknowledge our use of a multiplicative interaction term
does not assess for other possible modes of interaction (i.e., additive
effects); future studies involving a larger number of observations will
allow for more detailed analyses of more complex interactions. Lastly,
we acknowledge that, because our model is murine and examined only
female mice, the generalizability of our findings to both genders and
extrapolation to human metabolism is not known; thus, future
metabolomic investigations in both genders and using human populations
are warranted.
Materials and Methods
Murine model and sample collection
Details regarding the generation of Hhip ^+/− mice and the chronic
cigarette smoke exposure conditions have been previously
published^[128]7. All protocols were in compliance with NIH
recommendations for the Care and Use of Laboratory Animals; all
protocols were approved by the Harvard Medical Area Standing Committee
on Animals (Protocol #: 04833). Female Hhip ^+/− heterozygotes on a
C57/BL6 background and their female wild type littermates were
subjected to either mixed main-stream and side-stream cigarette smoke
(3R4F Kentucky Research cigarettes) for 5 days per week or filtered
room air starting at age 10 weeks for a total duration of 6 months.
Plasma, urine, and lung tissue from five mice from each experimental
condition (room air-exposed Hhip ^+/+, CS-exposed Hhip ^+/+, room
air-exposed Hhip ^+/−, and CS-exposed Hhip ^+/−) were subjected to
metabolite profiling. Urine was collected immediately prior to
euthanization by placing each mouse on a clean surface and aspirating
spontaneously voided urine using a sterile pipette. Urine was
transferred into a sterile 1.5 mL centrifugation tube and was
immediately stored at −80 °C until analysis. A new, clean surface was
used for each mouse. Euthanization by CO[2] narcosis and cervical
dislocation was then performed and 500 μL of blood was collected from
the right ventricle immediately using a 26-gauge needle. Blood was
transferred into 1.5 mL tubes coated with heparin and centrifuged
(2000 × g, 5 minutes, 4 °C) to separate plasma. Plasma aliquots were
transferred into new 1.5 tubes and stored immediately at −80 °C until
analysis.
Murine lungs were harvested after blood collection for mean alveolar
chord length (MACL) measurements as described previously^[129]8 (also,
see below) and were snap-frozen for metabolomic profiling. Lung tissue
was homogenized in 6 volumes of water using a bead mill (TissueLyser
II; Qiagen Inc.; Valencia, ca) and the aqueous homogenate (30 μL) was
subjected to protein precipitation using four volumes of 80% methanol
containing inosine-15N4, thymine-d4, and glycocholate-d4 internal
standards (Cambridge Isotope Laboratories; Andover, MA). Samples were
then centrifuged (9,000 × g, 10 minutes, 4 °C) and aliquots of the
supernatant (10 μL each) were subjected to LC-MS profiling methods
(below).
Metabolomic profiling
Untargeted metabolite profiling was performed using liquid
chromatography tandem mass spectroscopy (LC/MS-MS) as previously
described^[130]36; additional details are also provided in the Online
Supplement – Methods section. MultiQuant software (v1.2) was used for
automated peak integration; all peaks were manually reviewed and
compound identities were confirmed using reference standards and
reference samples^[131]37. Lung tissue profiles were adjusted for
tissue dry weight and urinary profiles were normalized by creatinine
levels (assayed as one of the metabolites included in our mass
spectroscopy-based platform).
Data cleaning and analysis
Data cleaning and formatting were performed in MetaboAnalyst 3.0^[132]9
and R (version 3.2.2, base package). Samples with >40% missingness and
metabolites with >30% missingness were removed; remaining missing
values were replaced with a small value (one half the minimum detected
peak area of the metabolite). Data were log transformed and Pareto
scaled prior to conducting univariate analysis by genotype (Hhip ^+/+
versus Hhip ^+/−) or smoke exposure (room air versus CS). Metabolites
which demonstrated a minimum fold change of 2 and a Students t-test
p-value < 0.05 were considered significant. Metabolites that
additionally met statistical significance following correction for
multiple testing using an false-discovery rate (FDR) < 0.05 were
denoted in the results table. Gene-by-smoking interactions and
associations between mean alveolar chord length and metabolites were
tested using empiric Bayes-mediated linear models as implemented in the
limma ^[133]38 package for R. An association p-value < 0.05 was
considered significant.
Metabolite Set Enrichment and Pathway Analysis
Metabolite set enrichment analysis (MSEA) and pathway analyses were
performed using MetaboAnalyst 3.0^[134]9. Over-representation analysis
(ORA) was performed using the hypergeometric test using metabolite sets
based on normal metabolic pathways and the reference pathway library
for Mus musculus available through the Kyoto Encyclopedia of Genes and
Genomes (KEGG)^[135]39, respectively. An FDR ≤ 0.05 was considered
significant.
Validation/replication of selected metabolites
Validation of several metabolites was performed on plasma and urine
samples collected from a partially overlapping group of mice (n = 20).
Cotinine concentrations were determined in the plasma and urine of Hhip
^+/+ and Hhip ^+/− mice using the Calbiotech Mouse/Rat Cotinine
enzyme-linked immunosorbent assays (ELISA) kit (Catalog Number
C096D-100, Spring Valley, CA, USA). Quantitative measurements of urine
creatine were made using the Abcam colorimetric/fluorometric creatine
assay kit (ab65339, Cambridge, MA) in Hhip ^+/+ and Hhip ^+/− mice
exposed to chronic cigarette smoke. Standard curves were generated
according to the manufacturer’s guidelines for each assay. Comparisons
between groups were made using a Student’s t-test with a p-value < 0.05
to denote significance.
Electronic supplementary material
[136]Online Supplement^ (543.4KB, pdf)
Acknowledgements