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JBC Papers in Press. Published on May 16, 2019 as Manuscript RA118.006074
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.006074
Extensive metabolic remodeling after limiting mitochondrial lipid burden
is consistent with an improved metabolic health profile.
Sujoy Ghosh1,2, Shawna E. Wicks3,4, Bolormaa Vandanmagsar4, Tamra M. Mendoza4, David S. Bayless4,
J. Michael Salbaum5, Stephen P. Dearth6, Shawn R. Campagna6, Randall L. Mynatt4,7, and Robert C.
Noland8
1
Laboratory of Computational Biology, Pennington Biomedical Research Center, Baton Rouge, LA,
70808; 2Program in Cardiovascular and Metabolic Disorders and Center for Computational Biology,
Duke-National University of Singapore Medical School, Singapore, 169857; 3Talaria Antibodies, Inc.,
Pennington Biomedical Research Center, Baton Rouge, LA, 70808; 4Gene Nutrient Interactions
Laboratory, Pennington Biomedical Research Center, Baton Rouge, LA, 70808; 5Genomics Core
Facility, Pennington Biomedical Research Center, Baton Rouge, LA, 70808; 6Department of Chemistry,
University of Tennessee, Knoxville, TN, 37996; 7Transgenic Core Facility, Pennington Biomedical
Research Center, Baton Rouge, LA, 70808; 8Skeletal Muscle Metabolism Laboratory, Pennington
Biomedical Research Center, Baton Rouge, LA, 70808
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Running title: Limiting fat oxidation improves health
*To whom correspondence should be addressed: Robert C. Noland: 6400 Perkins Road, Baton Rouge,
LA, 70808; Email: robert.noland@pbrc.edu; Tel: 225-763-2788; Fax: 225-763-0273
Keywords: fatty acid oxidation, mitochondria, peroxisome, genomics, metabolomics, proteomics
ABSTRACT precursors to meet energy demand; however,
Mitochondrial lipid overload in skeletal this doesn’t translate to enhance energy status as
muscle contributes to insulin resistance and Cpt1bM-/- mice have low ATP and high AMP
strategies limiting this lipid pressure improve levels, signifying energy deficit. Comparative
glucose homeostasis; however, comprehensive analysis of transcriptomic data with disease-
cellular adaptations that occur in response to associated gene-sets not only predicted reduced
such an intervention have not been reported. risk of glucose metabolism disorders, but were
Herein mice with skeletal muscle-specific also consistent with lower risk for hepatic
deletion of carnitine palmitoyltransferase 1b steatosis, cardiac hypertrophy, and premature
(Cpt1bM-/-), which limits mitochondrial lipid death. Collectively these results suggest
entry, were fed a moderate fat (25%) diet and induction of metabolic inefficiency under
samples were subjected to a multi-modal conditions of energy surfeit likely contribute to
analysis merging transcriptomics, proteomics, improvements in metabolic health when
and non-targeted metabolomics to characterize mitochondrial lipid burden is mitigated.
the coordinated multi-level cellular responses Moreover, the breadth of disease states to which
that occur when mitochondrial lipid burden is mechanisms induced by muscle-specific Cpt1b
mitigated. Limiting mitochondrial fat entry inhibition may mediate health benefits could be
predictably improves glucose homeostasis; more extensive than previously predicted.
however, remodeling of glucose metabolism
pathways pale in comparison to adaptations in INTRODUCTION
amino acid and lipid metabolism pathways, In the absence of increased energy demand,
shifts in nucleotide metabolites, and biogenesis heightened lipid pressure in a hyperlipidemic
of mitochondria and peroxisomes. Despite environment can overwhelm mitochondrial β-
impaired fat utilization, Cpt1bM-/- mice have oxidative demand, resulting in greater
increased acetyl-CoA (14-fold) and NADH (2- incomplete fatty acid oxidation (1-3). This
fold), indicating metabolic shifts yield sufficient excess mitochondrial lipid burden appears to be
1
a key contributor towards insulin resistance, biology approach to assess global molecular
whereas strategies that limit mitochondrial fatty differences in skeletal muscle from Cpt1bfl/fl vs.
acid entry improve glucose homeostasis and Cpt1bM-/- mice. Specifically, we analyzed
mitigate lipid-induced insulin resistance (3-9). skeletal muscle (mixed gastrocnemius) samples
Comprehensive examination of models that limit through datasets derived from transcriptomic,
skeletal muscle mitochondrial lipid overload is proteomic and metabolomic platforms and
likely to facilitate identification of novel interrogated the unique and common responses
mechanisms through which alleviating the among the different methodologies. This
mitochondrial lipid burden improves whole body integrative approach revealed substantial
glucose homeostasis. One such model that links remodeling of substrate metabolism pathways
improved glucose homeostasis with reduced which are consistent with our previous findings
mitochondrial fatty acid entry is the skeletal and provides insight into potential mechanisms
muscle-specific, Cpt1b-deficient (Cpt1bM-/-) that could contribute to the beneficial phenotype
mouse (8,9). that result when mitochondrial lipid entry is
Carnitine palmitoyltransferase 1 (Cpt1) is limited specifically in skeletal muscle.
localized to the outer mitochondrial membrane
and is the rate-limiting enzyme controlling RESULTS
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mitochondrial lipid entry. Cpt1 facilitates Global Changes in Gene, Protein, and
mitochondrial lipid entry by converting acyl- Metabolite Levels
CoAs to acyl-carnitines, which then enter the Transcriptome analysis via SAGE detected
mitochondrion by passing through the 26,639 genes, of which 13,602 were deemed as
transmembrane-spanning carnitine-acylcarnitine reliably identified (gene count >2 in at least 1
translocase (CACT). Once within the sample). 539 genes (~4% of all detected genes)
mitochondrial matrix, acylcarnitines are displayed an absolute fold-change of ≥1.5
converted back into acyl-CoAs, which then enter between genotypes, with 229 genes up-regulated
the β-oxidative pathway for use as metabolic and 310 genes down-regulated in Cpt1bM-/- mice
fuel. Given its critical role in this process, it is (Figure 1A). Proteomics analysis yielded
not surprising that inhibition of Cpt1 activity identifications for 3,036 peptides and 591
greatly limits mitochondrial fatty acid oxidation proteins across all samples. After curation for
(9-12). Three isoforms of Cpt1 have been high quality peptides, the final quantitative
discovered (Cpt1a, Cpt1b, and Cpt1c) with dataset was based on 2,955 peptides and 581
Cpt1b being predominant in skeletal muscle. proteins, with 408 proteins containing at least 2
Studies by Mynatt et al. showed Cpt1bM-/- mice unique peptides. 62 proteins (~11% of the
have reduced mitochondrial lipid oxidation and identified proteome) were >1.5-fold different
excess intramuscular lipids (triglycerides, between genotypes with 60/62 being
diglycerides, and ceramides); however, these significantly higher in Cpt1bM-/- mice (Figure
mice exhibit improved glucose homeostasis and 1B). Non-targeted metabolomics analysis
were resistant to diet-induced obesity (8,9). annotated 9374 m/z features displaying
These results support the concept that chromatographic peak-like qualities. Currently,
mitochondrial lipid overload contributes to 85 of these features have been identified in our
glucose intolerance and strategies that limit library. The remaining 9289 features likely
mitochondrial fat entry in skeletal muscle can correspond to other unidentified polar, water-
limit lipid-induced insulin resistance. As such, soluble metabolites, but may also contain
Cpt1bM-/- mice provide a valuable experimental isotope and adduct variant metabolites. Using a
model that can be used to investigate adaptations p
whereas 1637 metabolites were lower compared metabolites. Alternatively, nucleotides are also
to Cpt1bfl/fl littermates (Figure 1D). critical to the synthesis of key factors involved
Principal component analysis (PCA) was in energy metabolism pathways including
performed on SAGE, proteomics, and nucleoside triphosphates (ATP, GTP, CTP, and
metabolomics datasets to detect potential UTP), reducing equivalents (NAD and FAD),
outliers and to further investigate how global and coenzyme A (CoA). As a result, much of the
gene, protein and metabolite expression could analyses performed using Cpt1bM-/- mice
differentiate between experimental groups (inset indicate significant remodeling of energy
within Figures 1A-D). Whereas a moderate level metabolism pathways and many of these
of group separation was noted for transcriptomic alterations are discussed in greater detail in the
and proteomic data, PCA results from the context of relevant metabolic pathways below.
metabolomics dataset (both known and unknown
metabolites) revealed a clear separation. Gene Set Enrichment Analysis (GSEA) and
Additionally, 41 of the 50 identified metabolites Ingenuity Pathway Analysis (IPA)
that were significantly different between Cpt1fl/fl To gain understanding of the possible effects
vs Cpt1bM-/- mice were also significantly of transcriptomic changes on biological
correlated (p ≤0.0251 and FDR
changes, we constructed a regulator/effector notable differences in insulin signaling (8,9). In
network based on curated information available the present study, globaltest analysis of 118
from the IPA Knowledge Base. The network genes linked to the insulin signaling cascade
consists of three layers with the top layer being indicated significant alterations in this pathway
transcription regulators, the middle layer being (p=0.006) within the basal (i.e. not insulin-
observed gene expression changes, and the stimulated) state (Figure 3A). It is, however,
bottom layer being predicted phenotypic effects worth noting that roughly half of the
(Figure 2A). This network map indicated differentially regulated genes were increased,
changes in genes that regulate substrate while the other half were decreased. We next
metabolism and metabolic disease phenotypes investigated gene enrichment within glycolysis
were likely mediated by induction of (Figure 3B) and pyruvate handling (Figure 3C)
overlapping transcription factor networks and observed significant upregulation of genes
involving peroxisome proliferator-activated in both pathways. Mapping changes to a KEGG
receptor (Ppar) pathways, sterol regulatory pathway map suggests remodeling of the
element-binding transcription factor 1 (Srebf1), glycolytic pathway was modest and most likely
RAR-related orphan receptor alpha (Rora), driven primarily by adaptations in pyruvate
Krüppel-like factor 15 (Klf15), estrogen-related handling (Figure 3D). This coincides well with
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receptor alpha (Esrra), and PPAR gamma the KEGG map for the pyruvate handling
coactivator 1-alpha (Pgc-1α). Analysis of the pathway (Figure S4D) and provides mechanistic
portion of the network directly linked to Cpt1b insight helping explain our previous
(Figure S4A) revealed induction of genes observations showing increased pyruvate
involved in fatty acid metabolism (Figure 2B), dehydrogenase (PDH) activity in Cpt1bM-/-
glycerolipid metabolism (Figure 2C) and skeletal muscle (8,9).
peroxisomal metabolism (Figure 2D), which
were some of the most robustly upregulated Integrative Analysis: TCA Cycle
pathways noted in the GSEA (Table S1). GSEA revealed genes linked to TCA cycle
Peroxisomal adaptations showed particular function were increased in Cpt1bM-/- skeletal
enrichment of genes involved in fatty acid muscle (Figure 4A), which supports the
oxidation (Figure S4B). Additionally, while observed heightened pyruvate oxidation in these
individual PPAR isoforms did not appear to be mice (8,9). Analysis of the identified metabolites
regulated, the PPAR signaling network was indicated that while pyruvate levels were not
identified as strongly enhanced via GSEA different between genotypes, acetyl-CoA levels
(Figure 2E). Mapping the PPAR network were nearly 14-fold higher in Cpt1bM-/- skeletal
indicated that the genes regulated in Cpt1bM-/- muscle (Figure 4B). Heightened acetyl-CoA
mice were primarily involved in lipid transport production appears to be at least partially
and oxidation (Figure S4C). Proteomic analysis facilitated by a 4.5-fold increase in free CoA
further supported this finding through the (Figure 4B). Interestingly, enrichment of the
observed induction of several proteins involved CoA biosynthesis pathway was observed in
in lipid metabolism in Cpt1bM-/- skeletal muscle Cpt1bM-/- mice (Figure 4C), which included a
(Figure 2F). significant increase in expression of the gene
encoding the rate-limiting enzyme in CoA
Integrative Analysis: Insulin Signaling biosynthesis, pantothenate kinase 1 (Pank1).
Pathway, Pyruvate Handling, and Glycolysis This was confirmed by RT-PCR (Figure S5A)
Figure S1B shows Cpt1bM-/- mice have and CT values (22-24) indicate Pank1 is a fairly
improved glucose homeostasis as they have abundant transcript in muscle. Additionally,
lower baseline glucose levels and maintain perhaps the most compelling evidence for
lower blood glucose throughout a glucose enhanced CoA biosynthesis is that Cpt1bM-/-
tolerance test than Cpt1bfl/fl controls, which is mice exhibit 41-fold greater levels of the
consistent with previous findings (8,9,13). metabolite precursor for CoA synthesis,
However, using Akt phosphorylation as a dephospho-CoA (Figure 4B).
measure of insulin sensitivity, we did not detect
4
Differences in CoA and acetyl-CoA could visual summary depicting changes in genes and
also be impacted by a variety of other factors metabolites related to the TCA cycle and
including, but not limited to: changes in TCA reducing equivalents is shown in Figure 4F.
flux rates, acyl-CoA synthetase activity (Acss1-
3, Acsf1-4, Acsm1-5, Acsl1-6, Acsbg1/2, Fatp1- Integrative Analysis: Amino Acid Metabolism
6), carnitine acyltransferase activity (CrAT, Leucine oxidation is increased in Cpt1bM-/-
CrOT, Cpt1a, Cpt1b, Cpt1c, Cpt2), activity of mice (8,9), suggesting that when mitochondrial
dehydrogenase enzymes with a lipoic acid lipid entry is impaired, they become more reliant
moiety (PDH, a-KGDH, BCKDH), acetyl-CoA on amino acids as fuel. Results in Table S1
acyltransferase activity (Acat1/2, Acaa1/2), confirm substantial remodeling at the gene level
acyl-CoA thioesterase activity (Acot1-13, of several pathways involved in amino acid
Them4/5), acetoacetyl-CoA synthetase activity metabolism and heatmaps for each identified
(Aacs), acetyl-CoA consuming reactions (CS, pathway can be found in Figure S6. Using genes
ACC, HS2, etc), as well as activity of various that contributed to core enrichment in all of
acetyltransferase and acyltransferase enzymes these amino acid metabolism pathways, a
that regulate post-translational modification. heatmap was made showing commonality of all
Differential expression of several genes involved regulated genes amongst the amino acid
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in these pathways was observed in the SAGE metabolism pathways (Figure 5A). As shown,
dataset, so we attempted to validate these with little overlap between regulated genes is
RT-PCR. We previously reported CrAT, CrOT, apparent for most of these pathways (Figure
Cpt2, CS, Pdha1, Sdhb, Bckdh, and Fatp1 are 5A), indicating the adaptations identified by
significantly higher in Cpt1bM-/- skeletal muscle GSEA (Table S1) is due to expansive
(8,9), so we didn’t repeat these analyses. Newly remodeling of amino acid metabolism, rather
identified genes involved in CoA/acetyl-CoA than a result of changes within a small set of
metabolism that were differentially regulated in genes common to all amino acid metabolism
the SAGE dataset included: Acss1, Acss2, Acsl1, pathways. Since genes involved in numerous
Acot2, Acot8, and Acot13. Of these, only Acot2, amino acid metabolism pathways were
Acot8, and Acot13 were significantly different regulated, the metabolomics dataset was probed
(Figure S5B). to identify amino acids in skeletal muscle.
Elevated acetyl-CoA provides more carbons Results show decreases in L-arginine, L-
that can enter the TCA cycle in Cpt1bM-/- mice. glutamine, L-leucine, and L-valine levels in
While our metabolomics platform did not Cpt1bM-/- mice, whereas increased levels of L-
resolve citrate or isocitrate, earlier results show asparagine, L-aspartate, L-homoserine, L-lysine,
Cpt1bM-/- mice have increased complete L-ornithine, and L-threonine were identified
oxidation (i.e. CO2 production) of non-lipid (Figure 5B). While results from this non-
substrates (pyruvate and leucine) that provide targeted metabolomics screen did not provide a
acetyl-CoA precursors (8,9), suggesting greater comprehensive amino acid panel, the amino
potential for substrate entry into the TCA cycle. acids that were identified show very similar
Since Cpt1bM-/- mice likely have greater entry of patterns to those found in our previous targeted
carbons into the citrate pool, it is interesting to metabolomics analysis (9). Collectively, the
note that α-ketoglutarate and succinyl-CoA metabolomics platforms from both
M-/-
levels are substantially lower in these mice vs. investigations show Cpt1b mice have 13
littermate controls (Figure 4D). Alternatively, amino acids increased in skeletal muscle, 4
downstream of these points in the TCA cycle, amino acids are decreased, and 3 amino acids
succinate is similar between genotypes, while remain similar when compared to Cpt1bfl/fl
fumarate and malate are both significantly controls. Since amino acids can be used as
elevated in Cpt1bM-/- mice (Figure 4D). Also metabolic fuel, results from these datasets were
noteworthy is the fact that NAD+ levels were used to develop an integrative map showing
similar between genotypes and NADH levels points of entry of carbons derived from amino
were higher in Cpt1bM-/- mice, resulting in an acid substrates into the TCA cycle (Figure 5C).
increased NADH:NAD+ ratio (Figure 4E). A As shown, amino acids that can be metabolized
5
to pyruvate, fumarate or oxaloacetate are almost concept of enhanced mitochondrial biogenesis in
universally increased. Likewise, amino acids Cpt1bM-/- skeletal muscle. This is likely driven
that can be catabolized to produce acetyl-CoA by activation of Pgc-1α and transcription factor
are primarily increased, except leucine. A, mitochondrial (Tfam; Figure 6E). It is also
Alternatively, amino acid-derived metabolites worth noting that changes in the ETC appear to
that enter the TCA cycle as either α- be almost exclusively due to induction of
ketoglutarate or succinyl-CoA are not as nuclear-encoded subunits as none of the genes
consistently present at higher concentrations. identified by the SAGE analysis were encoded
Notably, the amino acids most commonly by mitochondrial DNA (mtDNA), while only
considered to be important in generating α- one protein encoded by mtDNA (ATP6) was
ketoglutarate are glutamate and glutamine, significantly altered.
which are unaltered and decreased, respectively,
in Cpt1bM-/- mouse muscle. Of additional AMPK Signaling
interest, the most robustly upregulated pathway Since flux through the TCA cycle generates
identified by GSEA was leucine, isoleucine, and reducing equivalents that support energy
valine degradation (Table S1) and two of these production, the increased NADH:NAD+ ratio
amino acids (leucine and valine) are reduced in (Figure 4F) suggests there are ample reducing
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Cpt1bM-/- mice, which likely represents greater equivalents to support ATP synthesis in Cpt1bM-
/-
branched chain amino acid utilization. mice. With this in mind, it is interesting that
analysis of adenosine nucleotides found ATP
Integrative Analysis: Oxidative content is actually lower in Cpt1bM-/- mouse
Phosphorylation skeletal muscle, while AMP levels and free
With wide-spread alterations in substrate adenosine are increased (Figure 7A). The
metabolism pathways in Cpt1bM-/- skeletal elevated AMP:ATP ratio in Cpt1bM-/- mice
muscle, it was important to determine if suggests they are in an energy-insufficient state.
alterations in electron transport chain (ETC) 5-aminoimidazole-4-carboxamide ribonucleotide
components were also present. The heatmap in (AICAR) is a naturally occurring intermediate in
Figure 6A shows genes encoding proteins the synthesis of inosine monophosphate (IMP),
involved in oxidative phosphorylation were which can be used to produce AMP and
enriched in Cpt1bM-/- mice. Additionally, guanosine monophosphate (GMP). As shown in
analysis of the proteomics dataset for Gene Figure 7B, IMP and GMP levels are also
Ontology-based enrichment of cellular significantly higher in Cpt1bM-/- mice, while a
compartments (Figure S7A) demonstrated clear >6-fold increase in the metabolic precursor,
enrichment for mitochondrial respiratory chain AICAR, approached statistical significance
components (Figure S7B). A graphical (p=0.057). Since both AMP and AICAR are
representation of specific subunits of each ETC known activators of AMP-activated protein
complex showing significant differences kinase (AMPK), the SAGE results were
between genotypes for SAGE (Figure 6B) and examined for evidence of activation of this
proteomics (Figure 6C) datasets reveal pathway. Figure 7C provides a visualization
significant remodeling of the ETC in Cpt1bM-/- showing changes in several genes downstream
mice. The SAGE dataset identified 55 genes in from AMPK, which is consistent with activation
the oxidative phosphorylation pathway to be of this energy-sensing pathway. These findings
regulated, while the proteomics screen identified provide a mechanism that helps explain the
24 proteins that were altered in the ETC increase in AMPK phosphorylation observed
complexes (despite limited coverage of the previously in Cpt1bM-/- mouse skeletal muscle
proteome). Importantly, there appears to be (9).
reasonable agreement between SAGE and
proteomics results as 18 of the regulated proteins DISCUSSION
were also increased at the gene level (Figure The purpose of this investigation was to
6D). All differentially expressed genes and integrate information yielded from three distinct
proteins were upregulated, which supports the ‘omics technologies (SAGE, proteomics, and
6
non-targeted metabolomics) to identify regulated catabolism pathways. Of interest, genes
metabolic systems in skeletal muscle from encoding peroxisomal proteins were identified
Cpt1bM-/- mice. We previously reported Cpt1bM-/- as the second most robustly enriched metabolic
mice exhibit changes in substrate oxidation pathway via GSEA and our previous data
(8,9); therefore, initial analyses of our ‘omics confirm that peroxisomal function is heightened
datasets were targeted toward pathways involved in Cpt1bM-/- skeletal muscle (8,9). Notably,
in carbohydrate, amino acid, and lipid genes involved in peroxisomal fat oxidation
metabolism. Results confirmed extensive were particularly enriched. This is potentially
remodeling of these pathways, with the most important as unesterified fatty acids can enter
pronounced adaptations being observed in amino peroxisomes; therefore, peroxisomal lipid entry
acid and lipid metabolism pathways, while shifts does not require a carnitine shuttle system.
in glucose metabolism were modest. Strikingly, Within their matrix, peroxisomes contain
a biofunction analysis, based on a large body of carnitine acetyltransferase (CrAT) and carnitine
published gene-disease associations, indicated octanoyltransferase (CrOT), which convert
the adaptations were consistent with decreased chain-shortened moieties of peroxisomal
predicted risk of glucose metabolism disorders, oxidation into acylcarnitines. These products can
hepatic steatosis, fatigue, cardiac hypertrophy, then be exported into the cytosol, where they can
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and premature mortality; thus signifying that the enter the mitochondrial matrix independent of
breadth of disease states to which muscle- Cpt1. As a result, peroxisomes are capable of at
specific Cpt1b inhibition may mediate least partially rescuing LCFA oxidation when
improvements in health may exceed that which Cpt1 is inhibited (7). With this in mind, defining
has previously been believed. whether recruitment of peroxisomes in skeletal
Cpt1b has a critical role in facilitating muscle of Cpt1bM-/- mice is a critical adaptation
mitochondrial long chain fatty acid (LCFA) that helps combat a potentially lipotoxic
entry; thus, inhibiting this enzyme markedly environment and/or contributes to the beneficial
reduces mitochondrial LCFA oxidation. A metabolic phenotype is an important
potentially underappreciated role of Cpt1b lies consideration that warrants further investigation.
in the fact that the acylcarnitine metabolites Cpt1b deletion limits the ability to use lipids
produced via Cpt1b are also readily exported to meet energy demand; therefore, Cpt1bM-/-
from the cell, which is particularly relevant in mice must adapt to rely more heavily on other
conditions of lipid excess (1-3). Limitations in substrates. In this regard, earlier studies show
the ability to both use lipids as fuel, as well as Cpt1 inhibition increases reliance on
export excess lipid from the cell in the form of carbohydrates (4,5,7,14-20). Similarly, we found
acylcarnitines, leads to increased intramuscular Cpt1bM-/- mice show enhanced whole body
lipid accrual in Cpt1bM-/- mice as verified by glucose oxidation (indirect calorimetry),
elevated triglycerides, diglycerides, and improved glucose tolerance, as well as increased
ceramide metabolites (9). Accordingly, it is not pyruvate dehydrogenase activity and pyruvate
surprising that a primary molecular response is oxidation in skeletal muscle homogenates (8,9).
to remodel lipid metabolism pathways. It is also important to note that Cpt1 inhibition
Regulator/effector network analysis of the mitigates lipid-induced insulin resistance in
SAGE data predicts this might occur through skeletal muscle, despite the fact that such
activation of Srebf1, Rora, Klf15, Esrra, Pgc-1α, interventions promote a potentially lipotoxic
and/or Ppar regulated pathways. Of note, some environment (3,8,9). Results from the present
adaptations induced by these transcriptional analysis support these concepts and suggest that
regulators may actually exacerbate ectopic lipid at least a portion of the adaptive increase in
buildup as evidence herein shows Cpt1bM-/- mice carbohydrate utilization in skeletal muscle of
have increased expression of genes and proteins Cpt1bM-/- mice is encoded at the molecular level,
that drive cellular lipid uptake, transport, and as GSEA revealed enrichment of pathways
storage. Conversely, despite the inability of involved in glycolysis, pyruvate handling and
mitochondria to directly utilize LCFAs, our insulin signaling. It is, however, worth
results show significant upregulation of lipid mentioning that skeletal muscle is predicted to
7account for ~20% of the basal metabolic rate upregulation of amino acid metabolism in this
(21) and this can increase greatly during exercise study likely contributes to lower lean mass in
(22), emphasizing skeletal muscle has Cpt1bM-/- mice as they age.
significant substrate requirements. Skeletal SAGE and proteomics datasets both
muscle of Cpt1bM-/- has limited ability to utilize predicted enhanced mitochondrial biogenesis in
the most prevalent substrate available (lipid); skeletal muscle from Cpt1bM-/- mice. AMPK and
therefore, the adaptations in glucose handling Pgc-1α are activated under conditions of energy
pathways actually seem relatively modest deficit and promote mitochondrial biogenesis.
compared to what we expected. In this regard, it Cpt1bM-/- exhibit an increased AMP:ATP ratio
is important to recall that glucose is an essential (Figure 7A), as well as activation of pAMPK
substrate for brain and blood cells and the body and upregulation of Pgc-1α (9); therefore, it is
goes to great lengths to protect against likely that activation of these pathways
hypoglycemia. As such, it is tempting to contributes to the observed mitochondrial
speculate that the relatively modest remodeling biogenesis. Skeletal muscle from traditional
of glucose handling pathways in skeletal muscle models of energy-deficit (caloric restriction,
of Cpt1bM-/- mice is necessary to protect against prolonged fasting, acute exercise, etc) exhibit an
the possibility of hypoglycemia. If true, this increased AMP:ATP ratio, as well as decreases
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would also require adaptations in tissues other in NADH and FADH2 reducing equivalents.
than skeletal muscle, which provides an Since mitochondrial ATP production is
interesting model to identify novel mechanisms supported by delivery of reducing equivalents to
of organ systems crosstalk that regulate whole the electron transport chain, it is surprising to
body metabolic health that is worth future study. note that lower ATP levels in Cpt1bM-/- mouse
Cpt1bM-/- mice also exhibit remarkable muscle occurs despite greater mitochondrial
remodeling of amino acid metabolism pathways. content and ample reducing equivalents
Earlier we reported Cpt1bM-/- mice have (increased NADH levels and greater
heightened leucine oxidation (8,9), suggesting NADH:NAD+ ratio). Roughly 95% of the total
adaptations occur that allow for greater protein cellular NADH pool is estimated to reside within
catabolism. Findings herein strongly support this mitochondria (23); thus, an apparent mismatch
as 1) pathways involved in amino acid between NADH availability and ATP content is
metabolism represented 9 of the 21 significantly seemingly paradoxical. One possibility is that
enriched pathways identified via GSEA, and 2) despite increased mitochondrial biogenesis, the
the metabolomics screen revealed the majority relative lack of induction of genes and proteins
of amino acids are significantly different encoded by mitochondrial DNA may have
between Cpt1bfl/fl vs. Cpt1bM-/- mice. In fact, created mitochondria with an impaired ability to
based upon results of the current study, it can be utilize reducing equivalents to generate the
argued that adaptations in amino acid proton motive force required to support ATP
metabolism pathways actually exceed those synthesis. While potentially compelling, the
directing carbohydrate metabolism. In this prospect that the newly formed mitochondria are
regard, futile protein cycling has been linked to inherently dysfunctional seems unlikely since
increased energy expenditure (11) and could isolated mitochondria from Cpt1bM-/- mice
potentially contribute to the metabolically exhibited similar functionality (oxygen
advantageous phenotype observed in Cpt1bM-/- consumption, respiratory control ratio, and
mice; however, the importance of adaptations in uncoupled respiration) as littermate controls (9).
amino acid metabolism pathways in this rodent This does not, however, rule out the possibility
model remain to be more fully understood. It is that other cellular alterations occur in Cpt1bM-/-
also worth noting that we previously reported mice that either 1) limit the ability to produce
that Cpt1bM-/- mice begin to exhibit lower lean ATP, and/or 2) increase ATP utilization. Finally,
mass at ~17-20 weeks of age (8,9). Mice in this reducing equivalents are involved in a myriad of
study were 14-16 weeks of age at the time of other redox reactions throughout the cell, so
harvest, which likely precedes significant another possibility is that the increased NADH
differences in lean mass; however, the detected levels are linked to regulation of cellular redox
8balance; however, identifying specific pathways diminishes ATP production (24,27,28), which
requires further study. resembles the energy-deficit phenotype observed
Cpt1b converts long-chain fatty acyl-CoAs in Cpt1bM-/- mice. Second, at the cellular level,
(LCFA-CoA) into long-chain acylcarnitines, acetylation of histone residues is a critical
resulting in the liberation of coenzyme A (CoA). posttranslational modification that regulates
As such, manipulation of Cpt1b activity has the gene transcription within the nucleus. It has been
potential to affect the cellular CoA pool. Since postulated that heightened acetyl-CoA drives
Cpt1b acts directly on LCFA-CoA, it stands to acetylation of histone residues to promote cell
reason that manipulating Cpt1 activity would growth and survival even under states of energy
affect the LCFA-CoA pool; however, previous deficit (26,29), which also resembles results
studies that have measured LCFA-CoA levels in from Cpt1bM-/- mice. Ultimately, however, the
models of variable Cpt1 activity have yielded potential role that enhanced CoA biosynthesis
inconsistent findings (13,14,19,24). At this time &/or acetylation play in the phenotype observed
we cannot offer a resolution to this debate as in Cpt1bM-/- mice remains speculative and
targeted acyl-CoA metabolic profiling in warrants further study.
Cpt1bM-/- mice has not been performed; In summary, despite variable coverage of
however, our results clearly indicate significant the proteome and identified metabolites, the
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differences in CoA metabolism. Substantial combined use of SAGE, proteomics and non-
increases in free CoA (4.5-fold), dephospho- targeted metabolomics identified regulation of
CoA (41-fold), enrichment of genes involved in several pathways that were consistent across
CoA biosynthesis, and upregulation of acyl-CoA multiple platforms. Furthermore, the fact that
thioesterases point toward a system that is robust differences in numerous metabolites were
attempting to increase CoA availability. Recent observed increases confidence that changes at
work suggests increased CoA biosynthesis the gene and protein levels represent functional
occurs in systems attempting to drive LCFA adaptations. Importantly, findings herein
oxidation (25), which is consistent with the revealed that the extensive remodeling of
extensive remodeling of lipid metabolism substrate metabolism pathways in Cpt1bM-/- mice
pathways in Cpt1bM-/- mice. The rise in CoA are consistent with metabolically healthy
availability almost certainly contributes to the phenotypes. With this in mind, the large
14-fold increase in acetyl-CoA levels in Cpt1bM- publically available datasets developed from this
/-
mice. Heightened acetyl-CoA levels are comprehensive systems biology approach are
usually found in states of energy surplus (26); expected to help investigators define specific
thus, the robust increase in acetyl-CoA in mechanisms that contribute to the notable
Cpt1bM-/- mice signifies yet another seemingly improvements in metabolic health when the
paradoxical metabolite signature in a mouse mitochondrial lipid burden is mitigated in
model that otherwise exhibits a phenotype skeletal muscle.
similar to models of energy deficit. With this in
mind, it is important to note that while acetyl- EXPERIMENTAL PROCEDURES
CoA can enter the TCA cycle for use as EXPERIMENTAL MODEL
metabolic fuel, it can also be used as a substrate Animals: Skeletal muscle-specific Cpt1b-
in other reactions within the cell. Of particular deficient mice (Cpt1bM-/-) and littermate controls
relevance, surplus acetyl-CoA and NADH are (Cpt1bfl/fl) were developed as described (8,9).
known to promote protein acetylation (26). Briefly, floxed Cpt1b mice were bred to mice
While analysis of the SAGE data did not reveal expressing Cre recombinase under the control of
enrichment of pathways involved in protein the Mlc1f promoter (Jackson Laboratories,
acetylation or deacetylation, there are Stock # 024713) to delete Cpt1b in skeletal
observations from this study that support this muscle. As validated in our previous studies
possibility. First, numerous proteins in the TCA (8,9), this deletes Cpt1b expression and function
cycle and oxidative phosphorylation system are specifically in skeletal muscle with no effect on
acetylated and it has been shown that cardiac muscle. Mice were group-housed at
hyperacetylation of mitochondrial proteins room temperature (RT) under a 12:12 h
9light:dark cycle and allowed ad libitum access to 0.2 mL of chloroform was added. The samples
food and water. In an effort to be able to best were shaken vigorously for 15 sec and allowed
compare results from this systems biology study to sit at RT for 2-3 min before they were
to previous findings (8,9,30), mice were fed the centrifuged (12,000 x g; 15 min; 4°C) to induce
same rodent breeder chow (Purina Rodent Chow phase separation. Roughly 600 µL of the upper
#5015, Purina Mills, St. Louis, MO), which aqueous supernatant containing RNA was
provides 20% of calories from protein, 26% transferred to a new microcentrifuge tube
from fat, and 54% from carbohydrate. At 12-14 whereupon 600 µL of 70% ethanol was added
weeks of age body weight was measured (Figure and the samples were vortexed. RNA was then
S1A) and a glucose tolerance test was performed isolated using an RNeasy kit (Qiagen, Valencia,
as described below to confirm the Cpt1bM-/- mice CA) with DNAse treatment per manufacturer’s
used in this study exhibited the established instructions. RNA content and quality (260/280
improvement in glucose homeostasis (Figure ratio range 1.9 - 2.1) were assessed using a
S1B). Tissue harvest occurred at 14-16 weeks of Nanodrop 1000 and used for downstream Serial
age, which is a time point at which fat mass has Analysis of Gene Expression (SAGE) studies.
consistently been shown to be lower in Cpt1bM-/- The lower organic phase developed following
mice, yet lean mass is typically not yet different Trizol extraction contained protein samples that
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(8,9). Following a 4h food pull, Cpt1bfl/fl (n=8) were shipped to the Duke Proteomics Core
and Cpt1bM-/- (n=8) male mice were euthanized Facility (Durham, NC). Following
by cervical dislocation and tissues were centrifugation, phase separation was performed
collected, snap-frozen in liquid nitrogen, and by adding chloroform, and the aqueous layer
stored at -80°C until subsequent analyses could was discarded and protein precipitated from the
be performed. Mixed gastrocnemius (MG) organic layer by methanol precipitation. After
skeletal muscle was powdered and used for all washing and sonication of the protein pellet, the
assays presented. The Pennington Biomedical precipitate was reconstituted in 50 µL of 0.25%
Research Center has an AALAC-approved Rapigest SF (Waters Corporation, Milford, MA)
Comparative Biology Core facility and in 50 mM ammonium bicarbonate, pH=8.0.
veterinary staff that continuously monitor the Bradford assays were performed to determine
health of the animals via a sentinel program and protein quantity and were used for subsequent
daily inspection. All animal studies performed proteomics analysis.
were approved by the Pennington Biomedical Serial Analysis of Gene Expression
Research Center Institutional Animal Care and (SAGE): A primary goal of this study was to
Use Committee. detect comprehensive changes in gene
expression in skeletal muscle from Cpt1bfl/fl vs.
Cpt1bM-/- mice, thus a serial analysis of gene
METHOD DETAILS expression (SAGE) was performed by the
Glucose Tolerance Test: Glucose tolerance Genomics Core at the Pennington Biomedical
tests were performed after a 4h fast as described Research Center (Baton Rouge, LA). The SAGE
(9,13). Briefly, after measuring baseline blood analysis was performed as reported earlier
glucose levels via tail vein, mice received a (31,32). Briefly, gene expression profiling was
0.2mL intraperitoneal injection of 20% D- performed by expression tag sequencing
glucose (40mg glucose per mouse) and blood (SAGE) on an AB SOLiD 5500XL next-
glucose levels were subsequently monitored at generation sequencing instrument using reagent
20min, 40min, and 60min post-injection. kits from the manufacturer (Applied Biosystems,
RNA and Protein Isolation: RNA and Foster City, CA). Sequence reads were aligned
protein were extracted from 20-30mg of to mouse reference RefSeq transcripts (version
powdered mixed gastrocnemius (MG) skeletal mm9), via SOLiDSAGE (Applied Biosystems).
muscle using Trizol (ThermoFisher Scientific, Only uniquely mapped sequence reads were
Waltham, MA) as previously described (8,9). counted to generate the expression count level
Briefly, samples were homogenized in 1 ml for each respective RefSeq gene. Changes in
Trizol, allowed to sit at RT for 5 min, and then certain genes detected via SAGE were
10confirmed by RT-PCR using described methods after which the analytical separation was
(8,9) and primers shown in Table S3. performed using a 1.7 µm Acquity BEH130 C18
Proteomics: Another goal of this study was 75 mm × 250 mm column (Waters) using a 90-
to detect broad-scale adaptations in proteins min gradient of 5-40% acetonitrile with 0.1%
involved in metabolism in Cpt1bM-/- mice, with a formic acid at a flow rate of 300 nL/min and a
particular interest in mitochondrial adaptations. column temperature of 45°C. Data collection on
As such, we had a proteomics analysis the Synapt G2 mass spectrometer was performed
performed by the Duke Proteomics Core in ion-mobility assisted data-independent
(Durham, NC). Trizol-extracted protein (20 µg) acquisition (HDMSE) mode, using 0.6 sec
from each sample (concentration range 1-7.5 alternating cycle time between low (6 V) and
µg/µL) was normalized to an equal volume (20 high (27-50 V) collision energy (CE). Scans
µL) to yield a 1 µg/µL sample. This sample was performed at low CE measured peptide accurate
reduced and denatured with 10 mM DTT and mass and intensity (abundance), while scans at
heating at 80°C for 10 min. Samples were elevated CE allowed for qualitative
subsequently alkylated with 20 mM identification of the resulting peptide fragments
iodoacetamide at room temperature in the dark via database searching. The total analysis cycle
for 30 min and then digested with 1:50 (w/w) time for each sample injection was
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trypsin:protein overnight at 37°C. After approximately 2 h. Following the 21 total
digestion, 25 fmol/µL trypsinized yeast alcohol analyses, data was imported into Rosetta
dehydrogenase 1 (MassPrep, Waters Elucidator v3.3 (Rosetta Biosoftware, Inc), and
Corporation, Milford, MA) was added as a all LC-MS runs were aligned based on the
surrogate standard to each sample, and samples accurate mass and retention time of detected
were acidified to 1% trifluoroacetic acid and ions (“features”) using the PeakTeller algorithm
heated to 60°C for 2 h to hydrolyze Rapigest. in Elucidator. Relative peptide abundance was
After centrifugation, samples were transferred to calculated based on area-under-the-curve (AUC)
liquid chromatography (LC) vials and a quality of aligned features across all runs. The overall
control (QC) pool was prepared by mixing equal dataset had 168,700 quantified features, and
volumes of each sample. Quantitative one- 108,834 high collision energy (peptide
dimensional liquid chromatography/tandem fragment) spectra that were subjected to
mass spectrometry (LC/MS/MS) was performed database searching. This MS/MS data was
on 1 µg of the peptide digests (described in searched against a custom RefSeq database with
Supplemental Methods). Individual peptides Mus musculus taxonomy which contained a
were scored using PeptideProphet algorithm reversed-sequence “decoy” database for false
(Elucidator), and the data was annotated at a discovery rate determination. ProteinLynx(Phenomenex, Torrance, CA) held at 25˚C. The combined supernatants were dried under
mass spectrometer was run in negative nitrogen gas and subsequently resuspended in
ionization mode similar to a previously reported 300 µL of sterile water. The samples were
method (33). The chromatographic elution was transferred to autosampler vials and immediately
introduced to the Exactive Plus Orbitrap mass placed in an Ultimate 3000 RS autosampler
spectrometer (Thermo Fisher Scientific, (Dionex, Sunnyvale, CA) cooled to 4˚C.
Waltham, MA) via an electrospray ionization Samples were analyzed using Ultraperformance
source through a 0.1 mm internal diameter fused Liquid Chromotography-High Resolution Mass
silica capillary tube. Instrumental parameters Spectrometry (UPLC-HRMS) run in negative
were as follows: 3 kV spray voltage, 10 nitrogen ionization mode similar to a previously reported
sheath gas flow rate (unitless), 320˚C capillary method (33). Metabolite identification was
temperature, 3e6 AGC target, 140,000 performed by first converting raw spectrum files
resolution, and a scan window of 85 to 800 m/z to the open-source mzML format using the
from 0 to 9 min and 100 to 1000 m/z from 9 to Proteowizard package (35). Total ion
25 min. Solvent A was composed of 97:3 chromatograms were automatically corrected
water:methanol, 10 mM tributylamine, and 15 based on retention times for each sample using
mM acetic acid. Solvent B was methanol. The MAVEN software (Princeton University)
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gradient was as follows: 0 to 5 minutes, 0% B; 5 (36,37). Metabolites were identified based on
to 13 minutes, 20% B; 13 to 15.5 minutes, 55% retention times and m/z (± 5 ppm) and
B; 15.5 to 19 minutes, 95% B; and 19 to 25 chromatographic intensities were integrated for
minutes, 0% B. The flow rate was maintained each sample. Unidentified spectral features were
200 µL/min for the duration. annotated using MAVEN’s automated peak
Metabolomics: A third goal of this study detection feature, selecting m/z features
was to get a comprehensive view of global exhibiting chromatographic peak-like qualities.
adaptations in metabolism in Cpt1bM-/- skeletal Peak detection settings were as follows: 10 ppm
muscle, while also providing detail on mass domain resolution, 10 scan time domain
metabolites involved in glucose and amino acid resolution, 11 scan EIC smoothing, 0.50 min
metabolism, as well as energy status and redox peak grouping, 5 scan baseline smoothing, 80%
balance. To achieve this goal, non-targeted chromatograph intensity dropped baseline, 1
metabolomics was performed by the Biological minimum peak per group, 3 signal to baseline
and Small Molecule Mass Spec Core at the and signal to blank ratios, 5 scan minimum peak
University of Tennessee (Knoxville, TN). width, and 10,000 ion minimum peak intensity,
Metabolite extraction was performed at 4˚C using default peak classifier model.
unless stated otherwise. To 1.5 mL
microcentrifuge tubes containing 10-15 mg of QUANTIFICATION AND STATISTICAL
powdered mixed gastrocnemius skeletal muscle, ANALYSIS
1.3 mL of extraction solvent was added Gene expression data analysis: Differential
consisting of a 40:40:20 HPLC grade methanol, analysis of RNA read count data was performed
acetonitrile, and water with 0.1 M formic acid using DESeq2 software (38), which models read
(34). Samples were thoroughly mixed by counts as a negative binomial distribution and
vortexing before extraction was allowed to uses an empirical Bayes shrinkage-based method
proceed for 20 min at –20˚C. Following to estimate signal dispersion and fold-changes.
extraction, samples were centrifuged (5 min at Gene expression signals were logarithmically
16.1 rcf) and supernatants were transferred to transformed (to base 2) for all downstream
new vials. Pellets were resuspended in an analyses (the lowest expression value being set
additional 50 µL of extractions solvent and to 1 for this purpose).
extraction was again allowed to proceed for 20 Pathway enrichment analysis: Pathway
min at –20˚C before being centrifuged. enrichment was conducted via three separate
Supernatants were again transferred to vials and approaches - (a) competitive gene-scoring based
the process was repeated once more with an on gene set enrichment analysis (GSEA) (39),
additional 50 µL of extraction solvent. The (b) over-representation analysis (ORA) based on
12a pre-filtered list of differentially expressed In addition to the above 2 ‘competitive test’
genes (Ingenuity Pathway Analysis (IPA), methods (44), we also queried gene expression
Qiagen, Redwood City, differences via a ‘self-contained’ test for gene-
www.qiagen.com/ingenuity), and (c) enrichment set enrichment known as the globaltest. The
analysis of pre-selected pathways via the globaltest method is based on regression
globaltest package in R (http://bioconductor.org) analysis of the association of a gene-set’s
(40). GSEA was performed by first ranking the component gene expression to a phenotype, and
expression of all genes in the Cpt1bfl/fl (WT) and tests the null hypothesis that no gene in the
Cpt1bM-/- (KO) groups via the signal-to-noise gene-set is associated with the phenotype (with
ratio (SNR) metric, and then employing a no additional consideration for genes in other
weighted Kolmogorov-Smirnov test to gene-sets). We applied globaltest to the same
determine if the gene SNRs deviate significantly gene expression data as used for GSEA analysis,
from a uniform distribution in a priori defined and queried KEGG specific gene-sets obtained
gene-sets (pathways) derived from the Kyoto from the package kegg.db (Carlson M.
Encyclopedia of Genes and Genomes (KEGG) KEGG.db: A set of annotation maps for KEGG.
database (41) via the Molecular Signatures R package version 3.0.0.). Gene annotations
Database repository (MSigDb, were provided as Entrez IDs to match the gene-
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http://software.broadinstitute.org/gsea/msigdb/, set annotations utilized by globaltest from
(42)). Statistical significance for the observed org.Mm.eg.db (release 3.3) (Carlson M.
enrichment was ascertained by permutation org.Mm.eg.db: Genome wide annotation for
testing over size-matched gene-sets. Significant Mouse. R package version 3.2.3).
gene-sets were selected by control of the false For visual representations of gene and
discovery rate (FDR) at 25% (43). The per- metabolite expression pattern differences in
sample expression profiles of genes contributing pathways, we used the Search and Color
to core enrichment of the significant pathways Pathway tool from the KEGG Mapper suite of
were visualized via row-normalized blue-red tools (www.genome.jp/kegg/mapper.html). A
heatmaps with blue representing lower, and red total of 389 genes with nominal p-value 1.5-fold between the
IPA analysis was carried out on Cpt1bfl/fl and Cpt1bM-/- groups were selected for
differentially expressed genes (with absolute representation on the KEGG pathway maps.
fold-change >1.5-fold and FDRwere uploaded excluded an outlier (KO8) that as an outlier from the metabolomics dataset.
was identified in both, while WT8 was removed
ACKNOWLEDGEMENTS
We thank Jaycob Warfel, Susan Newman, Claudia Kruger, and Richard Carmouche for critical advice,
reagents and/or technical assistance. We recognize Drs. Matthew Foster, J. Will Thompson and Arthur
Moseley from the Duke Proteomics Core Facility (Duke University School of Medicine) for performing
the proteomic analysis. This work utilized PBRC core facilities (Genomics, and Transgenic and Animal
Phenotyping) that are supported in part by COBRE (NIH 3 P30-GM118430) and NORC (NIH 2P30-
DK072476) center grants from the National Institutes of Health. This research was supported by NIH
3P30-GM118430 (R.C.N.), NIH 1R01DK103860 (R.C.N.), ADA grant # 1-10-BS-129 (R.L.M.), and
NIH 1R01DK089641 (R.L.M.). S.E.W. was supported by a T32 AT004094 fellowship and received a
pilot and feasibility grant from NORC (NIH 2P30-DK072476). J.M.S. is funded in part by a Botanicals
and Dietary Supplements Research Center grant (P50 AT002776). Dr. Sujoy Ghosh was supported in part
by 2 U54 GM104940 from the National Institute of General Medical Sciences of the National Institutes of
Health, which funds the Louisiana Clinical and Translational Science Center.
Downloaded from http://www.jbc.org/ by guest on November 8, 2019
DECLARATION OF INTERESTS
The authors have no conflicts of interest to report.
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