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Metabolomic and Transcriptomic Changes Induced by Overnight (16 h) Fasting in Male and Female Sprague Dawley Rats Donald G. Robertson,*,† Stefan U. Ruepp,‡ Steven A. Stryker,† Serhiy Y. Hnatyshyn,† Petia A. Shipkova,† Nelly Aranibar,† Colleen A. Mcnaney,† Oliver Fiehn,§ and Michael D. Reily† †
Applied and Investigative Metabolomics and ‡Discovery Toxicology, Bristol-Myers Squibb Co., Princeton, New Jersey, Hopewell, New Jersey, and Wallingford, Connecticut, United States § Department of Molecular and Cellular Biology, UC Davis Genome Center, Davis, California, United States
bS Supporting Information ABSTRACT: The overnight (16-h) fast is one of the most common experimental manipulations performed in rodent studies. Despite its ubiquitous employment, a comprehensive evaluation of metabolomic and transcriptomic sequelae of fasting in conjunction with routine clinical pathology evaluation has not been undertaken. This study assessed the impact of a 16-h fast on urine and serum metabolic profiles, transcript profiles of liver, psoas muscle, and jejunum as well as on routine laboratory clinical pathology parameters. Fasting rats had an approximate 12% relative weight decrease compared to ad libitum fed animals, and urine volume was significantly increased. Fasting had no effect on hematology parameters, though several changes were evident in serum and urine clinical chemistry data. In general, metabolic changes in biofluids were modest in magnitude but broad in extent, with a majority of measured urinary metabolites and from 1/3 to 1/2 of monitored serum metabolites significantly affected. Increases in fatty acids and bile acids dominated the upregulated metabolites. Downregulated serum metabolites were dominated by diet-derived and/or gut-microflora derived metabolites. Major transcriptional changes included genes with roles in fatty acid, carbohydrate, cholesterol, and bile acid metabolism indicating decreased activity in glycolytic pathways and a shift toward increased utilization of fatty acids. Typically, several genes within these metabolic pathways, including key rate limiting genes, changed simultaneously, and those changes were frequently correlative to changes in clinical pathology parameters or metabolomic data. Importantly, up- or down-regulation of a variety of cytochrome P450s, transporters, and transferases was evident. Taken together, these data indicate profound consequences of fasting on systemic biochemistry and raise the potential for unanticipated interactions, particularly when metabolomic or transcriptomic data are primary end points.
’ INTRODUCTION The overnight (16-h) fast is one of the most common experimental manipulations performed in rodent toxicity studies. The usual rationale for employing the overnight fast is to normalize rodents to a common nutritional state so that parameters that are affected by dietary factors are less influenced by the uncertainty of when food was last consumed prior to sampling. These parameters most typically include histological assessments such as liver glycogen content and clinical chemistry measurements including serum triglycerides and glucose. For many parameters, it might be theoretically better to normalize animals to a common fed state, but that is difficult if not impossible to do routinely in vivo. It is much easier to simply remove feeders at a given time (typically overnight). Of course, even then there is some variability as feeders are typically removed toward the end of the light cycle when rodents are less r 2011 American Chemical Society
prone to feed; therefore, what is supposed to be a 16 h fast may in fact be much longer. This paradigm, with feeders removed during the active feeding cycle of rodent (dark cycle), stands in contrast to human overnight fasts where most subjects are asleep anyway. While an overnight fast might make sense in clinical practice, it is less clear how translatable rodent data collected after overnight fasts is to clinical data collected under the same conditions. Fasting has been recognized as one of the most significant sources of variation in transcriptomic studies1 and several studies have investigated the effect of fasting in different organs of various species; however, the impact of an overnight fast on individual transcripts from multiple tissues and metabolite expression in common biofluids has not been comprehensively Received: February 16, 2011 Published: March 07, 2011 481
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evaluated.2,3 Therefore, the objective of this study was to assess the impact of a 16-h fast in Sprague Dawley rats on serum and urine metabolic profiles (metabolomics) and liver, muscle, and intestine transcript profiles (transcriptomics) in conjunction with routine clinical and clinical pathology assessments. It is hoped that these data will establish variation that should be anticipated when comparing “omic” data between studies conducted in the fasted vs nonfasted state. Additionally, the extent and magnitude of fasting-induced changes in individual metabolites or transcripts should help contextualize changes produced in response to toxic insults.
males and females, respectively). Serum alanine aminotransferase, alkaline phosphatase, and calcium were significantly decreased, while inorganic phosphorus and total bile acids (TBA, males only) were significantly increased in fasted animals (Supporting Information, Table S2). It should be noted that with the exception of serum triglycerides and glucose, the magnitude of the observed differences between fed and fasted animals was minimal in most cases, with resultant means of significantly varying parameters usually remaining within, or very close to, our laboratory reference range which is determined in fasted animals. Urine Metabolomics. Twenty-eight metabolites were unambiguously identified in urine by NMR spectroscopy, and the results are summarized in Supporting Information, Table S3. In general, the changes in urinary metabolites were modest in extent with no annotated metabolite varying by more than 2.6-fold between the fed and fasted groups with little difference in effects on metabolic profile between the sexes. Of particular note were changes in metabolites characterized as “usual suspects” in metabolomic studies.4 The “usual suspects” are a group of metabolites that are found to frequently change in response to treatment, without any obvious relationship to the efficacy and/ or toxicity of the compound under investigation. These included 1.5- to 2-fold decreases in citrate, 2-oxoglutarate, fumarate, and hippurate (males only); and 1.2 to 1.6 fold increases in taurine, lactate, creatinine (females), and succinate (males). Serum Metabolomics. A total of 48 metabolites were identified by LCMS and 153 by GCMS in the serum from fed and fasted animals. Complete listings of all annotated metabolites identified by LCMS and GCMS are provided in Supporting Information, Tables S4 and S5, respectively. A total of 21 annotated metabolites were common between the two technologies (Supporting Information, Table S6). With the possible exception of glutamine and glutaric acid, the observed changes in these metabolites were generally in good agreement between the two techniques, with changes in some metabolites reaching statistical significance by one technique but not the other. The top 10 upregulated and downregulated serum metabolites are provided in Table 1. Increases in fatty acids (measured by GCMS) and bile acids (measured by LCMS) dominated the upregulated metabolites (Supporting Information, Tables S7 and S8, respectively). The changes in bile acids were characterized by wide interanimal variation, and despite large increases (up to 35fold) in some group means, statistical significance was not always achieved. Downregulated serum metabolites were dominated by diet-related and/or gut-microflora derived metabolites. These include raffinose, glycerol-3-galactoside, 3-phenyllactic acid, sucrose, hydrocinnamic acid, saccharic acid, and salicylic acid. General Transcriptional Effects. A profound number of transcripts were affected by feeding status in both sexes and in all tissues assessed (Supporting Information, Table S9). Although the absolute number of differentially expressed genes was similar in the liver [7.3% in males (M); 10.9% in females (F)] and psoas (8.8% M; 10.1% F), and slightly smaller in the jejunum (7.7% M; 6.3% F), there were distinct differences in individual transcript levels in the three tissues assayed, clearly differentiating between the 3 organs. Biological pathways and processes that were altered in the liver by feeding status included a large number of genes with roles in lipid metabolism (Supporting Information, Table S10). Prominently represented were genes involved in β-oxidation, essentially representing the entire chain of biochemical events from
’ MATERIALS AND METHODS A full description of the materials and methods used in this article can be found in the Supporting Information. Briefly, 7 8 week old male and female Sprague Dawley rats were split into two groups with one group (5/sex) fasted from 4 p.m. to 8 a.m. and another group (5/sex) allowed ad libitum access to food over the same period. Urine was collected over the 16-h period of fasting, and blood was collected at termination for hematology, clinical chemistry, and metabolomic analysis (serum). Liver, muscle (posas), and jejunum were collected at termination for transcriptomic analyses. Urine metabolomic analyses were conducted using nuclear magnetic resonance spectroscopy, and serum was analyzed by both liquid chromatographic and gas chromatographic mass spectroscopy (LCMS and GCMS) methods. RNA was isolated from selected tissues and transcript analyses conducted using Affymetrix (Santa Clara, CA) HT_FOCUS partial rat genome arrays following standard protocols. Transcriptional data were analyzed with Resolver version 7.1 (Rosetta Biosoftware, Seattle, WA), using a false discovery rate of 1%, and overrepresented categories and pathways were identified using Gene Ontology terms (Gene Ontology Consortium). Array data have been submitted to ArrayExpress (http://www.ebi.ac. uk/arrayexpress; accession numbers: E-MTAB-572 (liver), E-MTAB573 (psoas), E-MTAB-574 (jejunum).
’ RESULTS Body Weight. On average, fasted male and female rats lost approximately 9% and 7% of their prefast weight, respectively, while ad libitum fed male and female rats gained approximately 3 to 4% of their prefast weight leading to a weight differential of approximately 12% between the fasted and fed groups of both sexes over approximately 24 h from the prefast bodyweight determination to the postfast (terminal) weight determination. Clinical Pathology. There was no effect of fasting on any measured hematology parameter with the exception of platelet count in males, which was significantly elevated (p < 0.05) in fasted males relative to that of their ad libitum counterparts (1475 ( 213 103/mL vs 1170 ( 122 103/mL). Both means were within the laboratory reference range (1004 1546 103/ mL). Urine and serum clinical chemistry changes are summarized in Supporting Information, Tables S1 and S2. Urine clinical chemistry changes were largely driven by the increased urine volume evident in fasted animals of both sexes (∼2-fold), though the effect was statistically significant only in males. The increased volume in fasted animals correlated with significantly decreased specific gravity, osmolality, and protein concentration in both sexes. Fasting had no effect on serum blood urea nitrogen, creatinine, cholesterol, total protein, albumin(A), globulin(G), and A/G ratio in either sex. As anticipated, decreases were noted in serum triglycerides (∼70% in both sexes) and glucose (33 and 52% in 482
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Table 1. Top 10 Upregulated and Downregulated Serum Metabolitesa male
female
fold change
p value
fold change
p value
platform
cholic acid
38.28
0.0141
13.08
0.0856
GCMS
cholic acid
30.36
0.0087
6.69
0.2247
LCMS
3-hydroxybutanoic acid
6.47
0.0001
5.25
0.0001
GCMS
compound
glycoursodeoxycholic acid
5.61
0.1288
35.22
0.0799
LCMS
N-methylalanine
5.36
0.0001
7.9
0.0017
GCMS GCMS
palmitoleic acid
5.26
0.0068
7.81
0.0003
Biuret
3.86
0.1050
2.2
0.1179
GCMS
elaidic acid creatine
3.86 3.22
0.1233 0.0048
3.97 4.09
0.0009 0.0018
GCMS LCMS GCMS
oleic acid
3.2
0.0001
4.34
0.0004
ribonic acid
3.07
0.2069
1.54
0.3295
GCMS
3-hydroxy-3-methylglutaric acid
2.61
0.0073
6.28
0.0060
GCMS
sulfoglycolithocholic acid
2.60
0.1901
3.66
0.0495
LCMS
creatinine
2.56
0.0043
2.96
0.0030
GCMS
2-hydroxybutanoic acid
2.45
0.0008
2.89
0.0041
GCMS
0.0000 0.0008
GCMS GCMS
threitol salicylic acid
2.45 2.87
0.0002 0.0044
2.52 3.84
idonic acid
2.89
0.0316
2.63
0.0632
GCMS
lactobionic acid
3.23
0.0001
3.79
0.0002
GCMS
saccharic acid
3.26
0.0029
3.72
0.0001
GCMS
hydrocinnamic acid
3.36
0.0110
7.78
0.0002
GCMS
R-ketoglutaric acid
3.67
0.0096
5.61
0.0177
GCMS
taurodeoxycholic acid
3.70
0.0876
sucrose 3-phenyllactic acid
5.48 9.31 25.34
0.0269 0.0012 0.0018
156.92
0.0371
glycerol-3-galactoside raffinose
1.52
0.6704
LCMS
1.76 8.95 23.73
0.0942 0.0029 0.0000
GCMS GCMS GCMS
378.26
0.0002
GCMS
a
Ordered by fold change in males; nonsignificant (p > 0.05) metabolites indicated but not included in the 10-count. Fold change indicates change in fasted animals relative to ad libitum fed animals. Bold faced numbers indicate signific upregulated metabolites, and italicized numbers indicate significantly downregulated metabolites (p < 0.05).
cholesterol 7 R-hydroxylase (CYP7A1), 3-hydroxy-3-methylglutaryl-Coenzyme A reductase, and mevalonate kinase, and the cholesterol-related transcription factors sterol regulatory element-binding proteins 1 and 2. Additionally, mRNAs for both the LDL-receptor and proprotein convertase subtilisin/kexin type 9 (PCSK9), which cleaves the LDL receptor, were reduced (Supporting Information, Table S14). Interestingly, there were numerous transcriptional changes relevant to drug metabolism observed and included effects on cytochrome P450s, transferases, and transporters. There was no general concerted up or downregulation, but individual members were clearly affected (Table 2). In the jejunum, the most prominent transcriptional pathway changes affected by feeding status were similar in nature to changes observed in the liver and included upregulation in fatty acid catabolism (Supporting Information, Table S10) and decreases in cholesterol and sterol metabolism (Supporting Information, Table S14). However, both the number of transcripts affected in these pathways and the magnitude of change were generally smaller than those in the liver. Other changes of interest included downregulation of genes encoding enzymes in the TCA cycle (Supporting Information, Table S11) and strong downregulation of cytochrome P4501A1 (16 20-fold in males and females, respectively, Table 2).
the formation of acyl-CoA from fatty acids by acyl-CoA synthetase through the transcriptional induction of rate-controlling enzymes such as carnitine palmitoyltransferase (CPT) I and CPT II to the ultimate generation of acetyl-CoA by acetylCoenzyme A acyltransferase which can be further metabolized in the TCA cycle. Several genes encoding enzymes in the TCA cycle were altered in both sexes and included increases in mRNAs for aconitase 1 (Aco1), aconitase 2 (mitochondrial, Aco2), and succinate-CoA ligase and reduced citrate synthase (Cs) (Supporting Information, Table S11). Key transcripts in carbohydrate and glucose metabolism including glucokinase and pyruvate kinase were downregulated by fasting (Supporting Information, Table S12). In addition to the multitude of metabolism-related transcriptional changes, changes related to cell maintenance or cell growth were also observed and included downregulation of mRNA’s for thymidine kinase 1 and Cyclindependent kinase 2 (CDK2) (Supporting Information, Table S13). Fasting induced clear differences in cholesterol metabolism with the majority of the cholesterol biosynthesis pathway significantly downregulated in livers from fasted males and females compared to that of their respective ad libitum fed controls. Among the affected genes were several genes encoding key enzymes considered to be rate-limiting in cholesterol biosynthesis and included 483
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Table 2. Effect of Fasting on Selected CypP450s, Transporters, and Transferasesa accession #
sequence description
Li M 1.69
Li F
Ps M
Ps F
Je M
Je F
1.87
U39208
cytochrome P450 4F6 (Cyp4f6)
X00469
cytochrome P450, family 1, subfamily a, polypeptide 1 (Cyp1a1)
NM_012753
cytochrome P450, family 17, subfamily a, polypeptide 1 (Cyp17a1)
M58041
cytochrome P450, family 2, subfamily c, polypeptide 70 (Cyp2c70)
AB008424
cytochrome P450, family 2, subfamily d, polypeptide 13 (Cyp2d13)
NM_019303
cytochrome P450, family 2, subfamily f, polypeptide 2 (Cyp2f2)
NM_023025
cytochrome P450, family 2, subfamily J, polypeptide 4 (Cyp2j4)
2.68
2.42
M33936 AA893326
cytochrome P450, family 4, subfamily a, polypeptide 14 (Cyp4a14) cytochrome P450, family 4, subfamily a, polypeptide 14 (Cyp4a14)
3.07 1.93
2.30 1.92
NM_016999
cytochrome P450, family 4, subfamily A, polypeptide 22 (Cyp4a22)
5.42
2.98
M29853
cytochrome P450, family 4, subfamily b, polypeptide 1 (Cyp4b1)
NM_012942
cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1)
NM_031241
cytochrome P450, family 8, subfamily b, polypeptide 1 (Cyp8b1)
NM_012941
cytochrome P450, subfamily 51 (Cyp51)
2.51
3.90
2.05
2.27
BG664123
cytochrome P450, subfamily 51 (Cyp51)
3.51
5.50
2.24
2.98
AI175666 BF281299
Homo sapiens solute carrier family 4, sodium bicarbonate cotransporter, member 4 (SLC4A4) ornithine decarboxylase 1 (Odc1)
4.25
3.75 1.25
NM_031576
P450 (cytochrome) oxidoreductase (Por)
2.56
3.55
AI407454
P450 (cytochrome) oxidoreductase (Por)
2.49
3.69
D50306
solute carrier family 15 (oligopeptide transporter), member 1 (Slc15a1)
BI289867
solute carrier family 16 (monocarboxylic acid transporters), member 6 (Slc16a6)
U76379
solute carrier family 22 (organic cation transporter), member 1 (Slc22a1)
NM_019269
solute carrier family 22 (organic cation transporter), member 5 (Slc22a5)
6.77
3.98
BI274649 J02612
solute carrier organic anion transporter family, member 5A1(Slco5a1) UDP glycosyltransferase 1 family, polypeptide A6 (Ugt1a6)
1.77
1.69
AF461738
UDP glycosyltransferase 1 family, polypeptide A6 (Ugt1a6)
1.63
1.57
16.0 5.31 2.16 1.30 1.35
1.87 1.34 1.34
1.65 5.55 3.19
19.7
10.4
1.96
2.54
5.20 2.90
1.45
1.58
1.15
1.79
2.67
2.30
1.78
1.57
2.02
1.62
2.01
1.83
a
The numbers in the table refer to fold change in transcript expression in fasted animals relative to ad libitum fed animals. Bold faced numbers indicate significantly upregulated transcripts, and italicized numbers indicate significantly downregulated metabolites (p < 0.01). Li = liver, Ps = psoas, and Je = jejunum
Fasting affected a large number of transcripts (similar to liver) in the psoas (Supporting Information, Table 9); however, the effects within biological pathways (and Gene Ontology categories) were not as comprehensive as those in the liver. For example, increases in transcripts related to fatty acid catabolism (Supporting Information, Table S10) and decreases in transcripts involved in cholesterol and sterol metabolism (Supporting Information, Table S14) were observed as in the other organs assessed, but only some members within these pathways met the statistical criteria as opposed to almost every pathway member in the case of the liver and jejunum. There were some other interesting transcriptional changes in the psoas which included the upregulation of pyruvate dehydrogenase kinase isozyme 4 (PDK4), a key enzyme linking glycolysis and the TCA cycle. Psoas PDK4 was induced 5- and 8-fold in males and females, respectively. Also, uncoupling protein 3 (UCP3), a protein reported to be necessary for the fasting-induced increase in rate and capacity of fatty acid oxidation, was substantially upregulated.5 Other noteworthy metabolism-related transcripts included uncoupling fatty acid binding proteins (Fabp4, 5, 7), mitochondrial acyl-CoA thioesterase 1, and diacylglycerol O-acyltransferase homologue 2 (Dgat2) (Supporting Information, Table S10).
SD rat. We conducted this work to establish what range of variation might be anticipated in commonly identified metabolites and in transcripts when these “omic” evaluations are included in toxicology studies. While it is well beyond the scope of this article to discuss all the changes, general observations with a few specific examples will be made to highlight how and why the collection of such data is important. The fasting-induced weight loss and clinical pathology data were, with a few exceptions, consistent with previous studies.6,7 The most obvious effect of fasting was increased urine volume. The fasting-induced increase noted in this study (approximately 2- fold) is actually on the low end of the increases we historically see in overnight fasts of rats in growth phase (typically 3 5-fold, unpublished observations). Fasting-induced polyuria has been ascribed to the downregulation of aquaporins in the kidney with a concomitant decrease in water resorption.8 The significance for metabolomic evaluations is that, with all other things being equal, urine concentrations of most metabolites are diluted in fasting animals relative to nonfasted animals. This necessitates appropriate normalization of urine metabolite data. There are a number of normalization approaches, and no single method can be considered appropriate for all metabolomic analyses. However, we find normalization to the total NMR signal gives us the best results for rat urine metabolic analysis. Therefore, the urine metabolite changes are indicative of the more toxicologically relevant changes in fractional metabolite excretion, not
’ DISCUSSION This study identified numerous biochemical and transcriptional changes induced by a standard 16-h (overnight) fast in the 484
dx.doi.org/10.1021/tx200074f |Chem. Res. Toxicol. 2011, 24, 481–487
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Figure 1. Biochemical effects of fasting. Diagram includes measured (boxed) and known effects of fasting. Italicized type indicates downregulated (or mostly downregulated in the case of transcripts) parameters or processes, and bolded type indicates upregulated ones. Individual transcript data can be found in the indicated Supporting Information tables. Inset: results of fasting on TCA cycle intermediates. Black arrows indicate serum results, and gray arrows indicate urine results. A horizontal arrow means the metabolite was measured but was not significantly affected. If the effect was noted in only one sex, the appropriate symbol is indicated under the arrow; otherwise, the effect was evident in both sexes.
urinary metabolite concentration, which, in this case, would almost all decrease in fasted animals because of the increased urine volume. While creatinine normalization is an approach typically used (particularly when untimed collections of urine are taken), our data suggest that that approach may be questionable in fasted rats as fasting induced significant increases in both serum creatine (3 4) and urine creatinine (∼1.3) excretion. Data comparing transcriptomic and metabolomic differences between fed and fasted animals are limited. Additionally, given that in this study urine was collected over a 16 h time period while serum and transcripts were collected at a single time point at termination, care must be taken in comparing urine data with the serum and transcript data. While it has been recognized for some time that fasting (or inappetence) causes changes in rat urine NMR spectral profiles,9 corresponding serum metabolomic data are largely absent in the literature. Overall, perhaps the most striking aspect of the metabolomic data was the breadth of the measured changes. While most changes were relatively small in magnitude, a majority of urine metabolites and 1/3 to 1/2 of serum metabolites were significantly affected by fasting. Published data documenting metabolic disruption by toxicants seldom indicate fractional disruption of the metabolome, especially as the extent of coverage of the metabolome will vary on a platform and laboratory basis. Anecdotally, on the basis of our internal experience, the number of metabolites significantly affected by fasting are similar to or greater than the number affected by toxicants inducing profound histopathological changes. While data on several metabolites appeared to vary between the sexes, the similarity of the data between the sexes
was more notable than the differences. From a toxicogenomic perspective, the liver is the most widely investigated tissue, and a large comparative data sets exists. The number of hepatic transcriptional changes (p < 0.01) in response to fasting alone was 7% in males and 10% in females. This magnitude of change exceeds effects seen in the vast majority of preclinical studies, including treatment with hepatotoxic compounds.10 For the most part, the observed changes in metabolic and transcriptomic profiles were consistent with what might be expected from the known sequelae of fasting or starvation as depicted in Figure 1. Logically, serum and urine constituents of direct dietary origin are rapidly decreased, and the absence of food in the gut leads to decreases in several metabolites of microfloral origin. Concurrently, decreased food intake rapidly leads to decreased serum glucose with resultant increases in serum glucagon. The observed decrease in serum glucose was exemplified by the downregulation of a variety of genes with roles in glucose metabolism. These effects were observed in all three organs assessed, but were most profound in the liver involving key genes including glucokinase, a pivotal regulator of glucose metabolism, pyruvate kinase, and glycogen phosporylase (Supporting Information, Table S12). One action of glucagon is the initiation of gluconeogenesis, which requires oxaloacetate (OAA) at the level of phosphoenolypyryvate carboxykinase. As OAA is tied up in gluconeogenesis, it is no longer available to bring acetyl CoA, generated as a result of fatty acid oxidation, into the TCA cycle via citrate synthase. Consequently, acetyl CoA is shunted toward the formation of ketone bodies (e.g., 3-hydroxybutyrate). Accordingly, 3-hydroxy-3-methylglutaryl-Coenzyme A 485
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synthase 2 (Hmgcs2), a rate-limiting enzyme in ketogenesis,11 was upregulated in the liver, jejunum, and psoas muscle tissues, and HMG-CoA lyase was upregulated in the liver (Supporting Information, Table S15). Upregulation of Hmgcs2 was most profound in the jejunum (11- and 15-fold in males and females, respectively). Hmgcs2 protein is present in the intestine, and its expression can be modulated by the intestinal flora,12,13 which, as revealed by the metabolomic data, was altered by fasting. Another action of glucagon is the activation of lipolysis via increases in lipase and perilipin phosphorylation, the result of which is an increase in serum fatty acids as noted in Supporting Information, Table S7. While the profile of changes in fatty acids may simply reflect the relative abundance of the respective fatty acids in fat, it has been recognized that not all fatty acids are equivalently mobilized from peripheral adipose stores.14,15 Consistent with the metabolomic data, a wide variety of transcripts encoding proteins with important roles in fatty acid oxidation were upregulated in the liver, muscle, and jejunum (Supporting Information, Table S10). In the liver, essentially the whole pathway from acyl-CoA synthesis, import through the outer and inner mitochondrial membranes by CPT-I and CPT-2, respectively, and further reactions by Acad (acyl-CoA dehydrogenase) and Ech (enoyl CoA hydratase) to the level of acetyl-CoA formation was upregulated. In the muscle and jejunum, some individual transcripts involved with fatty acid oxidation did not meet the statistical criteria (FDR
