Progressive adaptation of hepatic ketogenesis in mice

fed a high-fat diet

Nishanth E. Sunny, Santhosh Satapati, Xiaorong Fu, TianTeng He, Roshi
Mehdibeigi, Chandra Spring-Robinson, Joao Duarte, Matthew J. Potthoff, Jeffrey
D. Browning and Shawn C. Burgess
Am J Physiol Endocrinol Metab 298:E1226-E1235, 2010. First published 16 March 2010;
doi:10.1152/ajpendo.00033.2010

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Am J Physiol Endocrinol Metab 298: E1226–E1235, 2010.
First published March 16, 2010; doi:10.1152/ajpendo.00033.2010.
Progressive adaptation of hepatic ketogenesis in mice fed a high-fat diet
Nishanth E. Sunny,1* Santhosh Satapati,1,2* Xiaorong Fu,1* TianTeng He,1 Roshi Mehdibeigi,1
Chandra Spring-Robinson,1 Joao Duarte,1 Matthew J. Potthoff,3 Jeffrey D. Browning,1
and Shawn C. Burgess1,2,3
1
The Advanced Imaging Research Center, University of Texas Southwestern Medical Center; 2Department of Molecular and
Cell Biology, University of Texas at Dallas; and 3Department of Pharmacology, University of Texas Southwestern Medical
Center, Dallas, Texas
Submitted 13 January 2010; accepted in final form 10 March 2010
Sunny NE, Satapati S, Fu X, He T, Mehdibeigi R, Spring-
Robinson C, Duarte J, Potthoff MJ, Browning JD, Burgess SC.
Progressive adaptation of hepatic ketogenesis in mice fed a high-fat
diet. Am J Physiol Endocrinol Metab 298: E1226 –E1235, 2010. First
published March 16, 2010; doi:10.1152/ajpendo.00033.2010.—He-
patic ketogenesis provides a vital systemic fuel during fasting because
ketone bodies are oxidized by most peripheral tissues and, unlike
glucose, can be synthesized from fatty acids via mitochondrial !-ox-
idation. Since dysfunctional mitochondrial fat oxidation may be a
cofactor in insulin-resistant tissue, the objective of this study was to
determine whether diet-induced insulin resistance in mice results in
impaired in vivo hepatic fat oxidation secondary to defects in ketogenesis.
Ketone turnover ("mol/min) in the conscious and unrestrained mouse
was responsive to induction and diminution of hepatic fat oxidation, as
indicated by an eightfold rise during the fed (0.50#/$0.1)-to-fasted
(3.8#/$0.2) transition and a dramatic blunting of fasting ketone
turnover in PPAR%$/$ mice (1.0#/$0.1). C57BL/6 mice made obese
and insulin resistant by high-fat feeding for 8 wk had normal expres-
sion of genes that regulate hepatic fat oxidation, whereas 16 wk on the
diet induced expression of these genes and stimulated the function of
hepatic mitochondrial fat oxidation, as indicated by a 40% induction
of fasting ketogenesis and a twofold rise in short-chain acylcarnitines.
Together, these findings indicate a progressive adaptation of hepatic
ketogenesis during high-fat feeding, resulting in increased hepatic fat
oxidation after 16 wk of a high-fat diet. We conclude that mitochon-
drial fat oxidation is stimulated rather than impaired during the
initiation of hepatic insulin resistance in mice.
hepatic insulin resistance; liquid chromatography-tandem mass spec-
trometry; nuclear magnetic resonance
THE LIVER MAINTAINS MULTIPLE METABOLIC PATHWAYS critical for
the adaptive response to variable macronutrient consumption.
During feeding, liver accumulates excess carbohydrate as gly-
cogen or converts it to lipid for storage in peripheral adipose
tissue. In the postabsorptive state, glycogen breakdown (gly-
cogenolysis) buffers plasma glucose levels, and adipose-de-
rived nonesterified fatty acids (NEFA) are oxidized by the liver
to support endergonic fasting pathways such as gluconeogen-
esis and ureagenesis. During extended fasting or limited car-
bohydrate availability, the liver adapts to dwindling glycogen
stores by increasing !-oxidation and converting the resulting
acetyl-CoA to ketone bodies, primarily acetoacetate and its
chemically reduced analog !-hydroxybutyrate. Since hepatic
glucose production can drop by as much as 40% during
* These authors contributed equally to this article.
Address for reprint requests and other correspondence: S. C. Burgess,
Advanced Imaging Research Ctr. and Dept. of Pharmacology, Univ. of Texas
Southwestern Medical Ctr., 2201 Inwood Rd., Dallas, TX 75390-8568 (e-mail:
shawn.burgess@utsouthwestern.edu).
E1226 0193-1849/10 Copyright © 2010 the American Physiological Society
extended fasting (52), the &10-fold rise in plasma ketone
concentration that occurs under these conditions (19) provides
a critical alternative substrate for oxidation by central nervous
and other tissues normally dependent on glucose metabolism.
Ketogenesis occurs almost exclusively in liver mitochondria
(23) and during extended fasting can account for roughly
two-thirds of total hepatic fat oxidation in rodents, with
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terminal oxidation of acetyl-CoA in the citric acid cycle
accounting for the rest (31, 44, 48). Hepatic ketogenesis is
influenced by a number of mechanisms, including hormonal
signaling (31), direct substrate metabolism (31), and auto-
nomic control (7).
Abnormal mitochondrial fat oxidation is a key component of
numerous diseases, including inborn errors of metabolism (51),
infection (12, 37), and alcohol toxicity (27), and is closely
associated with insulin resistance and related pathophysiolo-
gies (29, 30, 36, 49). Insulin-resistant skeletal muscle contains
mitochondria that inefficiently metabolize fat (22, 25, 42, 50),
a defect that may precede the onset of insulin resistance (42)
and likely contributes to impaired insulin signaling. A similar
deficiency in mitochondrial fat oxidation may be present in the
insulin-resistant liver (43, 53). Evidence that hepatic fat oxi-
dation, and specifically ketogenesis, is affected by the meta-
bolic manifestations of insulin resistance is demonstrated by
elevated ketone bodies in humans with nonalcoholic fatty liver
disease (47) and impaired fasting ketosis in the Zucker Dia-
betic fatty (ZDF) rat, which develops fatty liver in conjunction
with impaired fasting hepatic fat oxidation and a fourfold
reduction in ketogenesis (48, 54). However, systematic inves-
tigation of whether hepatic fat metabolism is altered during in
insulin resistance has been limited because few in vivo ap-
proaches can be applied against mouse models.
Here, we report the effect of a high-fat diet-induced insulin
resistance on hepatic fat oxidation in mice, using stable isotope
tracers and liquid chromatography-tandem mass spectrometry
(LC-MS/MS) to monitor ketogenesis and acylcarnitine pro-
files. Ketone turnover increased robustly during longitudinal
fasting studies in mice and was markedly blunted in fed and
fasted PPAR%$/$ mice, a well-described model of impaired
hepatic fat oxidation (24). In contrast, mice fed a high-fat diet
to induce insulin resistance had normal ketone body turnover
after 8 wk, and elevated ketone body turnover after 16 wk of
high-fat feeding, in conjunction with increased hepatic expres-
sion of genes principal to fat oxidation and ketone body
formation. The data indicate a progressive increase in hepatic
fat oxidation in diet-induced obese mice and demonstrate that
impaired fat oxidation is not necessarily required for the
development of hepatic insulin resistance.
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 ALTERED HEPATIC FAT OXIDATION DURING INSULIN RESISTANCE
MATERIALS AND METHODS
Animals
All of the animal protocols were approved by the University of
Texas Southwestern Institutional Animal Care and Use Committee.
Comparison of 13C NMR and LC-MS/MS. Long-Evans rats (&250
g; n ' 5) were fasted for 24 h, and ketone body turnover was
determined by LC-MS/MS (described below) or by a 13C NMR
method previously used by our laboratory (48) and compared to
confirm that the two methods provide identical results.
Effect of fasting on ketone body turnover in mice. Serial measure-
ments of ketone body turnover during feeding and 16 and 24 h of
fasting were performed in 12-wk-old female C57BL/6 mice (n ' 5)
maintained on normal laboratory chow. Mice were infused in the late
afternoon while given full access to chow, the next morning (16 h
after chow was removed), and then again in the afternoon of the 2nd
day (24 h after chow was removed).
Effect of impaired fat oxidation on ketone body turnover in mice.
Ketone body turnover during the fed-to-16-h-fasted transition (n '
3–5) was studied in male PPAR%#/# (129S1/SvlmJ, Jaxlab 002448)
and PPAR%$/$ (Ppara-tm1Gonz/J, Jaxlab 003580) mice to determine
whether ketone body turnover responded to known defects in fat
oxidation.
Effect of diet-induced obesity on ketone body turnover in mice.
Four- to six-week-old male C57BL/6 mice (n ' 7–10) were main-
tained on either a control 10% fat calorie diet (TD06416; Harlan-
Teklad) or a 60% fat calorie diet (TD06414; Harlan-Teklad) for 8 or
16 wk to induce obesity and insulin resistance (40). Unless noted
otherwise, fasted measurements were made after an overnight, 16-h
fast, and fed measurements were made in the morning from mice with
normal access to food.
Tracer Infusion
Rats and mice were implanted with an indwelling jugular vein
catheter and allowed to recover for 5 days before stable isotope tracer
infusion while they were awake and unrestrained (11). Ketone body
turnover by NMR was determined using stable isotope tracer dilution
of [3,4-13C]acetoacetate and [1,2-13C4]!-hydroxybutyrate. The latter
stable isotope tracer was replaced with [1,2,3,4-13C]!-hydroxybu-
tyrate for LC-MS/MS analysis of ketone body turnover. Preparation
and infusion of these stable isotope tracers were carried out as
described previously (48). Briefly, &2 h prior to stable isotope
infusion, 28 mg (&212 "mol) of [3,4-13C]ethylacetoacetate was
dissolved in 4 ml of deionized water and 80 "l of 4 M NaOH. This
solution was incubated at 40°C for 75 min to hydrolyze the ethyl
acetoacetate ester. The solution was neutralized and placed on ice, and
21 mg (&164 "mol) of either [1,2-13C]- or [1,2,3,4-13C]!-hydroxy-
butyrate was added and the volume adjusted to 8 ml with saline.
Tracers were infused as bolus (2.25 ml/h for rats and 0.30 ml/h for
mice) for the initial 10 min and as continuous infusion (0.5 ml/h for
rats and 0.12 ml/h for mice) for the remaining 80 min. Animals were
allowed unrestrained movement within the cage during the entire
infusion period. For NMR analysis, blood samples from rats were
collected from the descending aorta until exsanguination. For LC-
MS/MS analysis, which required 25 "l of blood, samples were
collected after a tail clip. Samples were immediately frozen at $80°C
until analysis.
Hepatic Insulin Sensitivity Index
Hepatic insulin sensitivity index was calculated as described pre-
viously (15, 28) using the following equation: 1/(endogenous glucose
production ( fasting insulin concentration). Endogenous glucose
production was determined by steady-state dilution of infused [3,4-
13
C]glucose, as described previously (11), and expressed in "mol/
min. Plasma insulin was measured by enzyme-linked immunoassay
AJP-Endocrinol Metab • VOL
E1227
using the Mouse Insulin ELISA kit (ALPCO Diagnostics, Salem, NH)
and expressed in nanograms per milliliter.
LC-MS/MS Analysis of Ketone Body Tracer Dilution
Thawed samples were immediately spiked with one volume of 1 M
sodium borodeuteride and two volumes of acetonitrile to reduce
plasma acetoacetate to !-hydroxybutyrate. Sodium borodeuteride
converts labile acetoacetate to stable !-hydroxybutyrate but preserves
the acetoacetate information by tagging the resulting !-hydroxybu-
tyrate with a single deuterium (2H) (16, 17). The samples were then
centrifuged at 14,000 rpm for 10 min, and the supernatant was loaded
on a cation exchange column, washed off the column with 4 ml of
water, and lyophilized overnight. The lyophilized sample was dis-
solved in 50 "l of mobile phase, of which 1–5 "l was injected to
optimize !-hydroxybutyrate isotopomer abundances.
Analysis was done on an API 3200 triple quadrupole LC-MS/MS
mass spectrometer (Applied Biosystems/Sciex Instruments) in posi-
tive electrospray ionization mode. The mass spectrometer was
equipped with a Shimadzu LC-20AD liquid chromatograph and a
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SIL-20AC auto sampler. A reverse-phase C18 column (T3, 150 ( 2.1
mm, 3 "m; Waters Atlantis) was used with liquid chromatograph
mobile phase consisting of water-methanol (2:98, vol/vol) with
0.025% acetic acid (eluent A) and water-methanol (40:60, vol/vol)
with 0.025% acetic acid (eluent B). The gradient program was 0%
eluent B in eluent A, which was increased to 15% eluent B over 10
min, followed by an increase to 90% eluent B rapidly over 0.5 min.
This gradient was held for 2 min before being returned to 0% eluent
B in 0.5 min and for 7 min to equilibrate the column. The flow rate
was held constant at 0.17 ml/min throughout the run.
For qualitative mass measurements, full scan and product ion scan
analyses were performed by direct infusion of !-hydroxybutyrate
solution to the mass spectrometer. Acquisition parameters, including
ionization source voltage, source temperatures, and gas flow, were
adjusted to maximize the signals of ions of interest. Quantitative
measurements were performed in multiple reaction monitoring
(MRM) mode, where the protonated molecular ion [M # H]# was
monitored via the first quadrupole filter (Q1) and its product fragment
[M # H]# minus 18 via the third quadrupole filter (Q3). The source
was operated at 300°C, and the mass spectrometer was operated with
the following parameters: declustering potential 26 V; entrance po-
tential 2.5 V; collision cell entrance potential 7.5 V; collision cell exit
potential 8 V; collision energy 8.1 V; ion spray voltage 4,000 V;
channel electron multiplier 2,000 V. Nitrogen was used for the
nebulizing gas, and the ion source gas 1, ion source gas 2, curtain gas,
and collision gas were 30, 40, 20, and 5, respectively. The following
MRM transitions were monitored to quantify !-hydroxybutyrate iso-
topomers (Fig. 1, A and B): detection of !-hydroxybutyrate originat-
ing from !-hydroxybutyrate 105/87 (M # 0); reduced acetoacetate
106/88 (M # 1); [3,4-13C]!-hydroxybutyrate 107/89 (M # 2); re-
duced [3,4-13C]acetoacetate 108/90 (M # 3); [1,2,3,4-13C]!-hy-
droxybutyrate 109/91 (M # 4); reduced [1,2,3,4-13C]acetoacetate
110/92 (M # 5).
The peak areas of !-hydroxybutyrate isotopomers were quantified
by Analyst Software version 1.4.2. Tracer-to-tracee ratios were cal-
culated after correcting for isotope natural abundance. Tracer enrich-
ments measured by LC-MS/MS were also corrected using enrichment
curves constructed with increasing tracer-to-tracee ratios (Fig. 1C). A
spillover curve was also constructed to correct for the contribution of
the deuterated acetoacetate (M # 1) to the M # 2 and M # 3
!-hydroxybutyrate isotopomers by determining the M # 2/M and M #
1/M ratios in solutions containing varying proportions of acetoacetate
reduced with sodium borodeuteride and unlabeled !-hydroxybutyrate,
and these ratios were plotted to obtain a linear regression equation
(Fig. 1D). The corrected enrichments of the !-hydroxybutyrate mass
isotopomers and infusion rates were fit to a two-pool model to
determine !-hydroxybutyrate and acetoacetate turnover and exchange
298 • JUNE 2010 • www.ajpendo.org
E1228 ALTERED HEPATIC FAT OXIDATION DURING INSULIN RESISTANCE
rates (3, 9, 32). The sum of !-hydroxybutyrate and acetoacetate
turnover rates is reported as ketone body turnover.
NMR Analysis of Ketone Body Tracer Dilution
Sample processing and analysis were carried out as described
previously (48). Briefly, &5 ml of thawed plasma was deproteinized
with 70% perchloric acid and the supernatant passed through cation
(H#) resin and neutralized with LiOH. The plasma extract was then
lyophilized to &500 "l, and 100 "l of D2O was added for 13C NMR
analysis of acetoacetate and !-hydroxybutyrate multiplets on a 14T
spectrometer equipped with a 5-mm broad-band probe. Peak areas
were analyzed using 1D NMR software ACD/Labs 9.0 (Advanced
Chemistry Development, Toronto, ON, Canada). The enrichments and
infusion rates were fit to a two-pool model, as described previously (3,
9, 32), but modified slightly for NMR (48) to determine ketone body
turnover.
AJP-Endocrinol Metab • VOL
LC-MS/MS Analysis of Liver Acylcarnitines
Following tracer infusion and blood collection, free carnitine and
acylcarnitines were extracted from liver, derivatized, and quantified as
described previously (1, 34) with some minor modification. Detection
was performed after LC separation using API 3200 triple quadrupole
LC-MS/MS mass spectrometer (Applied Biosystems/Sciex Instru-
ments) in positive ionization MRM mode. Free carnitine was moni-
tored using the 176 to 117 MRM transition. Acylcarnitines were
monitored using a precursor of 99 Da. Acylcarnitines were quantified
by comparison of the individual ion peak area with that of an internal
13
C standard (Cambridge Isotope Laboratories).
Gene Expression Analysis
Primers were designed using Primer Express software (Applied
Biosystems, San Jose, CA) on the basis of GenBank sequence data.
Quantitative real-time PCR reactions (10 "l) contained 25 ng of
cDNA, 150 nM of each primer, and 5 "l of SYBR Green PCR Master
Mix (Applied Biosystems). Reactions were performed in triplicate on
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an Applied Biosystems Prism 7900HT Sequence Detection System,
and relative mRNA levels were calculated by the comparative thresh-
old cycle method by using cyclophilin as the internal control.
Reagents and Materials
[3,4-13C]ethyl acetoacetate (98%), [1,2-13C]sodium !-hydroxybu-
tyrate (98%), and [1,2,3,4-13C]!-hydroxybutyrate were purchased
from Isotec (St. Louis, MO). Ketone body, NEFA, and triglyceride
concentrations in plasma or tissue samples were determined using
commercially available analytical kits (Wako). Other common chem-
icals were obtained from Sigma (St. Louis, MO).
Statistical Analysis
Results are expressed as means ) SE. Statistical differences were
analyzed using ANOVA for multiple groups or an unpaired t-test
between two groups. Means were considered significantly different at
P ! 0.05. Pearson’s correlation and regression analysis was per-
formed to determine whether a linear or nonlinear relationship existed
between two variables of interest.
Fig. 1. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) de-
tection of plasma acetoacetate and !-hydroxybutyrate isotopomers. A: LC-
MS/MS spectrum of !-hydroxybutyrate isotopomers isolated from mouse
blood. B: relationship between infused ketone tracers and mass isotopomers
detected in processed blood. M # 2 acetoacetate and M # 4 !-hydroxybu-
tyrate were infused but are interconverted to M # 4 acetoacetate and M # 2
!-hydroxybutyrate by biological exchange through !-hydroxybutyrate dehy-
drogenase. Upon processing of the blood with sodium borodeuteride, plasma
acetoacetate is chemically reduced to !-hydroxybutyrate but with a single
mass unit increase in its molecular weight due to the addition of a deuterium.
Thus, detection of M # 0, M # 2, and M # 4 mass isotopomers of
!-hydroxybutyrate quantifies blood enrichment of !-hydroxybutyrate, whereas
detection of M # 1, M # 3, and M # 5 mass isotopomers of !-hydroxybu-
tyrate quantifies blood enrichment of acetoacetate. C: enrichment curves for
acetoacetate (M # 3) and !-hydroxybutyrate (M # 4) tracers constructed with
increasing tracer-to-tracee ratios for correction of measured enrichments by
LC-MS/MS. D: a spillover curve was constructed with varying proportions of
acetoacetate and !-hydroxybutyrate to calculate the contribution of M # 1
acetoacetate to the M # 2 of !-hydroxybutyrate after processing with sodium
borodeuteride. The M # 1/M on the x-axis is a function of acetoacetate/!-
hydroxybutyrate, whereas the M # 2/M on the y-axis is the resulting spillover
correction for M # 2. This correction was applied as a function of M # 1/M
in blood samples.
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 ALTERED HEPATIC FAT OXIDATION DURING INSULIN RESISTANCE E1229
RESULTS

LC-MS/MS Determination of "-Hydroxybutyrate Enrichment

All six !-hydroxybutyrate isotopomers were easily detected
and quantified from mouse blood (Fig. 1, A and B). Quantifi-
cation of !-hydroxybutyrate was linear over a wide range of
injected mass (10 pg to 50 ng); r2 ' 0.9993. Injection of &0.75
ng of !-hydroxybutyrate was enough to maintain a signal-to-
noise ratio of *25 in all the samples. Enrichment curves
constructed with [1,2,3,4-13C]!-hydroxybutyrate (M # 4) and
reduced [3,4-13C]acetoacetate (M # 3) were linear over a wide
range (0 –12.5 mole%, r2 ' 0.999; Fig. 1C) and were used to
correct the measured enrichments. Reduction of acetoacetate
with sodium borodeuteride introduces a significant spillover of
M # 2 enrichment that was corrected using a linear (r2 '
0.980) equation generated from standards of variable acetoac-
etate/!-hydroxybutyrate ratios (Fig. 1D).
Determination of Ketone Body Tracer Enrichment by

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LC-MS/MS and NMR Provides Equivalent Values
Carbon-13 NMR analysis of positional enrichment provides a
very accurate readout of ketone body tracer dilution without
interference from background enrichments or requirement for
spillover corrections (48). However, 13C NMR is several orders of
magnitude less sensitive and requires long scan times for analysis
compared with MS, and therefore, it is less practical for mouse
experiments. To confirm that ketone body tracer dilution mea-
sured by LC-MS/MS and NMR agree, we infused rats with
[1,2,3,4-13C]!-hydroxybutyrate and [1,2-13C]acetoacetate and
measured plasma enrichments by NMR and LC-MS/MS in sam-
ples from identical rats. Both methods provided similar quantifi-
cation of ketone body enrichment with a correlation coefficient
between the two methods of 0.91 and a slope equaling 0.90 (Fig.
2A). To further confirm that LC-MS/MS and NMR measurements
provide equivalent measurement of ketone body turnover, we
infused two separate groups of fasted rats with either NMR tracers
([3,4-13C]!-hydroxybutyrate, [1,2-13C]acetoacetate) or LC-
MS/MS tracers ([1,2,3,4-13C]!-hydroxybutyrate, [1,2-13C]aceto-
acetate). Ketone body turnover determined by both NMR and
LC-MS/MS gave identical values in these two groups of rats (Fig.
2B). These data demonstrate that the LC-MS/MS approach pro-
vides identical measurements of ketone body dilution and turn-
over compared with the NMR approach we reported earlier (48),
but with much improved sensitivity.
Steady-State Infusion of Ketone Body Tracers in Mice
Before application to mice, we confirmed steady-state enrich-
ment of ketone body tracers during a 120-min infusion (Fig. 3A).
The initial tracer bolus resulted in supra-steady-state enrichment
of the infused tracers (M # 3 and M # 4), but enrichment of the
conversion products (M # 2 and M # 5) was nearly identical to
steady-state enrichment. All tracers reached steady state within 75
min of the infusion being started. Subsequent experiments used
similar bolus infusion rates for 90 min.
Ketone Body Turnover in Mice is Stimulated During the
Fed-to-Fasted Transition
To determine whether apparent ketone body turnover in
mice is reflective of normal hepatic physiology associated with
induction of ketogenesis, we performed experiments in
Fig. 2. Ketone tracer enrichment measured by LC-MS/MS is corroborated by
NMR measurements. A: comparison of a previously validated NMR method
for determining ketone enrichment with the LC-MS/MS method demonstrates
that the 2 methods provide identical measurements of !-hydroxybutyrate and
acetoacetate tracer enrichments in rat plasma. B: ketone turnover in 24-h-fasted
Long-Evans rats was not different when determined by NMR or LC-MS/MS.
C57BL/6 mice during the fed-to-fasted transition. Mice fitted
with indwelling jugular vein catheters were infused with ke-
tone body tracers without food being withheld and then 16 and
24 h later in the same mice after food was removed. Ketone
body turnover was very low when mice had access to food but
was induced fivefold after a 16-h fast (Fig. 3B), in agreement
with the known effects of fasting on hepatic ketogenesis in
mice and concomitant with an increase in plasma ketone body
concentration from 173.9 ) 11.74 to 857.5 ) 30.49 "M. An
additional 8 h of fasting did not induce any further increase in
ketone body turnover, although ketone body concentration did
increase by an additional 50% to 1,365.3 ) 83.29 "M (Fig.
3C). These data indicate that apparent ketone body turnover in
mice accurately reflects the induction of hepatic fat oxidation
in response to fasting but that extending fasting from 16 to 24
h does not further induce ketogenic rates. Subsequent studies
used 16-h fasting protocols.
Ketone Body Turnover is Impaired in PPAR#$/$ Mice
To confirm that ketone body turnover is sensitive to a
pathophysiological impairment of hepatic fat oxidation and
ketogenesis, we performed experiments in PPAR%$/$ and
PPAR%#/# mice before and after a 16-h fast. Not surprisingly,
fasting ketosis was impaired in PPAR%$/$ mice, as indicated by
impaired fasting plasma ketone body concentration (Fig. 4A). As
expected, PPAR%$/$ mice also had a 40% reduction in ketone
body turnover in the fed state and a twofold reduction in the
fasted state compared with control mice (Fig. 4B). These
results are consistent with the known impairment of hepatic fat

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E1230 ALTERED HEPATIC FAT OXIDATION DURING INSULIN RESISTANCE
Fig. 3. Steady-state infusion of ketone tracers demonstrates induction of
ketogenesis during fasting in mice. A: enrichment curves of !-hydroxybutyrate
isotopomers in mouse blood during a 105-min infusion of ketone tracers
demonstrates that steady state is reached by 75 min of infusion. B: 3 separate
infusion experiments were repeated in the same C57BL/6 mice during the
fed-to-24-h-fasted transition. Ketone turnover was stimulated substantially
during the transition from feeding (n ' 6) to 16 h of fasting (n ' 5) but did
not increase further by 24 h of fasting (n ' 3) (attrition over the experiment
was due to loss of catheter patency). C: total ketone concentration continued to
rise throughout the fed-to-fasted transition despite a leveling of ketone turn-
over. The data are represented by means ) SE. *P ! 0.05.
oxidation in these mice (24) and, taken with the former results,
indicate that steady-state ketone body dilution reflects alter-
ations in hepatic ketogenesis within the physiological window
encompassed by feeding, fasting, and PPAR%-targeted impair-
ment of hepatic fat oxidation.
Effect of High-Fat Diet on Ketogenesis in Mice Depends on
Duration of Exposure
Since defects in hepatic fat oxidation and ketogenesis have
been implicated in insulin-resistant rat models (48) and in
humans (47), we investigated whether ketogenesis is altered in
a diet-induced mouse model of obesity and insulin resistance.
The metabolic characteristics of these mice are presented in
Table 1. After 8 wk of a 60% high-fat diet, mice were obese
AJP-Endocrinol Metab • VOL
(30.86 ) 0.962 vs. 40.5 ) 1.18 g, P + 0.0001), hyperinsu-
linemic (0.40 ) 0.039 vs. 1.93 ) 0.329 ng/ml, P + 0.001), and
glucose intolerant (Supplemental Fig. S2; Supplemental Mate-
rial for this article can be found on the AJP-Endocrinology and
Metabolism web site) and had a severely suppressed hepatic
sensitivity index (Fig. 5A) compared with mice on the control
diet. These data are consistent with previous reports on the
diet-induced obese C57BL/6 mouse (40). Despite hepatic in-
sulin resistance and substantial hepatic steatosis (Table 1),
fasting plasma ketone body concentration (1,220 ) 103.7 vs.
1,178 ) 119.2 "M) and turnover (Fig. 5B) were normal after
8 wk of a high-fat diet. Additionally, the liver acylcarnitine
profile was unremarkable (Fig. 5C and Supplemental Fig. S1).
These data are consistent with unaltered hepatic fat oxidation
during 8 wk of diet-induced insulin resistance in these mice.
In view of the fact that we previously found that hepatic fat
oxidation is impaired in severely insulin-resistant rats (48), we
investigated whether a longer duration of high-fat feeding
results in impaired hepatic ketogenesis. Mice maintained on a
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high-fat diet for 16 wk gained an additional 25% (47.4 ) 0.98
g) of body weight and had elevated insulin levels (3.24 ) 0.71
ng/ml), higher plasma NEFA and liver triglyceride levels
(Table 1), deteriorated glucose tolerance (Supplemental Fig.
S2), and a further suppression of hepatic insulin sensitivity
index compared with mice on the diet for 8 wk (Fig. 5A). This
deterioration of insulin sensitivity was accompanied by a 40%
increase in ketone body turnover (Fig. 5B), demonstrating that
hepatic fat oxidation through the ketogenic pathway is induced
Fig. 4. In vivo ketone turnover in mice reflects alterations of hepatic fat
oxidation associated with PPAR% loss of function. A: fasting for 16 h induced
an 8-fold rise in total plasma ketone bodies in wild-type mice, which were
severely blunted in PPAR%$/$ mice. B: ketone turnover measured by ketone
tracer dilution was likewise impaired in PPAR%$/$ mice after a 16-h fast,
consistent with the known effects of PPAR% loss of function and indicative that the
method accurately reports alterations of hepatic ketogenesis. The data are repre-
sented by means ) SE. *P ! 0.05 between fed and fasted groups; **P ! 0.05
between PPAR%#/# and PPAR%$/$.
298 • JUNE 2010 • www.ajpendo.org
 ALTERED HEPATIC FAT OXIDATION DURING INSULIN RESISTANCE
Table 1. Metabolic characteristics of mice maintained on control (10% fat calories) or high-fat (60% fat calories) diets for
either 8 or 16 wk
8 Wk
Control
Body weight, g 30.9 ) 0.96
Fasting glucose, mg/dl 67.7 ) 2.2
Fed total ketones, "mol/l 144.2 ) 12
Fasting total ketones, "mol/l 1,220 ) 104
Fasting NEFA, mEq/l 0.61 ) 0.036
Fasting liver triglycerides, mg/g 80.9 ) 16
Fasting insulin, ng/ml 0.40 ) 0.039
Data are means ) SE. NEFA, nonesterified fatty acids. *P + 0.05 between control and high-fat groups; †P + 0.05 between 8- and 16-wk groups.
during this period of diet-induced insulin resistance. To further
characterize hepatic !-oxidation in mice fed a high-fat diet for
16 wk, we assayed the C2–C16 acylcarnitine profile in liver
extracts of these mice. There were no differences in free
carnitines or medium- and long-chain carnitines (C5–C16)
(Supplemental Fig. S1). However, short-chain acylcarnitines,
particularly acetylcarnitine (Fig. 5C), were significantly ele-
vated in the livers of mice fed a high-fat diet for 16 wk,
consistent with increased fatty acid oxidation and in agreement
with findings in other mouse models with increased fat oxida-
tion (1, 25, 36, 38). Together, these data indicate that the
function of hepatic fat oxidation is normal after 8 wk of
high-fat feeding but induced after 16 wk of high-fat feeding in
mice.
To probe the molecular origin of these functional findings,
we measured the expression of genes that regulate fat oxidation
in the liver. High-fat feeding for 16 but not 8 wk significantly
induced PPAR, coactivator-1% (P ' 0.03) and a trend for
increased PPAR% (P ' 0.13) expression in liver (Fig. 5D),
suggesting that hepatic fat oxidation is molecularly upregulated
by 16 wk of a high-fat diet in these mice. To specifically
address ketogenesis, we measured hepatic expression of fibro-
blast growth factor 21 (FGF21), a newly described factor
critical for regulation of ketogenesis (2, 44) and hydroxylm-
AJP-Endocrinol Metab • VOL
E1231
16 Wk
High Fat Control High Fat
40.5 ) 1.2* 37.4 ) 0.61† 47.4 ) 0.98*†
101.8 ) 6.9* 72.5 ) 3.0 129.4 ) 13*
106.1 ) 6.5* 69.1 ) 3.6† 100.4 ) 4.9*
1,178 ) 119 854 ) 123 1,189 ) 161
0.67 ) 0.062 0.81 ) 0.071 1.05 ) 0.065*†
110.2 ) 19 94.1 ) 22 201.1 ) 43*†
1.93 ) 0.33* 1.02 ) 0.078† 3.24 ) 0.71*
ethylglutaryl (HMG)-CoA synthase, the rate-limiting step in
ketone formation. Whereas 8 wk of a high-fat diet had no effect
on the expression of either of these genes, 16 wk of a high-fat
diet resulted in a twofold induction of both FGF21 expression
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(P ' 0.08) and HMG-CoA synthase (P ' 0.03) (Fig. 5D).
Taken together with the flux measurements, these data dem-
onstrate a progression of both expression and function of
hepatic fat oxidation during high-fat diet-induced insulin re-
sistance.
DISCUSSION
Ectopic lipid accumulation is a primary risk factor for the
development of insulin resistance and related metabolic mala-
dies (30). The mechanistic link between intracellular lipid
accumulation and insulin resistance appears to involve sub-
strate level mitochondrial pathways of fat oxidation and the
malformation of lipid-derived intermediates that inhibit the
insulin-signaling cascade (22, 25, 36, 42, 49, 50). Inasmuch as
most investigations along these lines have focused on skeletal
muscle, it remains unclear whether the same factors prevail in
liver. Since the largest proportion of mitochondrial fat oxida-
tion in the fasted rodent liver is directed toward the synthesis
and efflux of ketone bodies (31), the aim of this study was to
Fig. 5. High-fat feeding induces insulin resis-
tance and increased hepatic fat oxidation in
mice. A: mice fed a high-fat diet for either 8 or
16 wk had a dramatically impaired hepatic
insulin sensitivity index. B: ketone turnover
was unchanged after 8 wk of a 60% high-fat
diet but was significantly elevated after 16 wk
of the high-fat diet. C: liver acetylcarnitine was
assayed by LC-MS/MS in mice fed a syn-
thetic control diet (10% fat calories) or a
high-fat diet (60% fat calories) for either 8
or 16 wk. D: high-fat feeding for 8 wk
resulted in normal expression of genes that
regulate hepatic fat oxidation relative to
cyclophilin, whereas 16 wk on the diet
induced expression of these genes. Data
are presented as means ) SE. *P ! 0.05
between control and high-fat diet. **P !
0.05 between 8 and 16 wk of diet intervention;
#P ! 0.1 between control and high-fat diet;
##P ! 0.1 between 8 and 16 wk of diet
intervention. HMGCS, hydroxylmethylglu-
taryl-CoA synthase; CPT Ia, carnitine palmi-
toyltransferase Ia.
298 • JUNE 2010 • www.ajpendo.org
E1232 ALTERED HEPATIC FAT OXIDATION DURING INSULIN RESISTANCE
determine whether hepatic mitochondrial function, as indicated
by ketogenic flux and acylcarnitine profiles, is altered in
insulin-resistant, high-fat-fed mice. We first confirmed that
ketone body tracer dilution in conscious and unrestrained mice
accurately reflects the anticipated changes in hepatic fat oxi-
dation with feeding, fasting, and PPAR% loss of function. The
most important finding was that hepatic insulin resistance
induced by 8 wk of a high-fat diet in mice had no remarkable
effect on the expression or function of hepatic fat oxidation,
although the livers of these mice were steatotic and clearly
insulin resistant. Furthermore, 16 wk of high-fat feeding wors-
ened hepatic steatosis, insulin resistance, lipidemia, and glyce-
mia but also resulted in the induction of hepatic fat oxidation
gene expression, elevated ketogenic flux, and increased short-
chain acylcarnitine content in the liver. These findings estab-
lish that impaired mitochondrial fat oxidation is not an early
feature of hepatic insulin resistance in diet-induced obese mice
and that, in fact, there appears to be an induction of hepatic fat
oxidation in advance of any decline in mitochondrial function.
The finding of increased ketone body production in high-fat
diet-induced insulin resistance was contrary to our initial ex-
pectation, which was based on impaired mitochondrial func-
tion in insulin-resistant skeletal muscle (22, 36, 42, 49) and
causative links between impaired mitochondrial fat oxidation
and diminished hepatic insulin signaling. Induction of hepatic
mitochondrial dysfunction by loss of long-chain acyl-CoA
dehydrogenase was sufficient to cause hepatic insulin resis-
tance (57), whereas increasing hepatic mitochondrial fat oxi-
dation by malonyl-CoA decarboxylase gain of function (1) or
acetyl-CoA carboxylase loss of function (14) ameliorated
PKC-dependent hepatic insulin resistance. In addition, hepatic
insulin resistance is improved by treatment with pharmacolog-
ical interventions, such as PPAR% agonists (56) or the newly
described hepatokine FGF21 (6), that induce flux through
hepatic ketogenesis (44, 48). With regard to hepatic energy
metabolism during insulin resistance, humans with type 2
diabetes mellitus were found to have impaired rates of hepatic
ATP synthesis (53), whereas insulin-resistant humans with
fatty liver disease have been reported to have both decreased
(41) and increased (47) hepatic mitochondrial metabolism,
with the latter indicated by elevated plasma ketone concentra-
tion. In contrast, the frankly diabetic ZDF rat has severely
blunted fasting ketone turnover, leading to impaired hepatic fat
oxidation during fasting (48). Taken together with our present
findings, these data suggest that impaired hepatic mitochon-
drial fluxes may occur with severe insulin resistance/diabetes
but not in the milder setting of diet-induced insulin resistance
studied here. It is possible that the induction of frank hyper-
glycemia itself could impair hepatic ketogenesis during severe
insulin resistance (45).
We postulate that induction of ketone body production
during high-fat feeding in mice reflects a metabolic compen-
sation in response to increased lipid delivery to the liver. Acute
lipid overload in rodent models, either by lipid infusion (26) or
short-term (3-day) high-fat diet (46), induces hepatic insulin
resistance through PKC-dependent phosphorylation of insulin
receptor substrates. Increased short-chain acylcarnitines in the
skeletal muscle of high-fat-fed rodents suggests that skeletal
muscle !-oxidation is increased secondary to mitochondrial
overload of fat (38), and similarly, mitochondrial fat oxidation
is increased the hearts of high-fat-fed mice (55). We found that
AJP-Endocrinol Metab • VOL
circulating NEFA levels increased between 8 and 16 wk of
exposure to the high-fat diet, corresponding to the timing in
which ketone production increased. The induction of mito-
chondrial fat oxidation in response to lipid overload provides a
plausible mechanism for the induction of oxidative stress and
mitochondrial damage during later stages of insulin resistance.
Indeed, the initial metabolic compensation for increased lipid
delivery is followed by pathological gene expression in skeletal
muscle (50). Likewise, hepatic gene expression of fat oxidation
initially increases with high-fat feeding but then decreases after
prolonged exposure to a high-fat diet in rodents (13). Our
findings of normal ketogenesis after 8 wk, followed by ele-
vated ketogenesis after 16 wk of high-fat feeding, demonstrate
that changes in hepatic mitochondrial metabolism evolve dur-
ing progressing insulin resistance and establish that impaired
hepatic fat oxidation is not a required feature of hepatic insulin
resistance. A caveat of the present study is that we do not
report mitochondrial fat oxidation in the TCA cycle, but given
the requirement of the TCA cycle for gluconeogenesis (10), it
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is unlikely that this pathway is impaired during insulin resis-
tance.
The measurement of in vivo metabolic flux in mice is
challenging because of blood volume and sampling limitations.
The LC-MS/MS method used here provided a measurement of
ketone body enrichment and turnover identical to the 13C NMR
approach we used previously (48) but required 300 times less
blood (20 "l vs. &6 ml), allowing ketone turnover to be
measured in awake and urestrained mice. Many previous
studies used gas chromatography (GC)-MS to measure ketone
body enrichment and turnover (3, 8, 9, 16, 17, 32, 35) with
similar sensitivity, but the approach has not been applied to
mice. Although there is no inherent reason that GC-MS could
not be used, an advantage of LC-MS/MS is that sample
derivatization is not required, resulting in lower molecular
weight fragments and thus less natural abundance background
enrichment and attendant correction factors than standard si-
lane derivatives used for GC-MS (3, 9, 16, 17, 32). We note
that other GC-MS derivatives provide similarly low back-
ground correction by splitting off in the source before detection
(20). The method used here also takes advantage of a technique
to sequester labile acetoacetate as stabile !-hydroxybutyrate by
treating plasma samples with sodium borodeuteride prior to
analysis (16, 17), allowing samples to be stored and analyzed
without risk of sample degradation while retaining acetoacetate
enrichment information as deuterium-tagged !-hydroxybu-
tyrate.
Regardless of the analytical technique used to measure ketone
body tracer enrichment, the relationship between apparent ketone
body turnover and hepatic ketogenesis may be complicated by
exchange reactions in extrahepatic tissue (termed pseudoketogen-
esis) that can artificially dilute ketone tracers (17, 18). Despite this
potential complication, ketone body turnover and net hepatic
ketone arterial-venous difference were equivalent in both normal
and diabetic dogs (3, 21, 32). Since a similar validation is not
possible in mice, we measured ketone body turnover during the
fed-to-fasted transition and in gene-altered mice with impaired
hepatic fat oxidation to determine whether ketone body turnover
reflects hepatic fat oxidation in mice as expected. Fasted mice had
an eightfold increase in ketone body turnover compared with fed
mice, approximately equal to the increase in ketone body concen-
tration with fasting. We also found that ketone body turnover was
298 • JUNE 2010 • www.ajpendo.org
 ALTERED HEPATIC FAT OXIDATION DURING INSULIN RESISTANCE
robustly blunted in PPAR%$/$ mice, consistent with impaired
hepatic fat oxidation in these mice (24). Although we cannot
ascertain the absolute contribution of pseudoketogenesis to ketone
tracer dilution, it appears that ketone body turnover is largely
reflective of hepatic ketogenesis under the conditions studied here.
To probe the factors that influence ketogenesis during diet-
induced obesity, we analyzed correlations between ketone
turnover and other metabolic parameters related to liver fat
oxidation. When ketone body turnover (57 mice: fed, fasting,
PPAR%$/$, 8- and 16-wk control, and high-fat diet mice) was
compared with plasma ketone concentration, we found a cur-
vilinear relationship (Fig. 6A) with a strong correlation at
ketone concentrations +1,000 "M that tended to level off at
higher ketone concentrations. This finding recapitulated the
data in individual mice during the fed-to-starved transition
shown in Fig. 3C, where ketone body turnover leveled off after
16 h of fasting, but ketone body concentration rose by another
50% between 16 and 24 h of fasting. A similar curvilinear
relationship between plasma ketone concentration and produc-
tion was identified in the dog during fasting ketosis and type I
diabetes (21). The origin of this effect is unclear but has been
explained as either a regulatory pacing of ketone body produc-
tion when plasma ketone concentrations rise above maximal
peripheral ketone utilization (5, 39) or inhibition of ketone
utilization at elevated plasma ketone concentrations (21). Ad-
ditionally, hepatic acetylcarnitine correlated positively with
ketone body turnover in mice from control and high-fat-fed
groups (Fig. 6B). This finding is significant because acetylcar-
nitine reflects cellular acetyl-CoA, the end product of !-oxi-
dation and precursor of ketone body synthesis. These correla-
AJP-Endocrinol Metab • VOL
E1233
Fig. 6. Correlations of ketone turnover with liver and plasma
metabolite concentrations. A: ketone turnover in all mice re-
ported in this study (57 mice) correlates with plasma ketone
concentration. The best correlation was obtained by a polyno-
mial trend line, indicating that the highest ketone concentrations
may be influenced by suppressed ketone utilization rather than
increased hepatic ketogenesis. B: liver acetylcarnitine levels
determined by LC-MS/MS correlated positively with ketone
turnover in mice fed control or high-fat diets for 8 and 16 wk.
C: ketone turnover correlated positively with nonesterified fatty
acids (NEFA) in plasma of mice fed a control or high-fat diet for
8 and 16 wk. D: ketone turnover and hepatic triglyceride content
trended toward a positive correlation but did not reach signifi-
Downloaded from ajpendo.physiology.org on January 18, 2011
cance from mice fed a control or high-fat diet for 8 and 16 wk.
Correlations were determined by Pearson’s correlation and
regression analysis. P + 0.05 was considered significant.
tions provide some additional support that ketone turnover in
mice is reflective of hepatic fat oxidation.
We also determined whether lipid metabolites correlate with
ketone turnover in control and high-fat-fed mice. Plasma
NEFA concentration strongly correlated with ketone turnover
(Fig. 6C), a finding that reflects both the dependence of
ketogenesis on hepatic fatty acid delivery and the reality that
factors governing hepatic fat oxidation also influence adipose
fatty acid release. In contrast, hepatic triglyceride content
tended to but did not strongly correlate with ketone turnover
(Fig. 6D). This finding suggests that altered ketogenesis during
diet-induced obesity is not strongly dependent on the develop-
ment of hepatic steatosis and that the onset of fatty liver is
dependent on other factors besides altered mitochondrial func-
tion.
In conclusion, apparent ketone body turnover in mice re-
flects alterations in hepatic fat metabolism within the metabolic
window of feeding, fasting, and PPAR% loss of function.
High-fat feeding for 8 wk induces insulin resistance without
altered hepatic fat oxidation, whereas 16 wk of high-fat feeding
induces both hepatic gene expression and in vivo function of
fat oxidation. These findings demonstrate that hepatic fat
oxidation adapts progressively to high-fat feeding and estab-
lishes that diet-induced insulin resistance is not accompanied
initially by impaired mitochondrial fat oxidation.
ACKNOWLEDGMENTS
We are grateful to Dr. Bob Stevens of Duke University for providing
guidance in implementing the acylcarnitine profile assay. Dr. Henri Brunen-
graber of Case Western University provided invaluable discussions and guid-
298 • JUNE 2010 • www.ajpendo.org
E1234 ALTERED HEPATIC FAT OXIDATION DURING INSULIN RESISTANCE
ance in assaying ketone body enrichment by mass spectrometry. Sreeraman
Katikaneni and Josh Liu provided excellent technical assistance.
GRANTS
Support for this work was provided by National Institute of Diabetes and
Digestive and Kidney Diseases Grant RO1-DK-078184 (S. C. Burgess) and
American Diabetes Association Grant 7-09-BS-24 (S. C. Burgess). N. E.
Sunny is supported by Postdoctoral Training Grant RL9-DK-081180. Core
support was provided by Grants UL1-DE019584 (Task Force for Obesity
Research), RR-02584 (National Center for Research Resources), and DK-
076269 (University of Texas Southwestern Mouse Metabolic Phenotyping
Center).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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