Download Lipotoxicity in steatohepatitis occurs despite an increase in

Am J Physiol Endocrinol Metab 310: E484–E494, 2016.
First published January 26, 2016; doi:10.1152/ajpendo.00492.2015.
CALL FOR PAPERS
Mitochondrial Dynamics and Oxidative Stress
Lipotoxicity in steatohepatitis occurs despite an increase in tricarboxylic acid
cycle activity
Rainey E. Patterson,1* Srilaxmi Kalavalapalli,2* Caroline M. Williams,3 Manisha Nautiyal,2
Justin T. Mathew,2 Janie Martinez,2 Mary K. Reinhard,4 Danielle J. McDougall,1 James R. Rocca,5
Richard A. Yost,1,9 Kenneth Cusi,2,6,7,8 Timothy J. Garrett,9* and Nishanth E. Sunny2*
1
Submitted 17 November 2015; accepted in final form 25 January 2016
Patterson RE, Kalavalapalli S, Williams CM, Nautiyal M,
Mathew JT, Martinez J, Reinhard MK, McDougall DJ, Rocca JR,
Yost RA, Cusi K, Garrett TJ, Sunny NE. Lipotoxicity in steatohepatitis occurs despite an increase in tricarboxylic acid cycle activity.
Am J Physiol Endocrinol Metab 310: E484 –E494, 2016. First published January 26, 2016; doi:10.1152/ajpendo.00492.2015.—The hepatic tricarboxylic acid (TCA) cycle is central to integrating macronutrient metabolism and is closely coupled to cellular respiration, free
radical generation, and inflammation. Oxidative flux through the TCA
cycle is induced during hepatic insulin resistance, in mice and humans
with simple steatosis, reflecting early compensatory remodeling of
mitochondrial energetics. We hypothesized that progressive severity
of hepatic insulin resistance and the onset of nonalcoholic steatohepatitis (NASH) would impair oxidative flux through the hepatic TCA
cycle. Mice (C57/BL6) were fed a high-trans-fat high-fructose diet
(TFD) for 8 wk to induce simple steatosis and NASH by 24 wk. In
vivo fasting hepatic mitochondrial fluxes were determined by 13Cnuclear magnetic resonance (NMR)-based isotopomer analysis. Hepatic metabolic intermediates were quantified using mass spectrometry-based targeted metabolomics. Hepatic triglyceride accumulation
and insulin resistance preceded alterations in mitochondrial metabolism, since TCA cycle fluxes remained normal during simple steatosis.
However, mice with NASH had a twofold induction (P ⬍ 0.05) of
mitochondrial fluxes (␮mol/min) through the TCA cycle (2.6 ⫾ 0.5
vs. 5.4 ⫾ 0.6), anaplerosis (9.1 ⫾ 1.2 vs. 16.9 ⫾ 2.2), and pyruvate
cycling (4.9 ⫾ 1.0 vs. 11.1 ⫾ 1.9) compared with their age-matched
controls. Induction of the TCA cycle activity during NASH was
concurrent with blunted ketogenesis and accumulation of hepatic
diacylglycerols (DAGs), ceramides (Cer), and long-chain acylcarnitines, suggesting inefficient oxidation and disposal of excess free
fatty acids (FFA). Sustained induction of mitochondrial TCA cycle
failed to prevent accretion of “lipotoxic” metabolites in the liver and
could hasten inflammation and the metabolic transition to NASH.
* R. E. Patterson and S. Kalavalapalli contributed equally to this work. T. J.
Garrett and N. E. Sunny contributed equally to this work.
Address for reprint requests and other correspondence: N. E. Sunny, Dept.
of Medicine, Division of Endocrinology, Diabetes and Metabolism, Univ. of
Florida, 1600 SW Archer Road, Gainesville, Florida 32610 (e-mail: nishanth.
[email protected]).
E484
steatosis; hepatic insulin resistance; mitochondria; nonalcoholic steatohepatitis
and patients with type 2 diabetes
mellitus (T2DM) are afflicted with fatty liver disease (25, 30),
of which mitochondrial dysfunction is a central feature (19,
42). About 30 – 40% of patients undergo the transition from
simple steatosis to nonalcoholic steatohepatitis (NASH) (1, 11,
38), which is projected to be the most common indication for
liver transplantation (8). Uncovering defects in hepatic mitochondrial metabolism assumes great importance toward understanding the transition to NASH and developing future prevention strategies. With progression of hepatic insulin resistance
and triglyceride accumulation, there is constant remodeling of
mitochondrial oxidative metabolism (19, 35, 41), which includes alterations in enzyme activities and molecular regulation of mitochondrial networks (e.g., mitochondrial ␤-oxidation, TCA cycle, ketogenesis, mitochondrial respiration, and
ATP synthesis). Apart from fueling gluconeogenesis and lipogenesis, these pathways are also tightly coupled to generating
reactive oxygen species (ROS) and mediating normal inflammatory responses.
During the pathophysiology of fatty liver disease, triglyceride accumulation is thought to exceed and impede oxidative
catabolism of free fatty acid (FFA) (3). Defects in mitochondrial lipid oxidation are shown to occur together with hepatic
insulin resistance and simple steatosis (29, 31, 47). Interestingly, recent studies (15, 19, 35, 41) also point to a simultaneous induction of hepatic mitochondrial oxidative metabolism
during hepatic insulin resistance and simple steatosis. In the
setting of simple steatosis, hepatic tricarboxylic acid cycle
(TCA) cycle flux was induced together with high rates of lipid
accretion (21) in both mouse models (35) and human subjects
(41). Furthermore, simple steatosis was also associated with
higher rates of mitochondrial respiration (19, 35), which, under
normal physiology, is closely coupled to the generation of
reducing equivalents from the TCA cycle. However, progressive severity of hepatic insulin resistance and onset of NASH
OVER 70% OF OBESE HUMANS
http://www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on August 1, 2017
Department of Chemistry, University of Florida, Gainesville, Florida; 2Division of Endocrinology, Diabetes, and
Metabolism, Department of Medicine, University of Florida, Gainesville, Florida; 3Department of Integrative Biology,
University of California, Berkeley, California; 4Animal Care Services, University of Florida, Gainesville, Florida; 5Advanced
Magnetic Resonance Imaging and Spectroscopy Facility, McKnight Brain Institute, University of Florida, Gainesville,
Florida; 6Division of Endocrinology, Diabetes, and Metabolism, Malcom Randall Veterans Administration Medical Center,
Gainesville, Florida; 7Division of Diabetes, the University of Texas Health Science Center at San Antonio, San Antonio,
Texas; 8Division of Diabetes, Audie L. Murphy Veterans Administration Medical Center, San Antonio, Texas; and
9
Department of Pathology, University of Florida, Gainesville, Florida
E485
HEPATIC MITOCHONDRIAL METABOLISM IN NASH
RESEARCH DESIGN AND METHODS
Animals and diets. Animal studies were approved by the Institutional Animal Care and Use Committee at the University of Florida.
Male mice (C57BL/6) purchased from Jackson Laboratories (Bar
Harbor, ME) at 6 – 8 wk were fed either a control diet (10% fat
calories, no. D09100304; Research Diets) or a high-trans-fat highfructose diet (TFD, 40% fat calories, no. D09100301; Research Diets)
for 8 or 24 wk. The TFD diet was enriched with 40% kcal fat
containing Primex partially hydrogenated vegetable oil shortening,
fructose (22% by wt), and cholesterol (2% by wt) (10). At 8 wk of
TFD feeding, mice had already developed simple steatosis, whereas
24 wk on TFD resulted in development of histological features of
NASH as previously reported (Refs. 10 and 46, also see RESULTS).
Euglycemic-hyperinsulinemic clamp (insulin stimulation). Mice
(n ⫽ 6 –7/group) were implanted with indwelling jugular vein catheters 5 days before the experiment. In conscious and unrestrained
mice following an overnight fast, insulin was infused at a constant rate
(2.5 mU·kg⫺1·min⫺1). A mixture of 30% glucose enriched to 2.5%
with [U-13C]glucose was infused to maintain euglycemia. Blood
glucose was monitored every 10 min using a glucose meter. Following
90 min of euglycemia, the animals were killed, and blood glucose
enrichments were determined as previously detailed for determination
of endogenous glucose production (EGP) (35). Severity of hepatic
insulin resistance was also evaluated from the hepatic insulin sensitivity index, calculated as the product of fasting plasma insulin and
fasting EGP (23).
Stable isotope tracer infusions and metabolic studies. Alterations in
liver metabolism were studied following 8 (simple steatosis) and 24
(NASH) wk of dietary challenge. Mice (n ⫽ 6 –7/group) were implanted with indwelling jugular vein catheters 5 days before metabolic
experiments. Following an overnight fast, mice were infused with a
mixture containing [13C3]propionate (37.5 mg/ml) and [3,413
C2]glucose (3.75 mg/ml) as a bolus (0.30 ml/h) for the first 10 min
followed by continuous infusion (0.12 ml/h) for the next 80 min.
Following 90 min of tracer infusion, mice were anesthetized, and
whole blood was collected from the descending aorta. Livers were
flash-frozen in liquid nitrogen and stored at ⫺80°C until further
analysis.
Analysis of EGP and mitochondrial TCA cycle metabolism by
13
C-nuclear magnetic resonance. Glucose in the mice plasma was
converted to the 1,2-isopropylidene glucofuranose derivative (monoacetone glucose) before 13C isotopomer analysis (35). 13C-nuclear
magnetic resonance (NMR) isotopomer experiments were conducted
on a 600-MHz (14.1 T/5.1 cm) Agilent NMR spectrometer available
at the National High Magnetic Field Laboratory, University of Florida, using a probe optimized for 13C detection sensitivity accommodating 1.5-mm tubes with a sample volume of 35– 40 ␮l (28). Peak
areas were analyzed using one-dimensional NMR software ACD/Labs
9.0 before metabolic analysis as reported previously (5, 16, 32).
Endogenous glucose production was determined for stable isotope
dilution of [3,4-13C2]glucose in mouse plasma. Furthermore, isotopomer analysis of the multiples arising from 13C labeling of carbon-2
of glucose was used to determine the functional activity of hepatic
TCA cycle metabolism (32, 34). Briefly, [13C3]propionate is taken up
by the liver and enters the TCA cycle as succinyl-[1,2,3-13C3]CoA.
The 13C multiplets in glucose arising from the incorporation of 13C
are reflective of the relative rates of mitochondrial anaplerosis, pyruvate cycling, and TCA cycle flux (17). These relative mitochondrial
fluxes were converted to absolute fluxes by normalizing with EGP
(16, 35). The [13C3]propionate tracer by itself does not alter EGP or
hepatic mitochondrial fluxes (33), and the utility of this method has
been extensively validated and discussed (6, 27).
Targeted metabolomics. Frozen livers were ground to fine powder
in liquid nitrogen before a representative aliquot (⬃20 –25 mg) was
weighed out to determine concentrations of individual classes of
metabolites, including DAGs, Cer, acylcarnitines, organic acids, and
amino acids. Following addition of their respective internal standards
and homogenization with ceramic beads, metabolites were extracted
and analyzed by mass spectrometry as detailed below for each
individual class. Metabolites were quantified by peak area comparison
with their respective or a representative internal standard.
Analysis of liver DAG and Cer by LC-MS/MS. Internal standards
[Cer(d18:1/17:0) and a d5-DAGs mix] were prepared in chloroformmethanol (1:2 vol/vol) and added to the liver aliquot before Folch
extraction (14). Separation was accomplished with a Sigma Aldrich
Supelco C18 column (75 ⫻ 2.1 mm, 1.9 ␮m pore size) following the
gradient information described in the following section. Hepatic
DAGs and Cer were identified using precursor ion scanning [mass-
Table 1. Metabolic characteristics of C57Bl/6J mice fed either a control diet or a TFD for 8 and 24 wk
8 Weeks
Body wt, g
Fasting glucose, mg/dl
Fasting plasma insulin, ng/ml
Plasma ketones, ␮M
Fasting free fatty acids, mM
Hepatic insulin resistance fndex
Liver triglyceride, mg/g liver
24 Weeks
C
TFD
C
TFD
25.0 ⫾ 1.2
103 ⫾ 6
0.22 ⫾ 0.06
1,206 ⫾ 187
0.46 ⫾ 0.08
0.43 ⫾ 0.08
44 ⫾ 15
27.1 ⫾ 0.8*
99 ⫾ 3
0.19 ⫾ 0.02
2,253 ⫾ 255*
0.37 ⫾ 0.01
0.50 ⫾ 0.13
159 ⫾ 20*
30.9 ⫾ 0.7
99 ⫾ 5
0.16 ⫾ 0.01
1,087 ⫾ 315
0.54 ⫾ 0.06
0.42 ⫾ 0.05
98 ⫾ 13
37.2 ⫾ 1.2*
104 ⫾ 7
0.30 ⫾ 0.01*
1,165 ⫾ 289
0.40 ⫾ 0.03
1.67 ⫾ 0.34
317 ⫾ 13*
Values are means ⫾ SE; n ⫽ 6 –7 mice/group. C, control diet; TFD, high-fructose high-trans-fat diet. *P ⱕ 0.05 between C and TFD-fed mice.
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00492.2015 • www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on August 1, 2017
and/or T2DM is shown to impair mitochondrial respiration and
ATP synthesis (19, 36, 44). Taken together, it is not clear
whether specific defects in mitochondrial oxidative metabolism
are characteristic during the progression of simple steatosis and
NASH.
Hepatic insulin resistance and triglyceride accumulation are
also associated with accumulation of several “lipotoxic” lipid
intermediates, including diacylglycerols (DAGs), ceramides
(Cer), and long-chain acylcarnitines (20, 24). These are often
considered as products of impaired/incomplete mitochondrial
oxidative metabolism and furthermore have the potential to
impair insulin signaling and mediate hepatocyte inflammation
(13, 39). However, it is not clear whether impaired flux through
hepatic TCA cycle occurs concurrently with elevated levels of
lipid byproducts and/or altered expression of genes involved in
mitochondrial oxidative metabolism in the liver.
We investigated whether impaired mitochondrial TCA cycle
activity is a central feature of NASH. By using a combination
of metabolic flux analysis, targeted metabolomics, and hepatic
gene expression profiles, we demonstrate that the hepatic TCA
cycle activity is induced during NASH. However, elevated
TCA cycle activity was inadequate to prevent lipotoxicity and
incomplete fat oxidation.
E486
HEPATIC MITOCHONDRIAL METABOLISM IN NASH
The MS was operated in positive mode with spray voltage at 3 kV,
capillary temperature at 350°C, and vaporizer temperature at 50°C.
The precursor ion scan was set to monitor the precursor ions of m/z
264.3, representative of Cer with d18:1 chain. Cer putative assignments were based on fragmentation, presence of [M ⫹ H]⫹ and [M ⫹
H ⫺ H2O]⫹ peaks, as well as exact mass comparison of Q-Exactive
data and LipidMaps database. DAG assignments included presence of
[M ⫹ NH4]⫹ peak, exact mass comparison, and matching fragmentation. The neutral loss scans included 245, 273, 271, 297, 299, 301,
and 321, corresponding to losses of common fatty acid chain lengths.
LC-MS (Thermo Q-Exactive Orbitrap) conditions for
quantification. Each sample was analyzed in positive mode full scan
with resolving power of 70,000 at m/z 200, spray voltage of 3.5 kV,
capillary temperature of 300°C, sheath gas of 30, heater temperature
of 350°C, and s-lens RF of 35. Chromatography was the same as on
the triple quadrupole; however, the column employed was a Waters
Acquity C18 BEH column (50 ⫻ 2.1 mm, 1.7 ␮m pore size) (Milford,
8 wk
24 wk
Control
A
40x
40x
TFD
40x
B
#
*
30
C
24
#
*
#
*
18
12
*
6
*
*
Endogenous Glucose
Production (µmoles min-1)
Relative mRNA expression
Fig. 1. High-fructose high-trans-fat diet (TFD)fed mice develop hepatic insulin resistance and
simple steatosis by 8 wk that then transitions to
nonalcoholic steatohepatitis (NASH) by 24 wk.
A: trichrome staining of liver slices reveals
normal hepatocyte morphology in mice fed a
control diet for 8 or 24 wk. Liver histology of
mice fed TFD for 8 wk reveals panacinar steatosis. Liver histology of mice fed TFD for 24
wk reveals microvesicular and macrovesicular
steatosis with stage 1b fibrosis. B: expression of
genes involved in fibrogenesis was significantly
higher in the 24-wk TFD-fed mice compared
with their 8-wk counterparts. C: endogenous
glucose production fails to get suppressed in 8-wk
TFD-fed mice during euglycemic-hyperinsulinemic clamp (insulin stimulation), indicating hepatic
insulin resistance. Values are means ⫾ SE (n ⫽
5–7 mice/group). *P ⱕ 0.05 between control (C)
vs. TFD-fed mice or C vs. C insulin stimulated.
#
P ⱕ 0.05 between 8-wk TFD and 24-wk TFDfed mice.
40x
4
3
2
*
1
0
Control
0
Col1a1
Timp1
Mmp13
8 wk C
8 wk TFD
24 wk C
24 wk TFD
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00492.2015 • www.ajpendo.org
Basal
TFD
Insulin stimulated
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on August 1, 2017
to-charge ratio (m/z) 264.3] and neutral loss scanning (for fatty acid
tails), respectively, on a Thermo TSQ Quantum Access triple-quadrupole mass spectrometer (Thermo Scientific, San Jose, CA) with
Accela 1200 pump and heated electrospray ionization (HESI) source
(positive ionization). Relative quantitation data were acquired on the
Thermo Q-Exactive Orbitrap (Thermo Scientific) combined with a
Dionex Ultimate 3000 liquid chromatography instrument. Data analysis was performed with Xcalibur software and expressed as nanomoles of targeted compound per gram of liver protein.
LC-MS (Thermo TSQ triple quadrupole) conditions for DAG and
Cer analysis. Each reconstituted liver sample was injected (2 ␮l) on
a Sigma Aldrich Supelco C18 column (75 ⫻ 2.1 mm, 1.9 ␮m pore
size) with the mobile phases as follows: 1) 60:40 acetonitrile-water
(mobile phase A) and 2) 90:10 isopropanol-acetonitrile (mobile phase
B), with 10 mM ammonium formate and 0.1% formic acid in both
phases A and B. The gradient started at 32% phase B and rose to 100%
phase B over 16 min, remaining isocratic from 16 to 18 min, before
returning to starting conditions after 21 min, at a 500 ␮l/min flow rate.
HEPATIC MITOCHONDRIAL METABOLISM IN NASH
Gene expression analysis. As previously reported (32), quantitative
real-time PCR mix contained 25 ng cDNA, 150 nmol/l of each primer,
and 5 ␮l SYBR Green PCR master mix (Bio-Rad). Samples were run
in triplicate on a CFX Real Time system (C1000 Touch Thermal
Cycler; Bio-Rad). The comparative threshold method was used to
determine relative mRNA levels with cyclophilin as the internal
control.
Biochemical measurements. Plasma ketone body and FFA concentrations were determined using an analytical kit (Wako Chemicals,
Richmond, VA). Plasma insulin was measured by enzyme-linked
immunoassay using the mouse Insulin ELISA kit (Crystal Chem,
Downers Grove, IL). Triglyceride concentrations were determined
using an analytical kit from Sigma (St. Louis, MO).
Materials and reagents. [3,4-13C]glucose (98%) was purchased
from Omicron Biochemicals (South Bend, IN). [U-13C]propionate
and acylcarnitine internal standards were purchased from Cambridge
Isotopes. Internal standards, including Cer (d18:1/17:0) and d5-DAGs
mix, were purchased from Avanti Lipids (Alabaster, AL). Other
common chemicals were obtained from Sigma.
Statistical analysis. Statistical analyses for the targeted metabolomics data were performed in R version 3.1.2 (45). To test the effects
of age and treatment on metabolite levels, we fitted Type III ANOVAs
with the fixed effects age, treatment, and the interaction between age
and treatment (asking which metabolites changed differently with age
in mice on a high-fat diet compared with controls) and ranked the
metabolites according to their F-statistic for treatment effect (to
illustrate which metabolites showed the strongest effect of treatment).
We controlled the experimentwide false discovery rate at Q ⬍ 0.05
(2). We next examined overall patterns in classes of metabolites
(DAGs, Cer, organic acids, amino acids, and acylcarnitines) by
summarizing each class of metabolites using a principal-components
analysis on z-scored data (pcaMethods package in R; see Ref. 37). We
used the first principal component of variation in each class of
metabolites to describe the overall pattern in that class and ran five
separate ANOVAs (one for each class of metabolites, with fixed
effects as described above plus liver triglycerides as a covariate) on
the scores of each of these principal components. All other results
were analyzed using ANOVA, and pairwise mean comparisons were
performed using a t-test. Results are expressed as means ⫾ SE and
were considered significantly different at P ⱕ 0.05.
RESULTS
Eight weeks of TFD feeding resulted in simple steatosis that
transitioned by 24 wk to NASH. The metabolic characteristics
of the mice are presented in Table 1. Feeding TFD resulted in
Table 2. Histological scoring of mice livers for NASH after 24 wk of TFD feeding
Animal
C1
C2
C3
C4
C6
TFD
TFD
TFD
TFD
TFD
TFD
TFD
TFD
TFD
2
3
4
5
6
7
8
9
10
Steatosis
(0–3)/Location
Lobular
Inflammation (0–3)
Ballooning
(0–2)
Summation Score
(0–8)
Fibrosis Stage/Microgranuloma/
Microsteatosis (Present-Absent)
0
0
0
0
0
Panacinar
Panacinar
Panacinar
Panacinar
Panacinar
Panacinar
Panacinar
Panacinar
Panacinar
1
0
0
0
0
1
1
1
1
0
2
2
2
1
0
0
0
0
0
2
2
2
2
2
2
2
2
2
1
0
0
0
0
6
6
6
6
5
7
7
7
6
0/Absent/absent
0/Absent/absent
0/Absent/absent
0/Absent/absent
0/Absent/absent
1b/Present/present
1b/Present/present
1b/Present/present
1b/Present/present
1b/Present/present
1b/Present/present
2/Present/present
1b/Present/present
1b/Present/present
3,
3,
3,
3,
3,
3,
3,
3,
3,
The scoring system was adapted from Kleiner et al. (18). Steatosis: grade 0, ⱕ5%; grade 1, 5–33%; grade 2, 34 – 66%; grade 3, ⬎67%. Lobular inflammation:
grade 0, 0/200X; grade 1, ⬍2/200X; grade 2, 2– 4/200X; grade 3, ⬎4/200X. Ballooning: grade 0, 0; grade 1, few; grade 2, many. Fibrosis/stage: 1a, zone 3/mild;
1b, zone 3/dense; 1c, portal only; 2, perisinusoidal and portal/periportal.
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00492.2015 • www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on August 1, 2017
MA) at 30°C. Pooled QC and blanks were injected every 12 samples
throughout the sequence.
Analysis of liver acylcarnitines by LC-MS/MS. Liver aliquots were
homogenized and deproteinized with cold acetonitrile containing
stable isotope-labeled internal standards (Cambridge Isotopes, Andover, MA). Following centrifugation at 13,500 revolutions/min
(rpm) for 15 min, the supernatant was dried under nitrogen gas at
30°C and reconstituted in 90:10 methanol-water for LC-MS/MS
analysis. Acylcarnitine data were collected using SRM mode on a
Thermo TSQ Quantum Access triple-quadrupole mass spectrometer
with an Accela 1200 LC pump and HESI source (positive ionization).
Reactions fragmenting to m/z 85.3 were monitored following a 5-␮l
injection on an ACE PFP-C18 column (100 ⫻ 2.1 mm, 2 ␮m particle
size) at 40°C, as reported previously (40).
Analysis of liver organic acids by GC-MS. Liver aliquots were
spiked in with stable isotope-labeled organic acid internal standards,
deproteinized with sulfosalicylic acid (10% final volume), and centrifuged at 13,500 rpm for 15 min. The supernatant was decanted to
clean screw-cap tubes, 5 mmol of hydroxylamine-hydrochloride were
added, and the pH was adjusted to seven to eight with 4 M potassium
hydroxide. Following sonication (15 min) and incubation (65°C for 1 h),
the pH was reduced to two by adding hydrochloric acid (6 M) and
saturated with sodium chloride. Organic acids were extracted with ethyl
acetate, dried under nitrogen gas, and converted to their tertiary-butyldimethylsilyl (tBDMS) derivatives before separation on a HP-5MS column
(30 m ⫻ 0.25 mm ⫻ 0.25 ␮m; Agilent) under electron ionization (HP
5973N Mass Selective Detector; Agilent).
Analysis of liver amino acids by GC-MS. Amino acid concentrations were determined by isotope dilution with GC-MS as previously
described (7). To the liver aliquot, a known concentration of internal
standard (hydrolyzed [U-13C,15N]algae protein powder) was added.
Samples were then deproteinized with sulfosalicylic acid, and the
supernatant passed over a cation exchange resin before elution of
amino acids with 2.5 M ammonium hydroxide. The lyophilized amino
acids were converted to the tBDMS derivatives before separation on
a HP-5MS column (30 m ⫻ 0.25 mm ⫻ 0.25 ␮m; Agilent) and
fragmentation under electron ionization mode to monitor amino acid
and internal standard ions (7).
Histology. Liver from overnight-fasted mice were fixed in 10%
neutral buffered formalin for 20 –24 h, washed, and stored in 70%
ethanol before embedding in paraffin at the Molecular Pathology Core
at the University of Florida. The liver sections from the control mice
and mice with simple steatosis and NASH were then stained with
Masson’s Trichrome to visualize collagen fibers. The liver slides were
blinded and scored by a veterinary pathologist using a previously
published and validated scoring system of liver biopsies (18).
E487
E488
HEPATIC MITOCHONDRIAL METABOLISM IN NASH
Control
8
*
6
D
15
4
2
0
8 wk
B
24 wk
*
10
5
8
0
6
24 wk
E
4
30
p < 0.001
2
0
8 wk
C
8 wk
*
24 wk
*
20
Anaplerosis
(µmoles min-1)
TCA cycle flux
(µmoles min-1)
Fig. 2. Hepatic mitochondrial tricarboxylic
acid (TCA) cycle activity is induced in mice
with NASH. Overnight fasting basal levels of
endogenous glucose production (A), TCA cycle flux (B), anaplerosis (C), and pyruvate
cycling (D), determined using 13C-nuclear
magnetic resonance (NMR)-based isotopomer
analysis, were all elevated in mice fed a TFD
for 24 wk compared with their age-matched
control counterparts. E: robust relationship between hepatic mitochondrial TCA cycle flux
and mitochondrial anaplerosis. Values are
means ⫾ SE (n ⫽ 6 –7/group). *P ⱕ 0.05
between C and TFD-fed mice.
TFD
Pyruvate cycling
(µmoles min-1)
Endogenous glucose
production (µmoles min-1)
A
insulin stimulation was also 70% lower in 8-wk TFD-fed
animals (16.7 vs. 53.5 ␮mol/min), reflecting muscle insulin
resistance. Furthermore, hepatic insulin resistance worsened
with 24 wk of TFD feeding as evident from the significantly
higher values of hepatic insulin resistance index (Table 1).
Because mice fed TFD for 24 wk developed histological
features of NASH, we selected the 8 vs. 24 wk on TFD to
investigate alterations in mitochondrial metabolism associated
with progressive disease severity.
Mitochondrial TCA cycle activity was induced in mice with
NASH. As elaborated above, 13C-NMR-based isotopomer analysis allowed us to determine the activity of multiple pathways
through the hepatic mitochondrial TCA cycle, during the
transition from simple steatosis to NASH. In spite of hepatic
insulin resistance, EGP (Fig. 2A), absolute TCA cycle flux
(Fig. 2B), total anaplerosis (Fig. 2C), and pyruvate cycling
r = 0.76
20
10
Anaplerosis
(µmoles min-1)
0
0
15
2
4
6
8
TCA cycle flux
(µmoles min-1)
10
Simple steatosis
5
& Controls
0
8 wk
24 wk
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00492.2015 • www.ajpendo.org
NASH
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on August 1, 2017
a mild but significant weight gain by the 8th wk that progressed
to obesity by 24 wk of TFD feeding. Liver triglyceride content
was significantly elevated by 8 wk of TFD feeding without any
histological evidence of inflammation and fibrosis (Fig. 1A)
and thus was not scored. Upon prolonged feeding of TFD (24
wk), the phenotype of simple steatosis transitioned to NASH,
demonstrated by the presence of inflammation, severe steatosis, and fibrosis in mice with histology scores in the range of
six to seven (Fig. 1A and Table 2). The transition to NASH was
further evident from the significant induction of genes involved
in fibrogenesis after 24 wk of TFD feeding (Fig. 1B). Onset of
hepatic insulin resistance was evaluated using euglycemichyperinsulinemic clamps following 8 wk of TFD feeding.
Insulin stimulation failed to suppress EGP (Fig. 1C) in 8-wk
TFD-fed mice, indicating hepatic insulin resistance. The glucose infusion rate required to maintain euglycemia during
E489
HEPATIC MITOCHONDRIAL METABOLISM IN NASH
A
Ceramides
B
Diacylglycerols
Altered acylcarnitine levels and hepatic gene expression
profiles substantiate inefficient fat oxidation during NASH. In
contrast to the general pattern of accumulation of DAGs and
Cer, acylcarnitines were lower in TFD-fed mice (Fig. 3C),
although this was driven partly by increased liver triglycerides
rather than directly by the effects of diet. However, examination of individual metabolites showed a striking dichotomy in the
responses of long-chain and short-chain acylcarnitines: while all
acylcarnitines decreased or showed a trend toward decreasing in
mice with simple steatosis, long-chain acylcarnitines accumulated
markedly in the liver concurrent with the transition to NASH (Fig.
4E). Hepatic gene expression also revealed a consistent pattern of
dysregulated mitochondrial fat metabolism wherein expression of
several genes (Pc, Pgc1a, Ppara, Cpt1a, Lcad, and Mcad) was
downregulated with onset of NASH (Table 2). This occurred
together with an induction on genes involved in pathways of
inflammation (Il6 and Tnfa) during NASH. Taken together, elevated levels of metabolic byproducts of lipid metabolism and
altered hepatic mitochondrial gene expression point to incomplete
and inefficient disposal of FFA by hepatic mitochondria during
simple steatosis and NASH.
When all of the metabolites were ranked by statistical significance (F-statistic) of the effect of diet, it revealed a unanimous
trend predominated by DAGs and Cer (Table 3). In summary,
while most of the organic acids and amino acids, which are
substrates fueling the hepatic TCA cycle, remained unchanged
during the transition to NASH (Fig. 3, D and E), Cer, DAGs, and
acylcarnitines were strongly affected by the TFD treatment.
DISCUSSION
Dysfunctional hepatic mitochondrial energetics in the setting
of obesity and hepatic insulin resistance plays a key role in the
development of simple steatosis. However, it is unknown
C
Organic acids
1
100
20
0
50
0
-1
0
-20
-2
-50
8 wk
24 wk
D
8 wk
Amino acids
3
PC1 scores
PC1 scores
40
24 wk
E
8 wk
Acylcarnitines
50
2
24 wk
Fig. 3. Principal component analysis provides a snapshot of the overall metabolite
profiles during the transition from simple
steatosis to NASH. Summary of hepatic profiles of diacylglycerols (A), ceramides (B),
acylcarnitines (C), organic acids (D), and
amino acids (E) represented as a function of
age (8 or 24 wk) and diet (black, TFD; gray,
C). Data are means ⫾ SE of metabolite
scores from the first principal component
(PC1) of variation in each metabolite class.
25
1
0
0
-1
-25
-2
-50
8 wk
24 wk
8 wk
24 wk
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00492.2015 • www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on August 1, 2017
(Fig. 2D) remained unchanged after 8 wk of TFD. Thus, during
early stages of diet-induced insulin resistance and simple
steatosis, mitochondrial fluxes could remain apparently normal
(35). We then tested the hypothesis that onset of NASH will
impair mitochondrial fluxes through the TCA cycle. As expected, onset of NASH was accompanied by higher rates of
EGP (Fig. 2A). Interestingly, mitochondrial TCA cycle metabolism was upregulated in mice with NASH. There was an
approximate twofold induction in absolute TCA cycle flux
(Fig. 2B), total anaplerosis (Fig. 2C), and pyruvate cycling
(Fig. 2D) in mice with NASH, relative to their age-matched
controls. Furthermore, a robust correlation between TCA cycle
flux and anaplerosis was evident, indicating the dependence of
anabolic pathways of glucose production on TCA cycle flux
(Fig. 2E).
Lipotoxic byproducts of fat oxidation, including Cer and
DAGs, increased concurrently with hepatic TCA cycle activity.
Lipid byproducts, including DAGs and Cer, showed significant
increases in both the 8- and 24-wk TFD mice (Fig. 3, A and B).
DAGs were elevated in mice with simple steatosis by 8 wk,
and the magnitude of this elevation increased markedly by 24
wk concurrent with the onset of NASH (Figs. 3A, 4A, and 4C).
Cer also increased in TFD-fed mice compared with controls
(Fig. 3B), but, unlike DAGs, this elevation was no more
pronounced in NASH compared with simple steatosis (Fig. 4,
B and D). However, the patterns were far less consistent in the
Cer compared with the DAGs, with elevation of some metabolites increasing with increasing severity of disease (e.g.,
Cer16:0, Cer24:0), whereas elevation of others was mitigated
with disease progression (e.g., Cer22:0, Cer23:0; Fig. 4D).
Taken together, the concurrent induction of hepatic TCA cycle
metabolism and elevated Cer and DAGs suggest inefficient and
incomplete fat oxidation during NASH.
E490
HEPATIC MITOCHONDRIAL METABOLISM IN NASH
B
*
15
4
Total ceramides
(µmoles g liver protein-1)
*
Control
10
TFD
5
3
2
1
0
0
8 wk
24 wk
8 wk
21
#
*
#
*
15
*
#
*
*
*
*
* *
* **
1.5
whether the pathogenic combination of high rates of triglyceride accumulation, inflammation, and fibrosis, observed during
NASH, is accompanied with impaired oxidative capacity of the
mitochondrial TCA cycle. Our results demonstrate for the first
time that the oxidative capacity of hepatic mitochondrial TCA
cycle is twofold elevated during NASH. The upregulation of
TCA cycle activity persisted in spite of clear impairments in
disposal/storage mechanisms of FFA, as evident from accumulation of a variety of lipid intermediates, blunted ketogenesis,
and altered mitochondrial gene expression. A combination of
upregulated oxidative flux and accretion of lipotoxic metabolites could provide a potent mitochondrial milieu during NASH
to fuel ROS production and hasten inflammation.
Hepatic metabolism constantly adapts and remodels with
progressive severity of insulin resistance and substrate over-
*
*
*
**
18:1_22:6
20:2_20:4
16:1_22:6
**
*
**
18:2_20:2
22:6/22:6
#
*
2.0
#
#
*
1.5
#
#
1.0
0.5
* **
*
C
C24:1
C24:0
C23:0
C22:0
C20:0
C18:0
C16:0
1.0
#
*
* ** *
C16
**
2.5
2.5
C14
*
E
C6
3.0
24 Week
C5
#
*
C4
*
3.5
#
*
16:0_20:4
18:1_18:2
18:1/18:1
16:1_18:2
#
#
C3
D
4.0
#
16:0_22:6
#
34:2
16:0_18:1
16:0_18:0
1
16:1/16:1
3
*
*
*
*
*
*
* * *
* **
18:1_20:4
** *
5
#
*
*
Acylcarnitines: Fold change from
age matched controls
*
7
#
#
18:2/18:2
#
18:0_22:6
*
#
9
18:0_20:4
11
#
18:2_20:4
13
C8
17
16:0_16:1
Diacylglycerols: Fold change from
age matched controls
Ceramides: Fold change from
age matched controls
#
*
28
8 Week
2.0
24 wk
load. A key initial aspect of this adaptation is the rapid
accretion of triglycerides in the liver (simple steatosis), from
plasma or dietary FFA and de novo lipid synthesis (12, 21).
Depending on the ability of the hepatocyte to buffer and store
excess lipids, the initial stages of adaptation may not be
accompanied by any apparent alterations in mitochondrial
oxidative metabolism (35). In fact, hepatic insulin resistance
and simple steatosis were evident in our mice fed TFD for 8 wk
(Fig. 1D) even before alterations in mitochondrial TCA cycle
metabolism were apparent (Fig. 2), as previously reported (35).
However, the 8-wk TFD-fed mice with simple steatosis also
exhibited a twofold elevation in plasma ketones (Table 1)
along with upregulation of Ppara and Fgf21 gene expression.
This suggests an early onset of hepatic compensatory mechanisms to dispose acetyl-coA units via ketogenesis. Fasting
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00492.2015 • www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on August 1, 2017
C
35
Fig. 4. Elevated levels of ceramides and diacylglycerols (DAGs) together with altered
acylcarnitine profile in the liver of TFD mice
indicate inefficient fat oxidation. Total hepatic
DAG content (A), total hepatic ceramide content (B), fold changes in individual hepatic
DAGs (C), fold changes in individual hepatic
ceramides (D), and fold changes in individual
hepatic acylcarnitines (E) relative to their
respective age-matched controls. Mice were
fed a TFD for either 8 or 24 wk. Values are
means ⫾ SE (n ⫽ 6 –7/group). *P ⱕ 0.05
between C vs. TFD-fed mice. #P ⱕ 0.05
between 8- vs. 24-wk dietary treatments.
*
*
C2
Total diacylglycerols
(µmoles g liver protein-1)
A
20
E491
HEPATIC MITOCHONDRIAL METABOLISM IN NASH
Table 3. Expression of genes related to carbohydrate, lipid,
and mitochondrial metabolism in liver of overnight-fasted
C57Bl/6J mice fed TFD for 8 and 24 wk compared with
their age-matched control counterparts
TFD
24 wk
0.9 ⫾ 0.04
0.4 ⫾ 0.06*
1.4 ⫾ 0.15*
0.8 ⫾ 0.10
0.9 ⫾ 0.11
1.2 ⫾ 0.15
0.6 ⫾ 0.15
2.2 ⫾ 0.51*
1.2 ⫾ 0.12
0.8 ⫾ 0.08
1.2 ⫾ 0.16
0.7 ⫾ 0.02*
0.5 ⫾ 0.06*
0.7 ⫾ 0.03*
0.5 ⫾ 0.03*
0.7 ⫾ 0.06*
0.8 ⫾ 0.05*
0.7 ⫾ 0.07
1.6 ⫾ 0.20*
0.9 ⫾ 0.07
0.9 ⫾ 0.04
2.5 ⫾ 0.19*
4.3 ⫾ 0.54*
1.0 ⫾ 0.06
1.0 ⫾ 0.05
2.0 ⫾ 0.32*
1.2 ⫾ 0.06*
1.0 ⫾ 0.07
0.8 ⫾ 0.09
1.9 ⫾ 0.53
1.6 ⫾ 0.38
2.0 ⫾ 0.23*
Values are means ⫾ SE; n ⫽ 5– 6 mice/group. *P ⱕ 0.05 between C and
TFD groups.
Table 4. Effect of the dietary regime (TFD or C) and age (8 or 24 wk) on metabolite concentrations
Diet
Diet ⫻ Age
Age
Metabolite
F
Q
F
Q
DAG (18:0_20:4)
Cer (d18:1/24:1)
DAG (18:1/18:1)
DAG (18:1_20:4)
Cer (d18:1/24:0)
DAG (18:1_18:2)
DAG (16:0_18:1)
Cer (d18:1/23:0)
DAG (16:1_22:6)
DAG (16:1_16:1)
Cer (d18:1/18:0)
DAG (18:2_20:4)
DAG (16:0_16:1)
DAG (34:2)
DAG (18:2/18:2)
DAG (16:1_18:2)
DAG (16:0_22:6)
Carnitine
Cer (d18:1/22:0)
DAG (18:1_22:6)
Alanine
Cer (d18:1/16:0)
Acetylcarnitine
DAG (20:2_20:4)
Succinate
DAG (16:0_18:0)
DAG (18:0_22:6)
DAG (22:6/22:6)
␣-Ketoglutarate
Cer (d18:1/20:0)
DAG (18:2_20:2)
n-Butyrylcarnitine
Isovalerylcarnitine
Glutamate
299.1*
214.7*
183.1*
135.8*
133.8*
110.5*
100.5*
95.3*
75.9*
73.7*
68.8*
68.8*
66.7*
65.8*
56.2*
54.5*
53.2*
51.6*
51.2*
49.3*
26.3*
25.9*
25.9*
24.5*
23.1*
22.5*
21.8*
19.4*
14.4*
13.2*
12.1*
10.7*
8.7*
6.0*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
⬍0.001*
0.002*
0.003*
0.004*
0.006*
0.012*
0.038*
59.1*
1.3
28*
20.1
2.9
9.1*
13.3*
0.1
6.1
5.9
1.3
9.0*
10.4*
5.1
0.6
3.8
1.3
10.7
3.1
1.3
1.7
0.5
2.5
1.8
74.5*
1.2
1.7
1.3
0.3
2.9
1.3
0.4
4.4
5.3
⬍0.001*
0.396
⬍0.001*
0.002*
0.268
0.039*
0.014*
0.724
0.091
0.091
0.396
0.039*
0.028*
0.119
0.539
0.204
0.396
0.028
0.268
0.396
0.372
0.547
0.312
0.372
⬍0.001*
0.421
0.372
0.396
0.604
0.268
0.396
0.59
0.157
0.114
F
80.7*
0.4
35.7*
27.9*
2.9
11.7*
14.9*
1.8
7.2
9.1*
0.1
21.6*
6.9
7.8
3.4
3.0
2.1
1.4
5.8
6.1
0.01
0.9
0.02
6.7
7.2
1.3
1.5
0.3
0.02
9.6
3.8
0.07
1.8
1.5
Q
⬍0.001*
0.668
⬍0.001*
⬍0.001*
0.281
0.023*
0.01*
0.362
0.072
0.047*
0.812
0.002*
0.078
0.071
0.247
0.281
0.334
0.405
0.097
0.094
0.923
0.492
0.909
0.078
0.072
0.405
0.398
0.675
0.909
0.043
0.224
0.857
0.362
0.398
DAG, diacylglycerol; Cer, ceramide. F and Q are F-statistics and false discovery rate-corrected P values, respectively, from linear models (see text).
Metabolites are ranked by statistical significance of the effect of diet. *Significant effects.
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00492.2015 • www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on August 1, 2017
Mitochondrial Metabolism
Pc
Pgc1a/Ppargc1a
Ppara
Cpt1a
Lcad/Acadl
Mcad/Acadm
Hmgcs2
Fgf21
Cs
Cycs
Ucp2
Lipogenesis
Srebp1c/Srebf1
Acc1/Acaca
Fasn
Inflammation
Il6
Tnfa
8 wk
ketogenesis can account for up to two-thirds of the total fat
oxidation in the liver, and further diversion of acetyl-coA
toward ketones prevents oxidative burden on the hepatic TCA
cycle. Thus, consistent with previous observations in both
diet-induced (35, 43) and genetic rodent models of hepatic
insulin resistance (32), upregulated ketogenesis could serve to
efficiently channel FFAs away from the TCA cycle. Interestingly, the apparently normal TCA cycle metabolism and elevated plasma ketones during simple steatosis occurred together
with significantly higher levels of hepatic DAGs and Cer. This
could be an early indication of the failure of lipid storage
mechanisms to optimally buffer and store excess FFAs in the
form of hepatic triglycerides.
Transition from simple steatosis to NASH combines additional factors, including cellular inflammation, ballooning, and
fibrosis, together with steatosis, thus aggravating hepatocyte
injury. Do these additional insults responsible for the transition
to NASH impair mitochondrial oxidative metabolism? In this
regard, mitochondrial oxidative metabolism encompasses several central pathways, including ␤-oxidation, hepatic TCA
cycle, ketogenesis, respiratory chain activity, and ATP synthesis, all of which work in concert to maintain cellular homeostasis. Consistent with the idea of an early compensatory
induction of mitochondrial oxidative metabolism, ␤-oxidation
(15, 22), ketogenesis, and TCA cycle activity (32, 35, 41) have
E492
HEPATIC MITOCHONDRIAL METABOLISM IN NASH
in mice with simple steatosis was completely abolished with
NASH. Furthermore, several genes associated with mitochondrial oxidative metabolism were downregulated in mice with
NASH, suggesting an inability to adapt and compensate in
spite of elevated nutrient flux. Taken together with the elevated
levels of lipotoxic intermediates in NASH, these data indicate
that the flux through ␤-oxidation could be considered “impaired” (29, 47), since it is inefficient to handle the chronic
FFA overload during NASH. Even then, considering the twofold induction in the hepatic TCA cycle, the FFA flux through
␤-oxidation, or from other nonlipid sources (40), could be high
enough to generate enough acetyl-coA (15, 35, 41). This
acetyl-coA is then being selectively partitioned into mitochondrial TCA cycle for complete oxidation during simple steatosis
(35, 41) and NASH, fueling biosynthetic pathways in the liver.
In summary, hepatic insulin resistance during NASH presents a unique environment where high rates of triglyceride
synthesis/storage coexist with high rates of mitochondrial TCA
cycle activity. Considering the tight relationship between hepatic TCA cycle flux and mitochondrial anaplerosis (Fig. 2E
and Refs. 32 and 41) to maintain gluconeogenesis, impairment
at the level of mitochondrial TCA cycle metabolism could be
implausible during NASH. Even then, sustained induction of
hepatic mitochondrial TCA cycle during NASH could be
reflecting combinations of 1) early compensatory response to
FFA overload and 2) a progressive “inflexibility” of mitochondrial metabolism to insulin action and macronutrients, with
severity of the disease. Constitutive induction of mitochondrial
TCA cycle together with inefficient disposal/storage of FFA in
the liver can be a chronic source of ROS and could hasten
inflammation and fibrosis during NASH. Targeted manipulation of hepatic oxidative TCA cycle metabolism could provide
an attractive prospective to alleviate mitochondrial dysfunction
during simple steatosis and NASH.
GRANTS
This work was supported by a University of Florida Research Opportunity
Seed Fund Award (00089467; to N. E. Sunny), a Southeast Center for
Integrated Metabolomics National Institute of Diabetes and Digestive and
Kidney Diseases Grant (U24-DK-097209), and a National Institutes of Health
and National Center for Research Resources Clinical and Translational Science
Award to the University of Florida (UL1-TR-00064).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
R.E.P., S.K., M.N., J.T.M., J.M., D.J.M., J.R.R., and N.E.S. performed
experiments; R.E.P., S.K., C.M.W., M.N., J.T.M., J.M., M.K.R., D.J.M.,
T.J.G., and N.E.S. analyzed data; R.E.P., S.K., C.M.W., and N.E.S. prepared
figures; R.E.P., S.K., K.C., and N.E.S. drafted manuscript; R.E.P., S.K.,
C.M.W., M.N., R.A.Y., K.C., T.J.G., and N.E.S. edited and revised manuscript; M.K.R., R.A.Y., K.C., T.J.G., and N.E.S. interpreted results of experiments; T.J.G. and N.E.S. approved final version of manuscript; N.E.S.
conception and design of research.
REFERENCES
1. Bazick J, Donithan M, Neuschwander-Tetri BA, Kleiner D, Brunt
EM, Wilson L, Doo E, Lavine J, Tonascia J, Loomba R. Clinical model
for NASH and advanced fibrosis in adult patients with diabetes and
NAFLD: guidelines for referral in NAFLD. Diabetes Care 38: 1347–1355,
2015.
2. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a
practical and powerful approach to multiple hypothesis testing. J Royal
Stat Soc Ser B 57: 289 –300, 1995.
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00492.2015 • www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on August 1, 2017
all been reported to be induced in several mice models and in
human subjects with simple steatosis. However, prolonged
nutritional overload and progressive severity of liver disease
(e.g., onset of NASH, T2DM) are associated with impairments
in ketogenesis (32, 35), mitochondrial respiratory chain activity (19), and rates of ATP synthesis (36, 44). In spite of the
above reports, our results indicate that hepatic TCA cycle
metabolism remains upregulated during NASH. Because generation of acetyl-coA through ␤-oxidation and its terminal
oxidation through hepatic TCA cycle are major sources of
energy generation, induction of these networks could be an
obligatory phenomenon for synchronizing the energy-demanding processes (e.g., gluconeogenesis, lipogenesis) during states
of substrate overload, such as obesity, simple steatosis, and
NASH.
Consistent with the idea that impaired mitochondrial TCA
cycle could be deleterious to cellular energy demand, all three
discrete pathways through mitochondrial TCA cycle metabolism, including absolute TCA cycle flux, anaplerosis, and
pyruvate cycling, were all elevated in mice with NASH.
Elevated TCA cycle flux provides evidence for the higher
activity of citrate synthase toward terminal oxidation of acetylcoA. Furthermore, high rates of anaplerosis are reflective of the
activity of pyruvate carboxylase enzyme, which is closely
coupled to phosphoenolpyruvate carboxykinase and cataplerosis to regulate biosynthetic pathways of gluconeogenesis and
lipogenesis (26). Pyruvate cycling, which involves carboxylation/decarboxylation of pyruvate, has a significant role in lipid
biosynthesis. Here, acetyl-coA generated from the cleavage of
citrate by ATP-citrate lyase is used for lipogenesis, whereas the
oxaloacetate (OAA) generated is returned back to the mitochondrial TCA cycle (by converting it to malate and then
pyruvate and back to OAA) (4). Taken together, the coordinated induction of mitochondrial TCA cycle fluxes helps to
sustain the bioenergetics of the insulin-resistant liver during
simple steatosis (35, 41) and NASH.
A current prevailing hypothesis dictates that, during hepatic
insulin resistance, long-chain acylcarnitines are diverted from
mitochondrial oxidation to biosynthesis of hepatic triglycerides, DAGs, and Cer (9, 13). While this was indeed the case in
our study, it is important to note that accumulation of liver
triglycerides, DAGs, and Cer occurred together with hepatic
insulin resistance and simple steatosis when no alterations in
mitochondrial TCA cycle fluxes were evident. Furthermore,
during NASH, elevated levels of these lipotoxic intermediates
coexisted with a twofold induction in mitochondrial oxidative
TCA cycle flux. Taken together, incomplete fat oxidation (20,
24, 29) may be an early hallmark of hepatic insulin resistance.
Several hepatic DAGs and long-chain acylcarnitines continued
to increase with triglyceride accumulation and high rates of
TCA cycle activity in NASH. This suggests that mitochondrial
fat oxidation is becoming increasingly inefficient at disposing
excess FFA. The plot of scores from the first principal component of variation for each metabolite class (Fig. 3) together
with the statistical ranking of individual metabolites (Table 4)
illustrates the high predictive power of DAGs and Cer in
distinguishing NASH from simple steatosis.
With prolonged TFD feeding, hepatic ketogenesis was
blunted in mice with NASH, indicating impaired flexibility of
this pathway to adapt to excess substrate supply (35). This is
evident because the compensatory induction in plasma ketones
HEPATIC MITOCHONDRIAL METABOLISM IN NASH
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
in an experimental model of non-alcoholic fatty liver disease induced by
monosodium L-glutamate in rats. Exp Mol Pathol 91: 687–694, 2011.
Lomonaco R, Ortiz-Lopez C, Orsak B, Webb A, Hardies J, Darland
C, Finch J, Gastaldelli A, Harrison S, Tio F, Cusi K. Effect of adipose
tissue insulin resistance on metabolic parameters and liver histology in
obese patients with nonalcoholic fatty liver disease. Hepatology 55:
1389 –1397, 2012.
Muoio DM, Newgard CB. Mechanisms of disease:Molecular and metabolic mechanisms of insulin resistance and beta-cell failure in type 2
diabetes. Nat Rev Mol Cell Biol 9: 193–205, 2008.
Musso G, Gambino R, Cassader M, Pagano G. Meta-analysis: natural
history of non-alcoholic fatty liver disease (NAFLD) and diagnostic
accuracy of non-invasive tests for liver disease severity. Ann Med 43:
617–649, 2011.
Owen OE, Kalhan SC, Hanson RW. The key role of anaplerosis and
cataplerosis for citric acid cycle function. J Biol Chem 277: 30409 –30412,
2002.
Previs SF, Kelley DE. Tracer-based assessments of hepatic anaplerotic
and TCA cycle flux: practicality, stoichiometry, and hidden assumptions.
Am J Physiol Endocrinol Metab 309: E727–E735, 2015.
Ramaswamy V, Hooker JW, Withers RS, Nast RE, Brey WW, Edison
AS. Development of a (1)(3)C-optimized 1.5-mm high temperature superconducting NMR probe. J Magnet Res 235: 58 –65, 2013.
Rector RS, Thyfault JP, Uptergrove GM, Morris EM, Naples SP,
Borengasser SJ, Mikus CR, Laye MJ, Laughlin MH, Booth FW,
Ibdah JA. Mitochondrial dysfunction precedes insulin resistance and
hepatic steatosis and contributes to the natural history of non-alcoholic
fatty liver disease in an obese rodent model. J Hepatol 52: 727–736, 2010.
Rinella ME. Nonalcoholic fatty liver disease: a systematic review. J Am
Vet Med Assoc 313: 2263–2273, 2015.
Samuel VT, Liu ZX, Qu X, Elder BD, Bilz S, Befroy D, Romanelli AJ,
Shulman GI. Mechanism of hepatic insulin resistance in non-alcoholic
fatty liver disease. J Biol Chem 279: 32345–32353, 2004.
Satapati S, He T, Inagaki T, Potthoff M, Merritt ME, Esser V,
Mangelsdorf DJ, Kliewer SA, Browning JD, Burgess SC. Partial
resistance to peroxisome proliferator-activated receptor-alpha agonists in
ZDF rats is associated with defective hepatic mitochondrial metabolism.
Diabetes 57: 2012–2021, 2008.
Satapati S, Kucejova B, Duarte JA, Fletcher JA, Reynolds L, Sunny
NE, He T, Nair LA, Livingston K, Fu X, Merritt ME, Sherry AD,
Malloy CR, Shelton JM, Lambert J, Parks EJ, Corbin I, Magnuson
MA, Browning JD, Burgess SC. Mitochondrial metabolism mediates
oxidative stress and inflammation in fatty liver. J Clin Invest 125: 4447–
4462, 2015.
Satapati S, Sunny NE, Kucejova B, Fu X, He TT, Mendez-Lucas A,
Shelton JM, Perales JC, Browning JD, Burgess SC. Elevated TCA
cycle function in the pathology of diet-induced hepatic insulin resistance
and fatty liver. J Lipid Res 53: 1080 –1092, 2012.
Satapati S, Sunny NE, Kucejova B, Fu X, He TT, Mendez-Lucas A,
Shelton JM, Perales JC, Browning JD, Burgess SC. Elevated TCA
cycle function in the pathology of diet-induced hepatic insulin resistance
and fatty liver. J Lipid Res 53: 1080 –1092, 2012.
Schmid AI, Szendroedi J, Chmelik M, Krssak M, Moser E, Roden M.
Liver ATP synthesis is lower and relates to insulin sensitivity in patients
with type 2 diabetes. Diabetes Care 34: 448 –453, 2011.
Stacklies W, Redestig H, Scholz M, Walther D, Selbig J. pcaMethods:
a bioconductor package providing PCA methods for incomplete data.
Bioinformatics 23: 1164 –1167, 2007.
Starley BQ, Calcagno CJ, Harrison SA. Nonalcoholic fatty liver disease
and hepatocellular carcinoma: a weighty connection. Hepatology 51:
1820 –1832, 2010.
Summers SA. Ceramides in insulin resistance and lipotoxicity. Prog Lipid
Res 45: 42–72, 2006.
Sunny NE, Kalavalapalli S, Bril F, Garrett TJ, Nautiyal M, Mathew
JT, Williams CM, Cusi K. Cross-talk between branched chain amino
acids and hepatic mitochondria is compromised in nonalcoholic fatty liver
disease. Am J Physiol Endocrinol Metab 309: E311–E319, 2015.
Sunny NE, Parks Elizabeth J, Browning Jeffrey D, Burgess SC.
Excessive hepatic mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease. Cell Metab 14: 804 –810, 2011.
Sunny NE, Parks EJ, Browning JD, Burgess SC. Excessive hepatic
mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease. Cell Metab 14: 804 –810, 2011.
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00492.2015 • www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on August 1, 2017
3. Birkenfeld AL, Shulman GI. Nonalcoholic fatty liver disease, hepatic
insulin resistance, and type 2 diabetes. Hepatology 59: 713–723, 2014.
4. Burgess SC, Hausler N, Merritt M, Jeffrey FM, Storey C, Milde A,
Koshy S, Lindner J, Magnuson MA, Malloy CR, Sherry AD. Impaired
tricarboxylic acid cycle activity in mouse livers lacking cytosolic phosphoenolpyruvate carboxykinase. J Biol Chem 279: 48941–48949, 2004.
5. Burgess SC, Jeffrey FMH, Storey C, Milde A, Hausler N, Merritt ME,
Mulder H, Holm C, Sherry AD, Malloy CR. Effect of murine strain on
metabolic pathways of glucose production after brief or prolonged fasting.
Am J Physiol Endocrinol Metab 289: E53–E61, 2005.
6. Burgess SC, Merritt ME, Jones JG, Browning JD, Sherry AD, Malloy
CR. Limitations of detection of anaplerosis and pyruvate cycling from
metabolism of [1-(13)C] acetate. Nat Med 21: 108 –109, 2015.
7. Calder AG, Garden KE, Anderson SE, Lobley GE. Quantitation of
blood and plasma amino acids using isotope dilution electron impact gas
chromatography/mass spectrometry with U-(13)C amino acids as internal
standards. Rapid Commun Mass Spectrom 13: 2080 –2083, 1999.
8. Charlton MR, Burns JM, Pedersen RA, Watt KD, Heimbach JK,
Dierkhising RA. Frequency and outcomes of liver transplantation for
nonalcoholic steatohepatitis in the United States. Gastroenterology 141:
1249 –1253, 2011.
9. Chavez JA, Summers SA. A ceramide-centric view of insulin resistance.
Cell Metab 15: 585–594, 2012.
10. Clapper JR, Hendricks MD, Gu G, Wittmer C, Dolman CS, Herich J,
Athanacio J, Villescaz C, Ghosh SS, Heilig JS, Lowe C, Roth JD.
Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical disease progression and methods of assessment. Am J Physiol Gastrointest Liver Physiol 305: G483–G495, 2013.
11. Clark JM, Brancati FL, Diehl AM. Nonalcoholic fatty liver disease.
Gastroenterology 122: 1649 –1657, 2002.
12. Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD,
Parks EJ. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest 115:
1343–1351, 2005.
13. Erion DM, Shulman GI. Diacylglycerol-mediated insulin resistance. Nat
Med 16: 400 –402, 2010.
14. Folch-Pi J, Lees M, Stanley GHS. A simple method for the isolation and
purification of total lipides from animal tissue. J Biol Chem 226: 497–509,
1957.
15. Iozzo P, Bucci M, Roivainen A, Nagren K, Jarvisalo MJ, Kiss J,
Guiducci L, Fielding B, Naum AG, Borra R, Virtanen K, Savunen T,
Salvadori PA, Ferrannini E, Knuuti J, Nuutila P. Fatty acid metabolism
in the liver, measured by positron emission tomography, is increased in
obese individuals. Gastroenterology 139: 846 –856, 2010.
16. Jin ES, Jones JG, Merritt M, Burgess SC, Malloy CR, Sherry AD.
Glucose production, gluconeogenesis, and hepatic tricarboxylic acid cycle
fluxes measured by nuclear magnetic resonance analysis of a single
glucose derivative. Anal Biochem 327: 149 –155, 2004.
17. Jones JG, Naidoo R, Sherry AD, Jeffrey FMH, Cottam GL, Malloy
CR. Measurement of gluconeogenesis and pyruvate recycling in the rat
liver: a simple analysis of glucose and glutamate isotopomers during
metabolism of [1,2,3-13C3]propionate. FEBS Lett 412: 131–137, 1997.
18. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, Unalp-Arida A, Yeh
M, McCullough AJ, Sanyal AJ, Nonalcoholic Steatohepatitis Clinical
Research Network. Design and validation of a histological scoring
system for nonalcoholic fatty liver disease. Hepatology 41: 1313–1321,
2005.
19. Koliaki C, Szendroedi J, Kaul K, Jelenik T, Nowotny P, Jankowiak F,
Herder C, Carstensen M, Krausch M, Knoefel WT, Schlensak M,
Roden M. Adaptation of hepatic mitochondrial function in humans with
non-alcoholic fatty liver is lost in steatohepatitis. Cell Metab 21: 739 –746,
2015.
20. Koves TR, Ussher JR, Noland RC, Slentz D, Mosedale M, Ilkayeva O,
Bain J, Stevens R, Dyck JR, Newgard CB, Lopaschuk GD, Muoio DM.
Mitochondrial overload and incomplete fatty acid oxidation contribute to
skeletal muscle insulin resistance. Cell Metab 7: 45–56, 2008.
21. Lambert JE, Ramos-Roman MA, Browning JD, Parks EJ. Increased
de novo lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease. Gastroenterology 146: 726 –735, 2014.
22. Lazarin Mde O, Ishii-Iwamoto EL, Yamamoto NS, Constantin RP,
Garcia RF, da Costa CE, Vitoriano Ade S, de Oliveira MC,. and
Salgueiro-Pagadigorria C.L Liver mitochondrial function and redox status
E493
E494
HEPATIC MITOCHONDRIAL METABOLISM IN NASH
43. 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.
44. Szendroedi J, Chmelik M, Schmid AI, Nowotny P, Brehm A, Krssak
M, Moser E, Roden M. Abnormal hepatic energy homeostasis in type 2
diabetes. Hepatology 50: 1079 –1086, 2009.
45. Team RC. R: A Language and Environment for Statistical Computing.
Vienna, Austria: R Foundation for Statistical Computing, 2014.
46. Trevaskis JL, Griffin PS, Wittmer C, Neuschwander-Tetri BA, Brunt
EM, Dolman CS, Erickson MR, Napora J, Parkes DG, Roth JD.
Glucagon-like peptide-1 receptor agonism improves metabolic, biochemical, and histopathological indices of nonalcoholic steatohepatitis in mice.
Am J Physiol Gastrointest Liver Physiol 302: G762–G772, 2012.
47. Zhang D, Liu ZX, Choi CS, Tian L, Kibbey R, Dong J, Cline GW,
Wood PA, Shulman GI. Mitochondrial dysfunction due to long-chain
Acyl-CoA dehydrogenase deficiency causes hepatic steatosis and hepatic
insulin resistance. Proc Natl Acad Sci USA 104: 17075–17080, 2007.
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on August 1, 2017
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00492.2015 • www.ajpendo.org