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InVivo Hyperpolarized Carbon-13 Magnetic Resonance
Spectroscopy Reveals Increased Pyruvate Carboxylase
Flux in an Insulin-Resistant Mouse Model
Philip Lee,1,2 Waifook Leong,1,3 Trish Tan,1,3 Miangkee Lim,1 Weiping Han,1,3,4 and George K. Radda1
The pathogenesis of type 2 diabetes is characterized by impaired insulin action and
increased hepatic glucose production (HGP). Despite the importance of hepatic metabolic
aberrations in diabetes development, there is currently no molecular probe that allows measurement of hepatic gluconeogenic pathways in vivo and in a noninvasive manner. In this
study, we used hyperpolarized carbon 13 (13C)-labeled pyruvate magnetic resonance spectroscopy (MRS) to determine changes in hepatic gluconeogenesis in a high-fat diet (HFD)induced mouse model of type 2 diabetes. Compared with mice on chow diet, HFD-fed mice
displayed higher levels of oxaloacetate, aspartate, and malate, along with increased 13C label
exchange rates between hyperpolarized [1-13C]pyruvate and its downstream metabolites,
[1-13C]malate and [1-13C]aspartate. Biochemical assays using liver extract revealed up-regulated malate dehydrogenase activity, but not aspartate transaminase activity, in HFD-fed
mice. Moreover, the 13C label exchange rate between [1-13C]pyruvate and [1-13C]aspartate
(kpyr->asp) exhibited apparent correlation with gluconeogenic pyruvate carboxylase (PC) activity in hepatocytes. Finally, up-regulated HGP by glucagon stimulation was detected by
an increase in aspartate signal and kpyr->asp, whereas HFD mice treated with metformin for
2 weeks displayed lower production of aspartate and malate, as well as reduced kpyr->asp and
13
C-label exchange rate between pyruvate and malate, consistent with down-regulated gluconeogenesis. Conclusion: Taken together, we demonstrate that increased PC flux is an
important pathway responsible for increased HGP in diabetes development, and that pharmacologically induced metabolic changes specific to the liver can be detected in vivo with a
hyperpolarized 13C-biomolecular probe. Hyperpolarized 13C MRS and the determination
of metabolite exchange rates may allow longitudinal monitoring of liver function in disease
development. (HEPATOLOGY 2013;57:515-524)
T
he coordinated actions of insulin and glucagon
ensure that glucose homeostasis is maintained
across a wide range of physiological conditions.
In obesity-associated type 2 diabetes, control of glucose
metabolism by these two regulatory hormones is
impaired, resulting in hepatic insulin resistance and excessive endogenous glucose production.1 To date, it
has not been possible to evaluate this metabolic dysfunction in the liver by a noninvasive in vivo method.
Carbon-13 (13C) magnetic resonance spectroscopy
(MRS) has been used to study hepatic gluconeogenesis
since the 1980s. However, its inherent low sensitivity
has largely limited its application to the study of
steady-state metabolism in perfused livers with long
Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; 13C, carbon-13; ChREBP, carbohydrate response element-binding protein; FAS, fatty
acid synthase; G-6-Pase, glucose-6-phosphatase; HFD, high-fat diet; HGP, hepatic glucose production; IHTG, intrahepatic triglyceride; IPGTT, intraperitoneal
glucose tolerance test; ITT, insulin tolerance test; IV, intravenously; kpyr->ala, 13C-label exchange rate between pyruvate and alanine; kpyr->asp, 13C-label exchange
rate between pyruvate and aspartate; kpyr->lac, 13C-label exchange rate between pyruvate and lactate; kpyr->mal, 13C-label exchange rate between pyruvate and
malate; kpyr->oaa, 13C-label exchange rate between pyruvate and oxaloacate; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; MRI, magnetic resonance
imaging; MRS, magnetic resonance spectroscopy; NAFLD, nonalcoholic fatty liver disease; OAA, oxaloacetate; PC, pyruvate carboxylase; PDH, pyruvate
dehydrogenase; PEP, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; SEM, standard error of the mean; SNR, signal-to-noise ratio; SREBP-1c,
steroid regulatory element-binding protein; TCA, tricarboxylic acid.
From the 1Singapore Bioimaging Consortium, Singapore; 2Clinical Imaging Research Centre, National University of Singapore, Center for Translation Medicine,
Singapore; 3Metabolism in Human Diseases, Institute of Molecular and Cell Biology, Singapore; and 4Department of Biochemistry, Yong Loo Lin School of Medicine,
National University of Singapore, Singapore.
Received April 16, 2012; accepted August 8, 2012.
This study was supported by an intramural funding from the A*STAR Biomedical Research Council.
515
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LEE ET AL.
acquisition times2 and is thus unsuitable for longitudinal studies. The recent development of hyperpolarized
13
C MRS addresses this problem by improving the signal-to-noise ratio (SNR) by more than 10,000-fold,3
making it possible to visualize uptake of 13C labeled
pyruvate in the liver and its subsequent metabolic conversion catalyzed by specific enzymes in real time.4,5
In gluconeogenesis, the conversion of pyruvate into
phosphoenolpyruvate (PEP) in the liver is accomplished
in two enzyme-mediated steps: anaplerosis of pyruvate
into oxaloacetate (OAA) catalyzed by pyruvate carboxylase (PC), followed by conversion of OAA into PEP
mediated by PEP carboxykinase (PEPCK). PEPCK is
commonly considered the control point for liver gluconeogenesis and its overexpression leads to hyperglycemia. However, deletion of PEPCK reduced gluconeogenic flux by only 40%,6 suggesting that PC may play a
more-central role in controlling gluconeogenesis.7
In this study, we investigated the effect of insulin
resistance on in vivo gluconeogenic flux in high-fat
diet (HFD)-fed mice with real-time measurements of
hyperpolarized [1-13C]pyruvate anaplerosis into the tricarboxylic acid (TCA) cycle by PC, as well as its transamination into [1-13C]alanine catalyzed by alanine
transaminase (ALT). We also evaluated the sensitivity
of hyperpolarized 13C MRS in detecting changes in
hepatic glucose production upon pharmacological
intervention. Such capability may facilitate longitudinal assessment of therapeutic response in diabetic drug
development, as well as a better understanding of the
mechanism of action of candidate compounds.
Materials and Methods
Animal Welfare. Starting at weaning age of 3
weeks, male C57/Bl6 mice received either a standard
chow diet with 6.0% (w/w) fat, 47.0% (w/w) carbohydrates, and 18.0% (w/w) protein, with metabolizable
energy of 3.1 kcal/g (Harlan Teklad, Madison, WI), or
an HFD containing 34.9% (w/w) fat, 26.3% (w/w)
carbohydrates, and 26.2% (w/w) protein, with metabolizable energy of 5.2 kcal/g (Research Diets, Inc.,
New Brunswick, NJ), for 24 weeks. Mice on these two
diets are referred to as Chow-fed and HFD, respectively, in this article. All animals were fasted 24 hours
HEPATOLOGY, February 2013
before examination. All procedures involving animals
were approved by the A-STAR Institutional Animal
Care and Use Committee (nos. 080351 and 090428).
Animal Handling. Anesthesia was induced with
2.0% isoflurane mixed with medical air. Body temperature was maintained at 37 C. A catheter was inserted
into the tail vein for intravenous (IV) administration
of the hyperpolarized 13C pyruvate inside the magnetic
resonance imaging (MRI) system. Mice were subsequently sacrificed for measurement of plasma metabolites and liver extraction.
Glucose and Insulin Tolerance Tests. The intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were conducted as described previously.8
Details are available in the Supporting Materials.
Body-Composition Measurement. Body compositions were measured with an EchoMRI 100 (Echo
Medical Systems, Houston, TX). Body fat mass and
body lean mass were measured within 1 minute.
In Vivo Proton MRS Determination of Fat Content in Hepatic Steatosis. Proton MRS measurements
were performed with a 7-T preclinical MRI system
(ClinScan; Bruker BioSpin MRI GmbH, Ettlingen,
Germany). The voxel of interest was a 64-mm3 volume placed in the right hepatic lobe of the liver, with
care taken to ensure exclusion of major blood vessels.
Intrahepatic triglyceride (IHTG) content was expressed
as a percentage of the fat signal peak area with reference to the combined signal with water9 (see Supporting Materials for more details).
In Vivo Measurements of Hepatic Metabolism
With Hyperpolarized 13C. [1-13C]pyruvic acid (40
mg; Cambridge Isotope Laboratories, Cambridge, MA),
mixed with 15 mM of trityl radical (OXO63; GE
Healthcare, Amersham, UK) and a trace amount of Dotarem (Guerbet, Birmingham UK), was polarized and dissolved in a hyperpolarizer (Oxford Instruments, Oxford,
UK), as described previously.10 Hyperpolarized
[1-13C]pyruvate (0.5 mmol/kg body weight) was injected
IV over 3 seconds, and 60 individual liver spectra were
acquired over 1 minute in a 9.4-T preclinical MRI scanner. MRS quantification and analysis protocols are
described in further detail in the Supporting Materials.
Glucagon and Metformin Stimulation. Chow-fed
mice were anesthetized and IV injected with glucagon
Address reprint requests to: Philip Lee, Ph.D., Singapore Bioimaging Consortium, 11 Biopolis Way, #02-01 Helios, 138667 Singapore. E-mail: philip_lee@sbic.
a-star.edu.sg; fax: þ65 64789957.
C 2012 by the American Association for the Study of Liver Diseases.
Copyright V
View this article online at wileyonlinelibrary.com.
DOI 10.1002/hep.26028
Potential conflict of interest: Nothing to report.
Additional Supporting Information may be found in the online version of this article.
HEPATOLOGY, Vol. 57, No. 2, 2013
(20 lg/kg), followed by hyperpolarized 13C MRS
measurements 10 minutes later. This interval was chosen specifically to coincide with the maximal increase
in blood glucose level (Supporting Fig. 1). HFD mice
were treated with metformin (200 mg/kg, once-daily)
for 2 weeks. In vivo measurements of hepatic metabolism were performed before and after treatment.
Ex Vivo Biochemical Enzyme Activity Assays. For
each enzyme-activity assay, 100 mg of liver tissue was
homogenized in 200 lL of ice-cold 100-mM Tris-HCl
buffer, then centrifuged for 10 minutes at 13,000g
to remove insoluble material. All assays were based on
continuous spectrophotometric rate determination.
Details are available in the Supporting Materials.
Statistical Analysis. All statistical analysis was performed with the Graphpad Prism software package
(GrapPad Software, Inc., La Jolla, CA). Data were presented as means 6 standard error of the mean (SEM).
Statistical significance in hyperpolarized 13C metabolite
signal ratios and ex vivo hepatic enzyme activity comparisons were assessed by using a two-tailed unpaired
Student t test. For the correlations between 13Cexchange rates and ex vivo enzyme activities, Pearson’s
product moment was computed, after which the twotailed Student t test was used to test for statistical significance. For the IPGTT, ITT glucose blood tests,
and insulin serum test, two-way analysis of variance,
followed by Bonferroni’s post-tests, were used. The significance limit was set at P < 0.05.
Results
Mice on Prolonged HFD Develop Hepatic Steatosis, Glucose Intolerance, and Insulin Resistance. We
first defined the pathophysiological effect of prolonged
HFD feeding. HFD-fed mice developed hepatic steatosis, with more than an 8-fold higher IHTG level than
Chow-fed mice (Table 1). HFD mice were also hyperglycemic and hyperinsulinemic. Hematoxylin and eosin and Oil Red O histology revealed massive lipid
deposits in the hepatocytes of HFD-fed mice (Supporting Fig. S2). Other physical characteristics
included a higher body weight and body fat composition, together with a lower body lean content. IPGTT
revealed impaired glucose tolerance in HFD mice, as
evidenced by delayed glucose clearance at 45, 60, 90,
and 120 minutes after infusion (Fig. 1A,B). In addition, there was simultaneous compensatory increase in
insulin secretion (Fig. 1C,D). ITT revealed a reduced
blood glucose decrease in HFD mice, compared to
Chow-fed mice (Fig. 1E,F), indicative of insulin resistance in HFD mice. Together, these results show that
LEE ET AL.
517
Table 1. Metabolic Characteristics of HFD-fed mice* (N 5 7
in each group)
Characteristics
Chow
HFD
P Values
Body weight, g
Body fat, %
Body lean, %
Intrahepatic TG, %
Fasting glucose, mmol/L
Fasting insulin, ng/mL
Fed glucagon, pg/mL
Plasma TG, mmol/L
36.6 6 0.9
14.2 6 1.9
81.2 6 1.6
5.0 6 0.5
6.56 6 0.42
0.37 6 0.01
189.6 6 12.3
1.72 6 0.04
56.0 6 0.9
39.9 6 0.8
58.7 6 1.5
42.7 6 5.1
9.25 6 0.13
3.94 6 0.95
266.6 6 27.1
2.06 6 0.05
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.05
<0.01
*N ¼ 7 in each group.
HFD mice were glucose intolerant, insulin resistant,
hyperglycemic, and hyperinsulinemic, clear indications
of pre–type 2 diabetes.11
Elevated Carbohydrate Anaplerosis in Mice With
Hepatic Steatosis as Detected by Hyperpolarized 13C
MRS. We next examined whether metabolic changes
in gluconeogenesis could be detected in vivo with
hyperpolarized [1-13C]pyruvate. Pyruvate is at a major
metabolic junction and generates four metabolite intermediates, each catalyzed by a distinct enzyme or
enzyme complex: lactate by LDH (lactate dehydrogenase); alanine by ALT; acetyl-coA by PDHC (pyruvate
dehydrogenase complex); and oxaloacetate by PC (pyruvate carboxylase). Because of the abundance of LDH
and ALT in the liver, rapid 13C label exchange from
[1-13C]pyruvate to [1-13C]lactate and [1-13C]alanine
rendered the lactate and alanine the two largest metabolite peaks in the MRS spectrum (Fig. 2A). PDH flux
could be assessed by the changes in [1-13C]bicarbonate
levels (Fig. 2A,B). The anaplerotic role of pyruvate was
observed by its conversion into OAA, a vital intermediate metabolite involved in gluconeogenesis and oxidative
phosphorylation. [1-13C]OAA can be rapidly converted
into
[1-13C]phosphoenolpyruvate,
[1-13C]malate,
13
13
[1- C]aspartate, and [6- C]citrate, catalyzed by
PEPCK, malate dehydrogenase (MDH), aspartate transaminase (AST), and citrate synthase, respectively. In the
MRS spectra, we were able to detect [1-13C]malate and
[1-13C]aspartate peaks, consistent with observations in
the perfused mouse liver.4 Because the conversion of
OAA to malate and aspartate are reversible reactions,
there is 13C label exchange between these three metabolites. In addition, reversible dehydration of
[1-13C]malate to [1-13C]fumarate, catalyzed by fumarase, resulted in the repositioning of the 13C label
between the C1 and C4 positions of fumarate. This, in
effect, gave rise to [4-13C]malate, [4-13C]aspartate, and
[4-13C]OAA peaks.4,12 A representative time course displaying the progression of metabolite signals is shown in
Fig. 2B. These results show that the major downstream
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HEPATOLOGY, February 2013
Fig. 1. Impaired glucose tolerance, increased insulin secretion, and reduced insulin sensitivity in HFD mice. (A) HFD mice exhibited glucose
intolerance, compared to Chow-diet fed mice. Mice were 27 weeks old. N ¼ 8 for each group. (B) Glucose AUC (area under curve) calculated
based on data in (A). (C) Plasma insulin levels in HFD (N ¼ 3) and Chow-diet fed mice (N ¼ 10) in IPGTT. (D) Glucose-induced insulin secretion calculated by integrating the AUC after baseline subtraction based on data in (C). (E) ITT revealed that HFD mice (N ¼ 4) exhibited lower
insulin sensitivity than Chow-diet fed control (N ¼ 10). (F) Insulin-induced glucose level in plasma calculated by integrating AAC (area above
curve) based on data in (E). Data are presented as mean 6 SEM. *P < 0.05; **P < 0.01.
pathways of pyruvate can be monitored with hyperpolarized [1-13C]pyruvate.
We next examined the metabolic changes in gluconeogensis in HFD mice. When compared to control
mice,
the
ratios
of
[1-13C]malate/tCarbon,
13
13
[4- C]OAA/tCarbon, [1- C]aspartate/tCarbon, and
[1-13C]alanine/tCarbon were significantly larger in fatty
livers of HFD-fed mice (Fig. 2C), whereas no significant
change in [1-13C]lactate/tCarbon and [1-13C]bicar/
tCarbon ratios was observed, suggesting higher PC and
ALT activities, but not LDH or PDH activities. Consistently, the rate of 13C label exchange between [1-13C]pyruvate and [1-13C]alanine, kpyr->ala, was increased by
more than 70% in HFD-fed mice, whereas no significant change was detected in its exchange with
[1-13C]lactate, kpyr->lac (Fig. 2D). The exchange rate
between [1-13C]pyruvate and [1-13C]malate, kpyr->mal,
which is dependent on both PC and MDH enzyme catalysis, increased significantly in fatty liver. The exchange
between [1-13C]pyruvate and [1-13C]aspartate, kpyr->asp,
mediated by both PC and AST enzymes, was also elevated in the steatotic liver. Exchange rate between
[1-13C]pyruvate and [4-13C]OAA, kpyr->oaa, showed a
more than 3-fold increase in the HFD group, relative to
Chow-fed mice. The corresponding time courses over
60 seconds illustrated the relatively faster production of
these four-carbon metabolites in the steatotic liver (Supporting Fig. S3). Together, the flux measurements showing increased PC activity in HFD-fed mouse livers suggest that PC may be a central player in enhanced
gluconeogenesis in the prediabetic stage.
Increased Malate, Aspartate, and Alanine 13C
Metabolite Signals in Fatty Liver. With the observation that [1-13C]malate and [1-13C]aspartate signals
were significantly increased in fatty liver, we next
sought to understand the mechanism underlying the
changes. Because each pathway involves two mediating
enzymes, PC/MDH and PC/AST, respectively, it is
essential to distinguish each enzyme’s contribution to
the 13C metabolite signal. Ex vivo enzyme-activity
assays of liver extracts obtained from both HFD- and
Chow-fed mice revealed a significant up-regulation of
PC activity in fatty liver (Fig. 3A). However, there was
no apparent increase in AST activity (Fig. 3B),
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519
Fig. 2. Elevated pyruvate anaplerosis in mice with hepatic steatosis is detected by hyperpolarized
13
C MRS. (A) Representation of the
in vivo measured hyperpolarized
13
C spectra in fatty liver. Metabolites that were detected include
(183.0
ppm),
[1-13C]lactate
(181.5
ppm),
[1-13C]malate
(180.2
ppm),
[4-13C]malate
[1-13C]pyruvate hydrate (179.1
ppm), [4-13C]aspartate (177.8
ppm), [1-13C]alanine (176.4 ppm),
(175.6
ppm),
[4-13C]OAA
[1-13C]aspartate (175.0 ppm),
[1-13C]pyruvate (170.8 ppm), and
[1-13C]bicarbonate (160.8 ppm).
(B) Representative time course
depicting the simultaneous production of downstream metabolites
upon infusion of hyperpolarized
[1-13C]pyruvate. (C) Normalized
metabolite
signal
comparison
between mice fed on Chow and
HFD. (D) Corresponding 13C label
exchange rates (N ¼ 8 for each
group). Data are presented as
mean 6 SEM. *P < 0.05; **P <
0.01. Values are displayed in Supporting Table 1.
indicating that the larger [1-13C]aspartate signal was
primarily the result of increased PC activity. Hepatic
MDH activity, on the other hand, was up-regulated in
diabetic mice (Fig. 3C). Therefore, the higher
[1-13C]malate signal could be attributed to a combination of increased PC and MDH activities. This combined effect probably led to increased 13C label
exchange between OAA, malate, and fumarate, thus
contributing to an elevated [4-13C]OAA signal (Fig.
2C). These results further support the critical role of
PC in gluconeogensis in the prediabetic stage.
Another key enzyme in gluconeogenesis, PEPCK,
was concomitantly up-regulated in the insulin-resistant
liver (Fig. 3D). This further corroborated the observation that elevated pyruvate anaplerosis was required to
support the increased hepatic glucose production in
diabetic mice. The higher exchange rate between
[1-13C]pyruvate and [1-13C]alanine indicated faster
transamination, which was confirmed in the biochemical ALT activity assay (Fig. 3E).
Hyperpolarized 13C Metabolic Fluxes as Potential
Biomarkers of Liver Function. We next determined
the potential of hyperpolarized 13C metabolic signals
as relevant diagnostic biomarkers of liver dysfunction
in the diabetic state by examining the relationship
between measured in vivo hyperpolarized 13C exchange
and actual hepatic enzyme activity. First, the exchange
rate between hyperpolarized [1-13C]pyruvate and
[1-13C]alanine, kpyr->ala, correlated with ex vivo hepatic
ALT activity (Fig. 4A). Because ALT is a key regulatory
enzyme in pyruvate recycling and urea production,
in vivo kpyr->ala could be used as an important biomarker of liver dysfunction. Second, faster 13C label
exchange between [1-13C]pyruvate and [1-13C]aspartate, kpyr->asp, correlated well with higher PC activity
in hepatocytes (Fig. 4B). Therefore, kpyr->asp could be
a potential biomarker to reflect in vivo gluconeogenic
flux in the liver. Together, these results demonstrate
that hyperpolarized 13C metabolic signals may be used
as relevant diagnostic biomarkers of liver dysfunction,
such as in diabetes.
Detecting Changes in Liver Metabolism Induced
by Glucagon and Metformin With Hyperpolarized
13
C MRS. To assess the detection sensitivity of hyperpolarized 13C MRS on changes in liver metabolism, we
first examined glucagon-induced glucose production in
Chow-fed animals. Higher aspartate, bicarbonate, and
OAA signals were recorded 10 minutes after IV glucagon injection (Fig. 5A). The corresponding 13C-label
exchanges rates (kpyr->asp, kpyr->bic, and kpyr->oaa) were
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HEPATOLOGY, February 2013
Fig. 3. Up-regulation of gluconeogenic fluxes in fatty liver is accompanied by increased intracellular hepatic enzyme activities. (A) Increased
anaplerotic influx of pyruvate was reflected in the elevated enzyme activity of PC. (B) No significant change was observed in AST activity. (C)
MDH activity was elevated in steatotic hepatocytes. (D) Augmented gluconeogenic enzyme PEPCK activity corroborated the state of increased gluconeogenesis in the insulin-resistant liver. (E) Abnormal liver function in diabetes was evident in the increased ALT flux (N ¼ 8 for each group).
Data are presented as mean 6 SEM. *P < 0.05; **P < 0.01.
also significantly increased (Fig. 5B). Elevated kpyr->asp
and kpyr->oaa are signatures of enhanced hepatic gluconeogenesis (see above), whereas higher kpyr->bic indicates
up-regulated pyruvate dehydrogenase (PDH) activity.13
Conversely, metformin treatment successfully reduced
hepatic gluconeogenesis, as evidenced by the significantly lower malate and aspartate signals, and the abatement of their corresponding exchange rates, kpyr->mal
and kpyr->asp (Fig. 5C,D). Blood glucose level was
decreased by 24% as well (Supporting Table 3). These
results show that hyperpolarized 13C MRS appears to
be sufficiently sensitive for measurement of induced
metabolic changes in the liver.
Discussion
Novelty in Measuring Liver Metabolism In
Vivo. Although it is recognized that glucose homeostasis
maintained by the tissue trio (muscle, liver, and fat) is
disturbed in diabetes,14 it has not been possible to detect
and measure the underlying hepatic metabolic aberrations noninvasively in real time. In this study, we demonstrate, for the first time, the novel use of hyperpolarized 13C MRS to quantify and assess enzyme fluxes
specific to the liver in a type 2 diabetes mouse model in
vivo. By measuring gluconeogenic fluxes, we identify PC
and that its downstream MDH activities are up-
Fig. 4. Correlation between PC and ALT
enzyme activities and the corresponding in vivo
hyperpolarized 13C metabolic flux biomarkers.
Faster [1-13C]pyruvate to (A) [1-13C]alanine,
and (B) [1-13C]aspartate exchanges correlate
to higher intracellular ALT and PC activities,
respectively.
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521
Fig. 5. Hyperpolarized 13C MRS detects
changes in hepatic gluconeogenesis upon
pharmacological intervention. (A) Significantly
higher [1-13C]aspartate and [4-13C]OAA signals and (B) exchange rates were detected 10
minutes after glucagon stimulation. (C) Significantly lower [1-13C]malate and [1-13C]aspartate signals and (D) exchange rates were
detected in metformin-treated mice (N ¼ 5 for
each group). Data are presented as mean 6
SEM. *P < 0.05; **P < 0.01. Values are displayed in Supporting Tables 2 and 3.
regulated and suggest the PC pathway as a critical component in the development of hyperglycemia and
diabetes.
Heightened Gluconeogenesis in Diabetes Drives
Pyruvate Anaplerosis. Through validation with spectrophotometric assays of liver tissue extracts, we demonstrate that in hepatic steatosis, the larger
[1-13C]aspartate metabolite signal may be attributed to
a higher PC flux, whereas the increased [1-13C]malate
signal is a combined effect of increased PC and MDH
activity.
Because
both
[1-13C]aspartate
and
13
[1- C]malate are derived from [1-13C]oxaloacetate
through anaplerosis from [1-13C]pyruvate, and a larger
[4-13C]oxaloacetate pool is indeed detected simultaneously, we hypothesize that the increase in these downstream metabolite pools is a manifestation of elevated
demand for the intermediate OAA, in response to
higher anabolic rates of gluconeogenesis and fatty acid
synthesis (FAS) (Fig. 6). A few factors may contribute
to this phenomenon in fatty liver, as described below.
Insulin resistance.. Insulin insensitivity in the fatty
liver is detrimental to the hormone’s inhibitory role in
gluconeogenesis, primarily through the inactivation of
the phosphatidylinositol 3-kinase/serine/threonine kinase–signaling pathway,15 thereby enfeebling the sup-
pression of key gluconeogenic enzymes PEPCK and glucose-6-phosphatase (G-6-Pase) expression.14 In
addition, previous studies utilizing radioisotopic analysis
also showed that carboxylation of pyruvate into OAA is
up-regulated in the diabetic rat liver, concomitant with
dramatic increases in PC,16 PEPCK, and G-6-Pase15
expression. These studies corroborate our finding that
both PC and PEPCK enzyme activities are increased in
the fatty liver, leading to larger 13C-malate, -aspartate,
and -OAA signals as well as higher rates of chemical
exchange with pyruvate. Indeed, higher hepatic PC activity correlated with increased PEPCK activity (r2 ¼
0.82; P < 0.0001) (Supporting Fig. 4), further supporting the hypothesis that both PC and PEPCK are important regulators in gluconeogenesis.7
Increased glucagon effect.. In diabetes, pathological
alteration of the precise balance between insulin and
glucagon action results in excessive hepatic gluconeogenesis and glycogenolysis, both of which induce
hyperglycemia. Moreover, inadequate suppression of
postprandial glucagon secretion by insulin in the diabetic state causes hyperglucagonemia and evokes elevated HGP, as observed in HFD mice. We previously
reported that combined defects in insulin secretion
and signaling were not sufficient to cause
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HEPATOLOGY, February 2013
Fig. 6. Demand for intermediate metabolite OAA drives higher anaplerotic pyruvate carboxylase flux. Anaplerotic flux through PC is augmented
in fatty liver as a result of increased demand for the intermediate metabolite, OAA. This phenomenon can be attributed to a series of molecular
events triggered by (1) insulin resistance, (2) dominant glucagon effect, and (3) obesity-induced de novo FAS (see text). Metabolites in red
boxes can be detected by hyperpolarized 13C MRS.
hyperglycemia in the absence of dysregulated glucagon
secretion in a mouse model with deletion of calciumsensing protein synaptotagmin-7.17 Indeed, glucagon
plays a major role in promoting gluconeogenesis in
enhancing G-6-Pase activity and PEPCK transcription
in the liver, likely through the protein kinase A–signaling cascade mechanism.18 Thereafter, up-regulated gluconeogenesis increases the demand for OAA. In this
work, we demonstrated up-regulated PC activity in
glucagon-stimulated HGP in Chow-fed animals, as
detected in vivo with hyperpolarized 13C MRS,
through the biomarker kpyr->asp. Concomitantly, glucagon increases PDH activity.19 This technology appears
to possess sufficient sensitivity to detect this phenomenon as well, as evident from the higher kpyr->bic
exchange rate. Treatment with a glucagon-receptor antagonist appears to alleviate HGP in the diabetic
liver,20 and reducing glucagon signaling is being
explored as a potential therapy for diabetes.21 It will
be interesting to measure corresponding changes in hepatic metabolism upon therapeutic intervention with a
glucagon-receptor antagonist in diabetic animals, and
that forms the next phase of our research.
Elevated FAS.. De novo FAS in the liver is regulated
by three known transcription factors: steroid regulatory
element-binding protein (SREBP-1c)22; carbohydrate
response element-binding protein (ChREBP)23; and
peroxisome proliferator-activated receptor gamma.24
Hyperinsulinemia induces hepatic SREBP-1c expression while hyperglycemia stimulates ChREBP activity.
These events lead to transcriptional activation of all lipogenic genes including adenosine triphosphate citrate
lyase, acetyl-CoA carboxylase, and FAS,25 effectively
increasing FAS flux. Because citrate formed in the
TCA cycle and shuttled to the cytosol is the primary
metabolite required in the production of fatty acids,
there is inevitably an increase in demand for this intermediate. Therefore, it is unsurprising that a recent 13C
isotopomer study found a 2-fold increase of hepatic
TCA cycle flux in patients with nonalcoholic fatty liver
disease.26 Because only pyruvate that enters the TCA
cycle through PC produces a net increase in cycle
intermediates, whereas pyruvate entering through
PDH is restricted to energy production only,27 the elevated PC flux and OAA pool observed in diabetic
mice must have also catered to the increased FAS
HEPATOLOGY, Vol. 57, No. 2, 2013
demand. This agrees with our recent observation in
the hypertrophied heart, in which a larger 13C-citrate
signal (from increased pyruvate anaplerosis) was
recorded.28 Moreover, detection of citrate pool with
hyperpolarized [2-13C]pyruvate substrate has recently
been demonstrated to be feasible in the study of myocardial TCA flux29; therefore, similar measurements in
the insulin-resistant liver will undoubtedly aid in validating the hypothesis that an enlarged citrate pool supports FAS. Metformin is used clinically to counter elevated FAS and gluconeogenesis in diabetes, primarily
through its activation of adenosine-monophosphate–
activated protein kinase.30 In this study, we demonstrated that metformin treatment leads to reduced
HGP by, at least in part, decreasing PC activity, as
well as production of malate and aspartate from
pyruvate.
The advent of hyperpolarized 13C MRS has enabled
visualization of real-time metabolism in the in vivo
mouse liver, in particular, the anaplerosis of pyruvate
into the TCA cycle. The distinct patterns in downstream metabolite progression suggest that hyperpolarized 13C MRS is sensitive to subtle differences in metabolic conversions. It is worth noting that LDH-,
ALT-, MDH-, and AST-mediated conversions are reversible. Therefore, the appearance of lactate, alanine,
malate, aspartate, and OAA peaks resulted from the
enzyme-mediated exchange of the hyperpolarized 13C
label, in which equilibrium is dependent on the concentrations of both substrate and product, as well as
the redox potential. Hence, these metabolite signals
reflect the concentration of each metabolite that already exists within the cellular environment and in the
plasma, rather than net metabolite production.31
Nonetheless, the validity of hyperpolarized 13C
exchange rates to investigate metabolic changes in pathology has been demonstrated in cardiac diseases28,32
and cancer.33
Biomarkers
of
Gluconeogenesis:
kpyr->asp,
kpyr->mal. Although both malate and aspartate signals
were increased in the fatty liver, kpyr->asp, rather than
kpyr->mal (data not shown), correlated linearly to PC
activity. In addition, kpyr->asp appeared to be more sensitive in detecting glucagon-induced up-regulation in
gluconeogenesis because it increased significantly upon
glucagon injection. However, with metformin treatment, the changes in gluconeogenesis were large
enough to manifest in both kpyr->asp and kpyr->mal. This
confounding phenomenon warrants further investigation, in particular, the influence of MDH activity. In
addition, from our experience, the metabolite peaks
measured in vivo are generally lower in the fed state
LEE ET AL.
523
than fasted, in part because of the abundance of other
alternative metabolic substrates in the circulation (e.g.,
unlabeled pyruvate), which limits the uptake of the
infused hyperpolarized 13C-labeled pyruvate. This observation is observed in the lower metabolite peaks in
the spectrum of fed mice (Supporting Fig. 3A).
In summary, we demonstrate the application of
hyperpolarized 13C MRS in probing metabolic events
in the liver and its correlation with enzyme activities.
We identify an important role of the PC pathway in
the development of hyperglycemia and diabetes. We
also demonstrated the capability of this technique to
probe changes in hepatic metabolism upon therapeutic
intervention, paving the way for longitudinal assessment. The significant correlation between 13C
exchange rates and hepatic enzyme activities illustrates
the potential of these indices as biomarkers of liver
function in diabetes and a wide range of other
diseases.
Acknowledgment: The authors thank Dr. Hongyu
Li for technical assistance in the glucose and insulin
tolerance tests.
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