Download Targeted Disruption of the SUCNR1 Metabolic Receptor

1154
Diabetes Volume 64, April 2015
Kenneth J. McCreath,1 Sandra Espada,1 Beatriz G. Gálvez,1 Marina Benito,2,3
Antonio de Molina,4 Pilar Sepúlveda,5 and Ana M. Cervera1,5
Targeted Disruption of the
SUCNR1 Metabolic Receptor
Leads to Dichotomous Effects on
Obesity
METABOLISM
Diabetes 2015;64:1154–1167 | DOI: 10.2337/db14-0346
A number of metabolites have signaling properties by
acting through G-protein–coupled receptors. Succinate,
a Krebs cycle intermediate, increases after dysregulated energy metabolism and can bind to its cognate
receptor succinate receptor 1 (Sucnr1, or GPR91) to activate downstream signaling pathways. We show that
Sucnr1 is highly expressed in the white adipose tissue
(WAT) compartment of mice and regulates adipose
mass and glucose homeostasis. Sucnr12/2 mice were
generated, and weight gain was monitored under basal
and nutritional stress (high-fat diet [HFD]) conditions.
On chow diet, Sucnr12/2 mice had increased energy
expenditure, were lean with a smaller WAT compartment, and had improved glucose buffering. Lipolysis
measurements revealed that Sucnr12/2 mice were released from succinate-induced inhibition of lipolysis,
demonstrating a function of Sucnr1 in adipose tissue.
Sucnr1 deletion also protected mice from obesity on
HFD, but only during the initial period; at later stages,
body weight of HFD-fed Sucnr12/2 mice was almost
comparable with wild-type (WT) mice, but WAT content
was greater. Also, these mice became progressively
hyperglycemic and failed to secrete insulin, although
pancreas architecture was similar to WT mice. These
findings suggest that Sucnr1 is a sensor for dietary
energy and raise the interesting possibility that protocols to modulate Sucnr1 might have therapeutic utility
in the setting of obesity.
G-protein–coupled receptors (GPCRs) constitute the major
and most diverse group of membrane receptors in
eukaryotes (1). Found in the plasma membrane, these
receptors are tasked with the recognition and transmission of messages from the external environment. An everincreasing number of GPCRs are now being identified as
receptors for metabolites or energy substrates (2),
expanding the repertoire of biological targets to diseases
associated with the metabolic syndrome, including hypertension and type 2 diabetes (3). Metabolites such as lactate (4), ketone bodies (5), and a-ketoglutarate (6),
known primarily to serve functions distinct to signaling,
have been shown to act as ligands for GPCRs. Representative of this new class of receptor is Gpr91, a receptor for
the Krebs cycle intermediate succinate, now termed
Sucnr1 for succinate receptor (7). The Krebs, or citric
acid, cycle occurs at the junction between glycolysis and
oxidative phosphorylation (8) and is a key component of
aerobic metabolism by providing reducing equivalents for
the electron transport chain. Interestingly, succinate has
been known for many years to increase in tissues during
hypoxia (9–11) and may act as a surrogate marker for
reduced oxygenation. Accordingly, Sucnr1 may be activated in times of hypoxic stress such as ischemic injury
in liver (12) or retina (13) or during cardiomyocyte crisis
(14). Moreover, dysregulated metabolism, akin to diabetes
or the metabolic syndrome, also has the potential to increase peripheral blood concentrations of succinate to
1Department
Corresponding author: Ana M. Cervera, [email protected] or amcerveraz@
gmail.com.
of Cardiovascular Development and Repair, Fundación Centro
Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain
2Advanced Imaging Unit, Department of Atherothrombosis, Imaging, and Epidemiology, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III,
Madrid, Spain
3CIBER de Enfermedades Respiratorias, Madrid, Spain
4Comparative Medicine Unit, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain
5Regenerative Medicine and Heart Transplantation Unit, Instituto de Investigación
Sanitaria La Fe, Valencia, Spain
Received 28 February 2014 and accepted 14 October 2014.
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0346/-/DC1.
© 2015 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and
the work is not altered.
diabetes.diabetesjournals.org
levels where activation of the Sucnr1 receptor will occur
(15,16). Indeed, the low levels (;2–80 mmol/L) of succinate found in blood plasma (15–18), in comparison with
that required for activation (in the region of 180 mmol/L
[2]), would suggest that Sucnr1 functions more as a sensor
of metabolic (or oxidative) damage (19) rather than
a physiological mediator of signaling.
Mounting evidence points to an important role for
Sucnr1 during periods of metabolic dysfunction, such as
diabetes-related hypertension, oxygen-induced retinopathy, and the metabolic syndrome (19). Interestingly,
obesity is a key component of many metabolic-related
diseases (20) and can lead to diabetes, hyperlipidemia,
and cardiovascular disease (20,21). The effect of Sucnr1
deficiency on body composition and metabolism has not
previously been studied. We reasoned that Sucnr1 could
be a potential modulator of metabolism since a recent
study indicated that this receptor participates in the
breakdown (lipolysis) of stored triglycerol (TG) in fat
(22). To test this hypothesis, we analyzed the expression
of Sucnr1 in mouse adipose tissue and observed high
expression in the white adipocyte compartment. To gain
insight into the (patho)physiological role of Sucnr1, we
created mice deficient for this gene using a knockout
(KO)-first approach. We show that deficiency of the
Sucnr1 gene has metabolic consequences for weight gain
and glucose homeostasis. Sucnr1 KO mice have a modest
lean phenotype under standard diet (SD) conditions, correlating with a reduction in the size of white adipose
tissue (WAT) depots, increased energy expenditure, and
an improved glucose clearance rate. Challenge with highfat diet (HFD) also resulted in a significant decrease in
body weight in Sucnr1 KO mice, but only during initial
periods. At later periods, body weight of Sucnr1 KO mice
was indistinguishable from wild-type (WT) littermates,
but WAT content was increased. This appeared to coincide
with a hyperglycemic profile and a reduction in insulin
secretion from the pancreas. Collectively, our results
point to a potentially important role for Sucnr1 in metabolic disease related with obesity.
RESEARCH DESIGN AND METHODS
Generation of Sucnr1-Mutant Mice, Feeding Regimes,
and Indirect Calorimetry
To create Sucnr1-mutant mice, we obtained two Sucnr1targeted embryonic stem (ES) cell clones from the European
Mouse Mutagenesis Consortium (EUCOMM), designated as
KO-first, conditional-ready ES cells (23). This flexible system
allows both for gene-trap (GT) reporter constructs and also
for conditional and null alleles to be generated by exposure
to the site-specific recombinases Cre and flippase (www
.knockoutmouse.org). ES cells were injected into C57BL/6
blastocysts, and chimeric males were bred to C57BL/6
females to produce germ line transmission. Heterozygous
animals were intercrossed, and genotyping of F2 progeny
detected homozygous mice at the expected Mendelian ratio.
Genotyping was performed after weaning by PCR assay
McCreath and Associates
1155
with genomic DNA extracted from tail biopsies using the
REDExtract-N-Amp Tissue PCR kit (Sigma-Aldrich, Saint
Louis, MO). Genotyping primers were primer 1, 59-ACTA
TAAGCATCACTTCACCACTTCC-39; primer 2, 59-CAACGG
GTTCTTCTGTTAGTCC-39; and primer 3, 59-AAGAACAA
ATGGTAGAACAGTCACGG-39. A PCR product of ;500
nucleotides (nt) was obtained from WT DNA, whereas
mutant DNA produced an amplification product of
;320 nt in size. These KO-first mice were designated GT
mice. In order to remove the critical exon of the Sucnr1 gene,
together with the neomycin cassette, GT mice were crossed
with Sox2-Cre mice (supplied by Dr. Miguel Torres, Centro
Nacional de Investigaciones Cardiovasculares [CNIC]) to produce whole-body KO mice. Genotyping was performed
as before using the primer 4, 59-ACTTCCAGTTCAACAT
CAGCCGCTACA-39; primer 5, 59-GTGATTCATCTGTATTAT
TAGATGAGC-39; and primer 6, 59-CAGCACAACCATCAGA
GAAACAATGGACTC-39. A PCR product of ;650 nt indicated that recombination had occurred to produce a KO
locus, and a product of ;300 nt identified a WT locus. All
experiments were carried out using these KO mice.
Mice were housed at the CNIC animal facility under
pathogen-free conditions. Male mice were used for all
studies. Mice were given ad libitum access to food and
water unless otherwise noted in experimental methods.
Environmental conditions were controlled to 12/12-h
light/dark cycles. Animal studies were approved by the
local ethics committee. All animal procedures conformed
to European Union Directive 86/609/EEC and recommendation 2007/526/EC regarding the protection of animals
used for experimental and other scientific purposes,
enacted under Spanish law 1201/2005. Male Sucnr1 KO
mice and WT mice (5–6 weeks old) obtained from heterozygous mating were fed an SD of rodent chow, containing
13.4% fat (16% energy; Diet 5K67, LabDiet, Saint Louis,
MO) or an HFD containing 34.9% fat (60.9% energy;
Diet 58Y1, TestDiet, Saint Louis, MO). Body weight
was recorded weekly. Separate cohorts of animals on
SD and HFD were used for metabolic cage analysis using
a 16-chamber indirect calorimetry system (PhenoMaster;
TSE Systems, Bad Homburg, Germany). Mice were individually housed in the chambers for 5 days; the first
2 days were used for acclimatization, and data were analyzed from the last 3 days. Oxygen consumption rate,
carbon dioxide production rate, feeding, and total locomotive activity were measured concurrently for each
mouse.
MRI and Spectroscopy Analysis
Imaging was performed in the Advanced Imaging Unit at
CNIC. Mice on SD or HFD were preanesthetized by
inhalation of isoflurane/oxygen (2–4%) and monitored by
electrocardiogram and breathing rhythm (Model 1025; SA
Instruments, Inc., NY). Animals were positioned supine in
a customized bed with a built-in nose cone supplying inhalatory anesthesia (1–2%) and kept at 35–37°C by a warm
airflow. Images were acquired with an Agilent/Varian 7
1156
SUCNR1 Effects on Obesity
Tesla system equipped with a DD2 console and an active
shielded 205/120 gradient insert coil with 130 mT/m maximum strength and a volume coil (Rapid Biomedical GmbH,
Rimpar, Germany) for transmit/receive. For MRI, data were
collected with a two- and three-dimensional (3D) spin-echo
sequence. T1 weighted two-dimensional images (repetition
time 380 ms, echo time 13 ms, number of averages 4) were
acquired to visualize the fat qualitatively in high resolution.
3D T1 weighted spin-echo acquisition was also acquired
to calculate the body fat–to–total body weight ratio using
100 ms repetition time, 8 ms echo time, 1 average. In both
sequences, the field of view was 90 3 45 3 45 mm3 and
the reconstruction matrix was 256 3 256 3 256. For
MRS, the 13C and 1H surface coil was placed over the
epididymal fat. 13C spectra were acquired with a repetition time of 1,000 ms, 512 accumulations, and a spectral
width of 18 kHz. The 1H spectra were acquired with 1,000
ms of repetition time, 256 scans, and a spectral width of
10 kHz. Images were processed using OsiriX (Pixmeo,
Geneva, Switzerland). Quantification of fat composition
from 3D images was estimated applying a semiautomatic
image thresholding segmentation plug-in included in
the software and referenced to the total (body) volumes
calculated by the same approach. 1H and 13C spectra
were processed with MestReNova (Mestrelab Research,
Santiago de Compostela, Spain). An exponential line broadening (20 Hz for 13C spectrum and 3 Hz for 1H spectrum)
was applied before Fourier transformation. Individual
contributions of the signals from carbonylic, olefinic,
methylene, and methyl moieties were quantified by integration after automatic baseline correction. The same process was applied in 1H spectrum. The areas under each
peak were used to calculate the fractional contribution
of saturated (130 ppm), monounsaturated (130 and
128 ppm), and doubly (poly) unsaturated (128 ppm) fatty
acids, taking as a reference the area under the carbonylic
peak (24). The areas under the peak in the 1H spectrum,
total lipids (1.3 ppm) and water peak (4.7 ppm), were used
to calculate the percentage of total lipids with respect to
water content (25).
Ex Vivo Lipolysis Assay
Mouse epididymal adipose tissue was dissected, weighed,
divided into equal pieces, and suspended in 400 mL KrebsRinger bicarbonate buffer (Sigma-Aldrich) containing 1%
fatty acid-free BSA (Sigma-Aldrich). The tissue samples
were treated with either PBS or 25 nmol/L isoproterenol
or pretreated with 100 mmol/L succinate (for 10 min) or
100 ng/mL pertusis toxin for 4 h before isoproterenol
treatment. Samples were incubated on a shaker at 37°C,
and glycerol release was measured using a Glycerol Determination kit (Cayman Chemical Company, Ann Arbor, MI).
In Vivo Lipolysis Assay
After an overnight fast, mice were injected intraperitoneally
with succinate (1.2 mg/kg) or the adenosine A1 receptor agonist (R)-N6-(2-phenylisopropyl)-adenosine (PIA; 0.15 nmol/g).
Diabetes Volume 64, April 2015
Blood was collected before and 15 min after injection.
Serum nonesterified fatty acid (NEFA) was measured
using the NEFA-HR(2) kit from Wako Chemicals
(Richmond, VA).
Tissue Collection and Blood Analysis
At the end of the study, mice were killed following an
overnight fast. Blood samples were collected by cardiac
puncture, and serum was separated by centrifugation.
Total serum cholesterol, triglycerides, free fatty acids,
hepatic aspartate aminotransferase (AST), and alanine
aminotransferase (ALT) were determined using an automated analyzer (Dimension RxL Max Integrated Chemistry
System; Siemens, Munich, Germany) and commercially
available kits from Siemens (total cholesterol, triglycerides,
ALT, and AST) and Wako Chemicals (free fatty acids).
Diabetes-related biomarkers in serum were analyzed using
the Bio-Plex Pro Mouse Diabetes standard 8-plex and
adiponectin kits (Bio-Rad Laboratories Inc., Berkeley, CA).
Selected tissues were collected and weighed. Tissues
were fixed in neutral buffered formalin solution and
processed for histological analysis or snap frozen in
liquid nitrogen and stored at 280°C for RNA and protein extraction.
Histological Analysis
Tissues were fixed in 10% buffered formalin (VWR International, Radnor, PA), embedded in paraffin, and sectioned at 5 mm. For morphometric analysis, sections were
stained with hematoxylin and eosin according to standard
protocols. Adipocyte size (from WAT) and islet cell area and
cell number (from pancreas) were determined with ImageJ
software (National Institutes of Health). Sections from six
animals per group were used, and at least 500 adipocytes
per animal were measured from multiple fields. Islet cell
area was quantified from at least four sections per pancreas
and expressed as the percentage of total pancreas area. Islet
cell density was assessed by counting the number of nuclei
in a 1,720 mm2 area from the same sections. Additional
sections were subjected to heat-induced epitope retrieval
in citrate-buffer, pH 6, followed by blocking of endogenous
peroxidase activity with hydrogen peroxide prior to the
immunostaining. Sections were incubated overnight with
antibodies specific for Sucnr1 (Novus Biologicals, Littleton,
CO), insulin (Cell Signaling Technology Inc., Danvers, MA),
glucagon (Abcam plc, Cambridge, U.K.), Ki67 (Master Diagnostica, Granada, Spain), or caspase-3 (R&D Systems Inc.,
Minneapolis, MN). For Sucnr1 immunodetection, a tyramide
signal amplification kit (Molecular Probes; Thermo Fisher
Scientific, Waltham, MA) was used. For insulin/glucagon
double staining, we used fluorescence-labeled secondary
antibodies. Insulin, Ki67, and caspase-3 were detected
using an automatic immunostainer (Dako Autostainer
Plus; Dako, Glostrup, Denmark) with a horseradishperoxidase-labeled polymer anti-rabbit as secondary
antibody (EnVision-HRP; Dako) and the chromogenic
substrate DAB (Dako), followed by counterstaining
with Mayer’s hematoxylin.
diabetes.diabetesjournals.org
Western Blot Analysis
For examination of phospho-Akt in WAT, skeletal muscle,
and liver samples, fasted mice were injected intraperitoneally with saline or insulin (1.5 units/kg body mass).
Extracts were prepared at 10 min postinjection as described (26). Briefly, tissues were homogenized in 500 mL
ice-cold lysis buffer (1% Triton X-100, 150 mmol/L NaCl,
10 mmol/L Tris-HCl pH 7.5, 1 mmol/L EDTA, 1% Nonidet
P-40, and complete mini protease inhibitor cocktail; Roche,
Mannheim, Germany) with a mechanical homogenizer, and
debris was cleared by centrifugation at 10,000g at 4°C for
10 min. The supernatant was collected and the quantity of
protein was measured with the BCA kit (Thermo Fisher
Scientific). Protein extracts (40 mg of protein) were examined by protein immunoblot analysis by probing with antibodies to Akt and phospho-Ser473 Akt (Cell Signaling
Technology Inc.). Immunocomplexes were detected with
the enhanced chemiluminescence reagent (Amersham, GE
Healthcare Bio-Sciences AB, Uppsala, Sweden).
Glucose Tolerance Test and Insulin Tolerance Test
For the intraperitoneal glucose tolerance test (IPGTT), mice
were fasted overnight (16–18 h). Conscious mice were
then injected intraperitoneally with glucose (1.5 g/kg
body weight). Blood glucose was monitored in samples
obtained from tail punctures before and after glucose
administration using a handheld glucometer (Ascencia
Elite; Bayer AG, Leverkusen, Germany). To measure insulin levels in response to glucose, blood was collected
at 0 and 10 min postinjection through the submaxilar
vein, and the resulting serum was used to measure insulin by ELISA (Millipore Corporation, Billerica, MA).
For the intraperitoneal insulin tolerance test (IPITT),
mice were fasted for 2 h and injected with insulin
(0.75 units/kg).
Flow Cytometry Analysis, Stromal Vascular Fraction
Isolation, and Preadipocyte Differentiation
The stromal vascular fraction (SVF) was isolated from
WAT by incubating the tissue for 1 h at 37°C in Krebs
buffer (1 g tissue/3 mL solution) containing 2 mg/mL
collagenase A (Roche Diagnostics, Basel, Switzerland) and
20 mg/mL fatty acid–free BSA (Sigma-Aldrich). Digested
tissue was centrifuged at 250g, and the SVF pellet and
adipocyte fractions were collected for RNA extraction.
For preadipocyte differentiation, the same procedure was
followed using subcutaneous WAT. Once the SVF fraction
was collected, SVF cells were differentiated to adipocytes ex
vivo using a modification of a published protocol (27).
Cells were plated (50,000 cells/cm2) and maintained in
culture medium (DMEM/F12, 8% FBS, 2 mmol/L L-glutamine,
50 units/mL penicillin, and 50 mg/mL streptomycin) for
5 days, and differentiation was induced by incubating with
culture medium supplemented with 5 mg/mL insulin, 1
mg/mL dexamethasone, 25 mg/mL isobutylmethylxanthine,
and 1 mmol/L rosiglitazone. After 2 days, cells were incubated for an additional 2 days in culture medium containing
insulin and rosiglitazone only and were then maintained in
McCreath and Associates
1157
culture medium supplemented with only insulin for the
remaining duration of differentiation (a further 4 days).
For flow cytometry analysis of adipocytes, the same enzyme dissociation procedure was followed for epididymal
WAT digestion. Then adipocytes were recovered by centrifugation (250g, 10 min, 4°C), collected in 1 mL of icecold PBS containing 0.25 mL of Draq5 (BioStatus Limited,
Leicestershire, U.K.) to stain the cells, and analyzed on
a LSR Fortessa flow cytometer (Becton Dickinson, San
Jose, CA). Data were analyzed with FlowJo 1.6 software.
Triglyceride Measurement
Hepatic and WAT triglyceride content was measured using
tissue from mice fasted overnight as previously described
(28). Briefly, total lipids were extracted from samples
(50 mg) by using an 8:1 mixture of chloroform and methanol
(4 h). The extracts were mixed with 1 N sulfuric acid and
centrifuged (10 min). Triglyceride content was measured
with a kit purchased from bioMérieux Inc. (Marcy l’Etoile,
France). Oil Red O staining and measurement of intracellular triglyceride levels were performed as previously described (27). Fixed cells or frozen liver sections (10 mm)
were stained with Oil Red O (Sigma-Aldrich) for 15–20 min
at room temperature. For quantification, cell monolayers
were washed extensively with water to remove unbound
dye, and 1 mL of isopropyl alcohol was added to the
culture dish. After 5 min, the absorbance (510 nm) of
the extract was measured by spectrophotometry.
Total Pancreatic Insulin Content
Total pancreatic insulin content was measured as described (29). Pancreata were harvested from fed mice,
Polytron homogenized (30 s) on ice in 5 mL cold 2 N
acetic acid, boiled for 5 min, transferred to ice, and centrifuged at 15,000g and 4°C for 15 min. Supernatant
extracts were then assayed for protein content and insulin
content (ELISA, Millipore Corporation).
RNA Isolation and Gene Expression Analysis
Total RNA extraction, cDNA synthesis, and quantification were performed by standard methods. The expression of mRNA was examined by quantitative reverse
transcription–polymerase chain reaction (qRT-PCR) using a 7900 Fast Real Time thermocycler and Fast SYBR
Green assays (Applied Biosystems, Thermo Fisher Scientific). Three technical replicates were performed per sample, and relative expression was normalized against
mouse Srp14. Primer sequences are given in Supplementary Table 1.
Statistical Analysis
Calculations were performed using Biogazelle qBase+ and
GraphPad Prism 5.0 software (San Diego, CA) and reported as
mean 6 SEM. MRI and MRS data were reported as mean 6
standard deviation. Statistical comparisons were made using
repeated-measures ANOVA with Bonferroni posttests for
comparisons between groups at individual time points and
Student t test for independent samples, as appropriate. Significance was established as a value of P , 0.05.
1158
SUCNR1 Effects on Obesity
RESULTS
Sucnr1 Is Highly Expressed in WAT
Gene transcription analysis of C57BL/6 WT mouse tissues
revealed high Sucnr1 expression in kidney and liver, with
little or no expression in lung tissue (Fig. 1A). These
results are consistent with previous work demonstrating
high Sucnr1 expression in kidney medulla (7) and also hepatic stellate cells (12). Interestingly, significant expression
was also detected in WAT from epididymal fat (Fig. 1A),
ratifying the recent grouping of this gene to an adipose
cluster in the adult mouse (22). Analysis of fractionated
WAT demonstrated Sucnr1 expression in the mature adipocyte but not the stromal cell fraction, which contains
preadipocytes (Fig. 1B). In contrast to WAT, Sucnr1 expression was minimal from brown adipose tissue (BAT)
deposits (Fig. 1C).
Generation of Sucnr1GT/GT and Sucnr12/2 Mice
To investigate the in vivo function of Sucnr1, we generated
Sucnr1-mutant mice by using KO-first targeted ES cell clones
obtained from EUCOMM. The targeting vector, directed to
intron 1 of the Sucnr1 gene (Fig. 2A), contained a splice
acceptor (SA) module from the engrailed-2 (En2) gene, followed by an internal ribosome entry site, which directs
translation of a fusion reporter protein with b-galactosidase
(LacZ), which, in turn, is dependent on transcription of the
endogenous Sucnr1 promoter. A separate transcription unit
contained a promoter-driven neomycin gene for antibiotic
selection. These two modules were flanked by flippase recognition target sites, permitting flippase-mediated recombination. Additionally, exon 2 of the gene, which was included
in the targeting vector, was flanked by LoxP sites, allowing
conditional ablation of the exon following Cre-mediated recombination (30). A third LoxP site bordered the neomycin
cassette (Fig. 2A), ensuring its removal with exon 2. Thus,
depending on mating regimes, either whole-body KO mice
or conditional KOs can be produced (23). In this work, we
describe only results from the GT and whole-body KO mice.
Two targeted ES cells clones were injected into mouse
blastocysts, and germ line transmission was confirmed
Diabetes Volume 64, April 2015
after mating. All mice were on a C57BL/6 background.
Mouse genotypes were evaluated by PCR using genomic
DNA from tail biopsies, with primers designed to detect
correctly targeted events (Fig. 2B, primers 1, 2, and 3).
Homozygous offspring from heterozygous Sucnr1GT/+
male and female intercrosses were viable and exhibited
normal development. Whole-body KO mice, distinct
from GT mice by the complete removal of exon 2, were
produced following a crossing regimen with Sox2-Cre
mice. Removal of exon 2 and the neomycin module was
confirmed by PCR using primers 4, 5, and 6, giving the
expected amplification products for WT and KO (Fig. 2C).
Because intronic insertions of GT vectors may allow normal mRNA splicing to some degree, we analyzed endogenous Sucnr1 levels in GT mice to determine whether
residual transcripts were expressed. Using primers e1
and e2, which bridge exons 1–2 (Fig. 2A), PCR amplification of cDNA from different tissues of WT mice yielded
a single product with the expected size of ;190 nt
(Fig. 2D). Unexpectedly, amplification of cDNA from
Sucnr1GT/GT mice with the same primer pair yielded
a larger single product of 300 nt, with no detectable WT
Sucnr1 expression (Fig. 2D, left panel) suggesting that the
SA is not skipped. Introducing an internal ribosome entry
site–specific reverse PCR primer (p7), together with primer
e1, resulted in the amplification of an intermediate-sized
fragment (220 nt), consistent with the proper functioning
of the SA module (Fig. 2D, middle panel). PCR across exons
1–2 in Sucnr1KO/KO (Sucnr1 KO) mice failed to amplify
a reaction product, consistent with the loss of exon 2
(Fig. 2D, right panel). To determine the origin of the unanticipated 300 nt product in Sucnr1GT/GT mice, and exclude the possibility of PCR artifacts, reactions were
performed on mRNA from different tissues of mice, and
the products were sequenced. Results from sequencing determined precisely in all cases that the fusion transcript
generated by the proper joining of exon 1 to the SA sequence had caused activation of a cryptic splice donor site
located 115 nt downstream in the En2 sequence, which
spliced to the SA of Sucnr1-exon 2 (Fig. 2E). This splicing
Figure 1—Sucnr1 tissue distribution. A: Relative expression of Sucnr1 in mouse tissues, determined by qRT-PCR. Expression levels (log10)
were relative to that in lung tissue arbitrarily chosen as 1 (n = 3 mice). B: Relative expression of Sucnr1 in the SVF and adipocytes isolated
from epididymal adipose tissue, examined by qRT-PCR. Adiponectin and macrophage scavenger receptor 1 served as positive controls for
adipocyte and stromal fractions, respectively (n = 4 mice). C: Relative expression of Sucnr1 in WAT and BAT compartments, examined by
qRT-PCR (n = 3 mice). Data represent mean 6 SEM. ADIP, adipocytes; Adp, adiponectin; H, heart; K, kidney; Li, liver; Lu, lung; Msr1,
macrophage scavenger receptor 1.
diabetes.diabetesjournals.org
McCreath and Associates
1159
In accord with mRNA expression, strong and homogenous b-galactosidase staining was observed throughout
WAT from KO mice but not WT littermates (Fig. 2F, top
panels), indicating robust b-galactosidase activity in this tissue driven by the endogenous Sucnr1 promoter. Subsequently, immunofluorescence analysis of paraffin-embedded
WAT sections revealed that Sucnr1 is present in the plasma
membrane of WT adipocytes but is not detected in equivalent samples of KO mice (Fig. 2F, bottom panels). Collectively, these results identify the white adipocyte as a
significant reservoir of Sucnr1 expression and suggest that
it might have a functional role in adipocyte metabolism.
Sucnr1 Deficiency Results in Release From SuccinateInduced Inhibition of Lipolysis
Figure 2—Generation of Sucnr1-deficient mice. A: Diagram of the
EUCOMM targeting vector including the LoxP-flanked exon and
Sucnr1 WT locus. Primers for genotyping correct targeting events
are shown by large arrows (1–7); exon-spanning primers are shown
by small arrows. Black boxes represent exons. B and C: PCR analysis of GT, KO, and WT alleles. D: Amplification of exons from
mRNA produced from liver, kidney, and WAT of WT, GT, and KO
mice. Larger product amplified with primers e1+e2 was extracted
for sequencing analysis. E: Schematic representation of alternative
splicing around the KO-first cassette. Correct splicing is denoted by
a solid line; the alternative transcript is denoted by a broken line (not
to scale). F: Representative b-galactosidase staining of intact adipose tissue from WT and KO mice (2003 magnification) demonstrating high LacZ expression in KO mice, which was absent in WT
mice. WAT was counterstained with nuclear fast red; immunohistochemical staining of WAT (2003 magnification) with an antibody to
SUCNR1 confirms its expression in WT but not KO tissue. ACT,
actin; FRT, flippase recognition target; IR, internal ribosome entry
site; NEO, neomycin.
resulted in a frameshift with premature stop codons within
exon 2, predicting termination of a truncated Sucnr1 translation product. Curiously, this cryptic splice element in the
En2 gene has been previously described in the literature
(31,32), and it seems rather puzzling that a large-scale genetic KO platform would use the En2-SA unit with a documented alternative splicing phenomenon. Because of this
incongruity, only KO mice were characterized further.
Hydrolysis of fat stored as TG in adipose tissue, termed
lipolysis, is tightly controlled by hormones and metabolites that are secreted according to nutritional status (33).
Enzymatic breakdown of TG occurs through a series of
steps (34) to release one molecule of glycerol and three
molecules of fatty acid. Released fatty acids enter the
circulation and can be taken up and oxidized by peripheral
tissues. Interestingly, succinate has been shown to inhibit
lipolysis in isolated adipose tissue, presumably through
engagement with Sucnr1. To investigate whether lipolysis
is perturbed in Sucnr1 KO mice, we dissected WAT from
epididymal fat regions and measured lipolytic activity in
explant tissue. Glycerol release, as a metric of lipolysis,
was similar in WT and Sucnr1 KO mice, both under basal
and b-adrenergic agonist (isoproterenol)-stimulated conditions (Fig. 3A). In accord with previous findings (22),
addition of succinate (100 mmol/L) induced an inhibition
of isoproterenol-stimulated glycerol release in WAT explants from WT mice, and pretreatment of adipocytes
with pertussis toxin blocked the antilipolytic effect of
succinate (Fig. 3B). As anticipated, inhibition of lipolysis by
succinate was absent in equivalent WAT from Sucnr1 KO
mice (Fig. 3C). Moreover, intraperitoneal injection of
succinate (1.2 g/kg) provoked a significant decrease in
serum levels of NEFA in WT mice, whereas the decrease
in Sucnr1 KO mice was not significant (Fig. 3D). Further,
intraperitoneal injection of the adenosine A1 receptor agonist PIA could inhibit lipolysis both in WT and Sucnr1
KO mice (Fig. 3E), demonstrating that the response to
general GPCR-mediated antilipolysis was not perturbed
in Sucnr1 KO mice. Taken together, these results establish
that succinate exerts its antilipolytic actions through
Sucnr1 coupled to Gi-type G proteins.
Disruption of Sucnr1 Results in a Moderately Lean
Phenotype With Reduced WAT Compartments
Given that expression of Sucnr1 in WAT alluded to an
involvement in energy homeostasis, we assessed weight
gain in WT and Sucnr1 KO mice on an SD. Total body
weight did not differ at weaning (13.1 6 1.1 g for male WT
vs. 13.2 6 0.6 g for male KO; n = 8), and both genotypes
displayed similar weight gains, although a discernible
trend for decreased weight gain in Sucnr1 KO mice was
1160
SUCNR1 Effects on Obesity
Diabetes Volume 64, April 2015
Figure 3—Lipolysis, body weight measurement, fat distribution, and adipocyte content of Sucnr1-deficient mice A: Measurement of
lipolysis in WAT explants from Sucnr1-deficient mice (KO) and WT littermates. Lipolysis was determined by measuring glycerol released
from adipocytes in the absence (–) or presence (+) of 25 nmol/L isoproterenol for 4 h (n = 4 mice per group). Results are expressed as mg
glycerol per liter released per mg tissue. B: WAT explants of WT mice were pretreated or not for 4 h with pertussis toxin (100 ng/mL21) prior
to the addition of succinate (100 mmol/L), followed 10 min later by isoproterenol (25 nmol/L). Lipolysis was determined by glycerol release
as in A. A representative experiment from four is shown. C: Lipolysis was measured in WAT explants from Sucnr1-deficient and WT
littermates. Explants were incubated with isoproterenol (25 nmol/L) plus/minus succinate (100 mmol/L), and glycerol release was determined after incubation for 3 h. Results show the percentage of activity with succinate (n = 4 mice per group). D: Fasted mice were injected
intraperitoneally with succinate (1.2 g/kg). Serum NEFA was measured before and 15 min after injection (n = 6 mice per group). E: Fasted
mice were injected intraperitoneally with PIA (0.15 nmol/g). Serum NEFA was measured before and 15 min after injection (n = 6 mice per
group). F: Body weight changes in male Sucnr1-deficient mice (KO) and WT littermates fed an SD for 16 weeks (n = 8–12 mice per group).
G: Body weights of male Sucnr1-deficient mice (KO) and WT littermates fed an SD for 50 weeks (n = 8–12 mice per group). H: WAT weight
measurements after 20 weeks on diet (n = 8–12 per group). I: Total fat weight taken as a percentage of body weight after 20 weeks on diet
(n = 8–12 per group). J: NMRI analysis of body fat content and quantification of several measurements (n = 7 mice per group). Results were
expressed as proportion of fat volumes vs. total mouse volume (fat volume/total volume). K: Lipid quantification. 1H spectra of abdominal
compartments from WT and Sucnr1 KO mice (n = 7 mice per group). Areas under the curve were used to calculate the portion of lipids with
respect to water content. L: Representative hematoxylin and eosin–stained sections (2003 magnification) of paraffin-embedded epididymal
WAT from mice after 18 weeks of diet and average adipocyte size (n = 8 mice per group). M: Flow cytometry analysis of epididymal
adipocytes (n = 6 mice per group). N: Triglyceride (TG) content in epididymal fat from SD-fed mice (n = 6 mice per group). O: Relative
expression of adipogenesis genes in epididymal WAT after 18 weeks on SD, determined by qRT-PCR analysis: Pparg, Cebpa, and Fabp4
(n = 6 mice per group). P: Representative images of Oil Red O–stained adipocytes differentiated from stromal vascular cells collected from
WT and Sucnr1-deleted mice and absorbance measurement (510 nm) of Oil Red O staining (n = 4 mice per group). Q: Relative expression of
adipogenesis genes in differentiated adipocytes from SD-fed mice, determined by qRT-PCR analysis; Pparg, Pref1, and Srebp1c (n = 4
mice per group). Data represent mean 6 SEM. Body weight analysis was assessed by repeated-measures ANOVA. *P < 0.05; **P < 0.01;
***P < 0.001. epi, epididymal; FSC-A, forward scatter-A; iso, isoproterenol; OD, optical density; PTX, pertusis toxin; ret, retroperitoneal;
sub, subcutaneous; succ, succinate.
apparent from 10 weeks of diet (Fig. 3F), which was significant when measured at 50 weeks (41.6 6 1.5 g for WT
vs. 35.5 6 1.9 g for Sucnr1 KO mice; P , 0.05) (Fig. 3G).
Analysis of body composition was performed by dissection of organs and fat pads, nuclear MRI (NMRI), and
MRS. After 18–20 weeks on the diet, mice were killed and
organ weights were measured. No differences between
genotypes were found in the weights of heart, spleen, liver,
and BAT at the end of the study (not shown); however,
evident differences were found in the WAT depots. Accordingly, retroperitoneal, subcutaneous, and epididymal fat
mass was significantly reduced in Sucnr1 KO mice relative
to WT littermates (Fig. 3H). Further, compared with total
body weight, cumulative fat content was significantly reduced in Sucnr1 KO animals (Fig. 3I). Visual inspection of
NMRI images of WT and Sucnr1 KO animals suggested
a greater percentage of abdominal fat mass in WT animals
relative to Sucnr1 KO littermates (Fig. 3J), and quantification analysis of axial slices demonstrated a trend for reduction (Fig. 3J). In the same experimental setting, 13C-NMR
spectra of glycerides were recorded over the entire abdominal region (Supplementary Fig. 1A). Quantification of the
spectra (24) showed that the fractional contribution of
saturated fatty acids to the total pool of glycerides was
diabetes.diabetesjournals.org
significantly increased in Sucnr1 KO animals (0.583 6
0.292 vs. 0.481 6 0.189; P , 0.05 for Sucnr1 KO vs.
WT, respectively), whereas monounsaturated and polyunsaturated fatty acids were comparable between genotypes.
Finally, 1H-MRS were also acquired from the abdominal
region to assess lipid content (25). As depicted in representative spectra of mice, the ratio of water to fat content
was altered in Sucnr1 KO mice relative to control littermates (Supplementary Fig. 1B), and area under the curve
measurements revealed a significant decrease in fat content
in Sucnr1 KO mice (Fig. 3K). Collectively, these results are
consistent with the fat weight analysis and strongly suggest
that Sucnr1 KO mice have a reduced WAT content.
In agreement with the decreased size of the different
anatomical WAT compartments in SD-fed Sucnr1 KO mice,
histological analysis of hematoxylin and eosin–stained
WAT established a significant decrease in adipocyte size
relative to WT mice (Fig. 3L). Further, flow cytometry analysis of disaggregated adipocytes from epididymal WAT
revealed a significant decrease in adipocyte size, measured
as forward scatter (Fig. 3M). These changes were mirrored
by a reduction in the concentration of adipose triglycerides
(Fig. 3N). As the decrease in adipose mass in Sucnr1 KO
mice can result from a reduction in adipocyte size and/or
decreased differentiation, we examined the expression of
McCreath and Associates
1161
genes related to adipocyte differentiation. Although expression levels of the adipogenic transcription factors, peroxisome proliferator-activated receptor g (Pparg) and CCAAT/
enhancer-binding protein a (Cebpa), together with the late
differentiation marker fatty acid binding protein 4 (Fabp4),
tended to be slightly lower in WAT taken from Sucnr1 KO
mice, this decrease was not significant (Fig. 3O). Moreover,
the differentiation ability of preadipocytes extracted from
the SVF of adult mice was comparable between both
groups, as measured by Oil Red O staining of neutral lipids
(Fig. 3P) and by expression of the adipocyte marker genes
Pparg, protein delta homolog-1 (Pref1), and SREBP-1c
(Srebp1c), although a nonsignificant increase in Pparg
and Srebp1c expression was observed in adipocytes from
Sucnr1 KO mice (Fig. 3Q). These results, taken together
with the findings of low levels of Sucnr1 expression in SVF,
a reduction in adipose tissue mass and adipocyte size, and
decreased lipid content, collectively suggest that Sucnr1 deletion does not alter adipogenesis but rather results in
decreased lipid accumulation and smaller adipocytes.
To further evaluate the metabolic changes caused by
Sucnr1 deletion, we measured energy expenditure by indirect calorimetry. Interestingly, the rate of VO2 consumption in the Sucnr1 KO group was consistently
higher than WT counterparts (Fig. 4A). The respiratory
Figure 4—Indirect calorimetry and UCP expression. Age-matched mice after 20 weeks of SD were individually placed in metabolic cages
(PhenoMaster) and allowed to acclimatize for 48 h before readings were taken (n = 7–8 mice per group). A: Mean whole-body oxygen
consumption rate. B: RER (VCO2/VO2). C: Mean food intake (g). D: Mean energy expenditure (kcal$h21$kg21). E: Mean RER over dark and
light cycles. F: Mean food intake over dark and light cycles. G: Mean energy expenditure over dark and light cycles. H: Total ambulatory
activity (sum of horizontal and vertical counts) over dark and light cycles. Measurements were recorded on a cycle of dark (8:00 P.M. to 8:00
A.M.) and light (8:00 A.M. to 8:00 P.M.). I: Relative expression of UCP1, UCP2, and UCP3 in tissues (n = 5–7 per group). Data represent mean 6
SEM. *P < 0.05; **P < 0.01; ***P < 0.001. D, dark; EE, energy expenditure; L, light; SM, skeletal muscle.
1162
SUCNR1 Effects on Obesity
exchange ratio (RER; VO2/VCO2) of Sucnr1 KO mice was
also significantly higher than that of WT mice during the
dark cycle (Fig. 4B and E), indicating that perhaps less tissue
fat is available for oxidation in these mice. Alternatively,
the increased RER values may suggest an increase in
whole-body utilization of carbohydrates and protein
over lipids. Surprisingly, food intake was increased in
Sucnr1 KO mice compared with WT (Fig. 4C and F),
and energy expenditure was greater during the dark cycle
(Fig. 4D and G), with no change in locomotor activity
between the two genotypes (Fig. 4H). This increase in
food intake together with the reduction in fat mass could
be due to evident increases in energy expenditure. Though
we were unable to find differences in BAT expansion between WT and Sucnr1 KO mice, dysregulated energy expenditure often results in overexpression of uncoupling
proteins (UCPs) to dissipate energy. However, analysis of
UCP expression revealed comparable levels of UCP2 and
UCP3 in skeletal muscle and liver (Fig. 4I). Interestingly,
although changes did not reach significance, there was
a trend for lower UCP1 expression in BAT tissue of Sucnr1
KO mice, together with elevated levels of UCP1 and UCP2
expression in WAT tissue (Fig. 4I), suggesting perhaps
compensatory mechanisms. Collectively, these results suggest that deletion of Sucnr1 results in substantial alterations in energy expenditure and disposition.
HFD Feeding Leads to Increased Fat Deposition in
Sucnr1 KO Mice and Hepatic Steatosis
We next challenged mice with HFD to provoke obesity.
HFD feeding led to increased weight in both genotypes;
however, Sucnr1 KO mice gained weight at a slower rate
than WT littermates, which was significant after only
;3 weeks of feeding (25.7 6 0.3 vs. 28.6 6 0.5 g; P =
0.0006). Variance in body weight widened until week 14
when differences became less pronounced and weight
curves were superimposable (Fig. 5A). Predictably, animals on HFD were substantially heavier than SD-fed animals at the end of the study (compare Fig. 5A and Fig.
3F), and this correlated with an increase in the weight of
WAT and BAT compartments and also liver. Surprisingly,
although Sucnr1 KO mice on HFD were generally leaner
than WT counterparts, the weight of WAT stores at the
end of the study was significantly increased (Fig. 5B),
whereas adipocyte size was comparable (Supplementary
Fig. 2). This finding was in contrast to that observed under SD, where Sucnr1 KO mice had significantly less WAT
(Fig. 3H and I). In a second cohort of HFD-fed mice used
for NMRI analysis, body weights of Sucnr1 KO mice after
20 weeks on diet were found to be significantly lower than
equivalent WT mice (Fig. 5C), in keeping with the overall
observations. Although no differences were found in the
weight of different anatomical WAT compartments between groups in this cohort (Fig. 5D), the proportion of
fat as a percentage of total body weight was significantly
higher in Sucnr1 KO animals, as determined by measurement of combined fat depots (Fig. 5E). Inspection of
Diabetes Volume 64, April 2015
NMRI images indicated comparable abdominal fat volume
in both groups (Fig. 5F), and quantitative assessment of
fat from axial slices revealed no differences (Fig. 5F). A
more accurate quantification was carried out using
1
H-MRS to determine lipid content. In this case, area under
the curve measurements revealed a significant increase in
fat content in Sucnr1 KO mice (Fig. 5G), in keeping with
the findings from the first cohort.
Results from indirect calorimetry at 8 weeks on HFD
revealed no significant differences in VO2 consumption or
RER (Fig. 5H) in Sucnr1 KO mice compared with WT
control, although ingestion of the HFD reduced RER values in both groups relative to SD. Again, total food consumption was modestly increased in Sucnr1 KO mice,
although this did not reach significance (Fig. 5H). Furthermore, locomotor activity was not significantly different
between groups (data not shown). These results indicated
that the initial slower rate of weight gain in Sucnr1 KO
mice under HFD was not attributable to alterations in
energy expenditure, food intake, or activity.
Changes in adipose mass were reflected in alterations
of adipokine levels in serum of animals. Compared with
WT littermates, the reduced quantity of adipose tissue in
Sucnr1 KO mice under SD was paralleled by decreased
circulating levels of the obesity indicator leptin (Fig. 5I).
This finding is in keeping with the increased feeding of
Sucnr1 KO animals on SD (Fig. 4C and F) and the satiety
effect of this hormone (35). In contrast, HFD feeding
induced a .10-fold increase in serum leptin, which was
not significantly different between Sucnr1 KO and WT
mice (Fig. 5I). In addition, a small but significant decrease
in serum levels of the insulin-sensitizing hormone adiponectin was observed for Sucnr1 KO mice under both dietary regimes (Fig. 5I). However, serum levels of the
adipokines resistin and plasminogen activator inhibitor1, although increased in HFD, were not statistically different between genotypes (Fig. 5I). Obesity in humans
and animal models is often associated with increased circulating lipids and ectopic lipid accumulation in nonadipose tissues including liver (36). Consistent with this
phenotype, both fasted serum levels of NEFAs and cholesterol were elevated in WT mice and Sucnr1 KO mice fed
HFD for 18 weeks, whereas TG concentrations remained
in the normal range (Fig. 5J). In accord with the measured
lipolytic rates in explants (Fig. 3A), no differences in
plasma parameters were observed between genotypes. Although no morphologic alterations were found in liver
between groups fed an SD, hepatic steatosis was induced
in both genotypes under HFD, evidenced by hematoxylin
and eosin staining of abundant vacuolar vesicles in liver
sections, which were confirmed as lipid droplets by Oil
Red O staining (Fig. 5K). Additionally, hepatic triglyceride
content in both genotypes was significantly increased under HFD, consistent with the fatty changes to liver (Fig. 5L);
however, no significant differences in liver TG content were
found between Sucnr1 KO and WT mice under either diet.
Further, HFD levels of AST and ALT were found to be
diabetes.diabetesjournals.org
McCreath and Associates
1163
Figure 5—Analysis of mice under HFD. A: Body weight changes in male Sucnr1-deficient mice (KO) and WT littermates fed HFD for 16
weeks (n = 8–12 mice per group). B: Organ weight measurements after 18 weeks on diet (n = 8–12 per group). C: Body weight changes in
male Sucnr1-deficient mice (KO) and WT littermates fed HFD for 20 weeks (n = 8 mice per group). D: WAT weight measurements after 20
weeks on diet (n = 8 per group). E: Total fat weight taken as a percentage of body weight after 20 weeks on diet (n = 8 per group). F: NMRI
analysis of body fat content and quantification of several measurements (n = 7 mice per group). Results were expressed as proportion of fat
volumes vs. total mouse volume (fat volume/total volume). G: Lipid quantification. 1H spectra of abdominal compartments from WT and
Sucnr1 KO mice (n = 7 mice per group). Areas under the curve were used to calculate the portion of lipids with respect to water content. H:
Indirect calorimetry of age-matched mice after 8 weeks of HFD was performed as in Fig. 3. Mean whole-body oxygen consumption, mean
RER over dark and light cycles, and mean food intake over dark and light cycles. I: Serum levels of adipokines measured in fasted mice at
end of study (n = 8–12 mice per group). J: Serum levels of NEFAs, triglycerides, and total cholesterol from Sucnr1-deficient mice (KO) and
WT littermates after 18 weeks on diets (n = 8–12 mice per group). K: Representative hematoxylin and eosin–stained and Oil Red O sections
of paraffin-embedded liver taken from WT and KO mice after 18 weeks on diets (1003 magnification). L: Hepatic triglyceride (TG) content
after 16 h fasting (n = 8–12 mice per group). M: Serum measurement of ALT and AST from Sucnr1-deficient mice (KO) and WT littermates
after 18 weeks on HFD (n = 8–12 mice per group). Body weight analysis was assessed by repeated-measures ANOVA. Data represent
mean 6 SEM. *P < 0.05; **P < 0.01; ***P < 0.001. CHOt, total cholesterol; epi, epididymal; ret, retroperitoneal; sub, subcutaneous.
significantly increased in Sucnr1 KO mice compared with
WT mice, suggesting increased hepatocyte damage (Fig. 5M);
the origin of these differences is not known. Consistent with
the increased lipid load in liver, expression analysis revealed
changes in relevant genes (Supplementary Fig. 3), including
the lipogeneic transcription factor sterol regulatory binding
protein 1c (Srebp1c), and its target stearyol CoA desaturase-1
(Scd1), which were induced under HFD in both genotypes;
however, other genes involved in triglyceride synthesis, fatty
acid synthase (Fas) and stearyol CoA desaturase (Acc),
were unaltered in HFD-fed mice. Similarly, expression of
the fatty acid transporter CD36 was upregulated under
HFD in both genotypes, although expression was significantly reduced in Sucnr1 KO mice compared with WT
littermates on SD. Additionally, a modest but significant
increase in the expression of a second fatty acid transporter, Fabp4, was found in Sucnr1 KO but not WT mice
under HFD diet, whereas expression of the fatty acid
1164
SUCNR1 Effects on Obesity
transport protein 2 (Fatp2) was downregulated in Sucnr1
KO mice under HFD. In contrast to these changes, no
differences were found between genotypes for the expression of genes related to fatty acid oxidation, although
carnitine palmitoyltransferase 1a and 1b (Cpt1a and
Cpt1b) were downregulated under HFD in both genotypes. Collectively, these data indicate that although the
absence of Sucnr1 results in subtle changes to liver function metrics compared with WT mice, HFD appeared to
cause comparable steatosis.
Sucnr1 KO Mice Are Hyperglycemic Under HFD With
Reduced Insulin Secretion
Although the metabolic phenotype of Sucnr1 KO mice
appeared modest, the evident differences in adiposity and
weight gain prompted us to evaluate glucose and insulin
homeostasis. These parameters were measured in mice
after 16 weeks on diet. Compared with SD, HFD led to an
increase in both fasted and fed levels of serum glucose in
Sucnr1 KO mice and WT littermates (Fig. 6A). Serum
glucose was comparable between genotypes in SD; however, despite their similar final body weights in HFD,
Sucnr1 KO mice had significantly higher levels of fasting
blood glucose, whereas fed glucose was unaltered (Fig. 6A,
right panel). Nevertheless, Sucnr1 KO mice showed similar levels of fasting insulin as those from WT mice in
HFD, which were elevated compared with those in SD
(Fig. 6B). To directly assess whether glucose homeostasis
was dysregulated in Sucnr1 KO mice, we performed glucose tolerance tests and insulin tolerance tests. Interestingly, in the SD group, intraperitoneal challenge with
1.5 g/kg glucose resulted in faster clearance in Sucnr1
KO mice, as glucose concentrations remained significantly
lower compared with WT mice (Fig. 6C, left panel). This is
in keeping with the general leanness of KO animals and
higher RER values (Fig. 4B and E), and suggested improved insulin sensitivity that promotes carbohydrate utilization. As anticipated, glucose clearance deteriorated in
the HFD group (Fig. 6D, left panel). However, in marked
contrast to the findings under SD, Sucnr1 KO mice fed an
HFD developed a clear impairment in glucose elimination
compared with WT mice, with manifest hyperglycemia
120 min after glucose injection. Notably, this decreased
ability to lower blood glucose was not apparent in Sucnr1
KO mice at 11 weeks on HFD (Supplementary Fig. 4),
suggesting a progressive glucose intolerance concurring
with the increased WAT weight. Abnormal glucose homeostasis could result from either a defect in insulin sensitivity of the peripheral tissues or a defect in insulin
secretion. Insulin tolerance tests showed that although
both genotypes exhibited decreased insulin sensitivity under HFD, Sucnr1 KO mice exhibited comparable insulin
sensitivity to WT littermates, as demonstrated by a quick
decline in plasma glucose after an acute insulin injection
(Fig. 6C and D, right panels). Furthermore, insulin was
shown to induce phosphorylation of the major insulinsignaling protein Akt (on Ser473) in liver, WAT, and skeletal
Diabetes Volume 64, April 2015
muscle to similar intensities in Sucnr1 KO and WT mice on
HFD (Fig. 6E). These results pointed to a defect in insulin
secretion as the origin of the hyperglycemia in HFD-fed KO
mice. To consider this, we repeated the glucose tolerance
test and measured circulating insulin levels at baseline and
10 min after a glucose load (1.5 g/kg). Whereas WT mice
showed a characteristic increase in insulin levels at t = 10 min,
Sucnr1 KO mice failed to secrete insulin, although GLP-1
levels were similar in both genotypes (Fig. 6F). Collectively, these results imply that while Sucnr1 KO mice
have significantly enhanced glucose tolerance under SD,
the HFD causes a sufficient deficiency in insulin secretion
to cause glucose intolerance.
Guided by these findings, we examined islet anatomy
and endocrine cell size in pancreatic tissue sections of
HFD-fed mice (Fig. 6G). As expected, morphometric analysis revealed that high-fat feeding increased islet cell size,
and this increase was comparable in both genotypes
(Fig. 6H). Moreover, b-cell number was also similarly increased in Sucnr1 KO and WT mice under HFD, compared
with SD (Fig. 6I). Also, total (acid-extracted) pancreatic
insulin concentration was similar between genotypes on
HFD (Fig. 6J). Immunohistochemistry staining with an
antibody to insulin showed that the morphologic features
of WT and KO islets were comparable (Fig. 6K, left panels), and detailed analysis of the islets revealed a normal
cell architecture shared between genotypes, with positive
immunostaining for glucagon (a-cells) in the periphery,
while the area of anti-insulin staining was observed
mostly in the central region (Fig. 6K, right panels). Examination of b-cell proliferation with an antibody to Ki67
indicated a similar and low (,1%) frequency of Ki67positive cells in both WT and KO mice, and very few
apoptotic (caspase-3 positive) b-cells were detected in either genotype (Fig. 6L). Thus our results are consistent
with an insulin secretory defect in Sucnr1 KO mice.
DISCUSSION
Impaired cellular metabolism is a defining hallmark of
many disease states and can affect homeostatic signaling
processes. As the Krebs cycle is the central metabolic
pathway in aerobic organisms, it is particularly significant
that succinate, an intermediate metabolite of this pathway, has an alternative occupation as a signaling molecule
for Sucnr1 coupling.
Information regarding the function of Sucnr1 is limited.
Previous studies have shown that Sucnr1 is distributed in
organs with high metabolic demand, including the kidney
and liver (19). Signaling of Sucnr1 appears to be an early
response to hyperglycemic conditions in the kidney and
leads to activation of the intrarenal renin-angiotensin system, resulting in hypertension and potential tissue injury
(37). Here we show that Sucnr1 is expressed in high levels
in the adipocyte and that loss of Sucnr1 leads to dichotomous effects on body weight and metabolism. Although
not conspicuously different during early growth, after
16–20 weeks on SD (at 21–25 weeks of age) Sucnr1 KO
diabetes.diabetesjournals.org
McCreath and Associates
1165
Figure 6—Sucnr1 KO mice show diet-dependent alterations in glucose homeostasis. A: Plasma levels of fasting (16 h) and fed glucose in
Sucnr1-deficient mice (KO) and WT littermates under SD (left) and HFD (right) (n = 8–12 mice per group). B: Plasma insulin levels after 16-h
fast (n = 8–12 mice per group). Plasma glucose during IPGTT (left) and IPITT (right) in SD (C) and HFD (D) (n = 8–12 mice per group).
E: Phosphorylation of Akt (Ser473) in liver, WAT, and skeletal muscle of HFD mice. After an overnight fast, mice were injected with PBS (–)
or insulin (+) (1.5 units/kg) for 10 min. F: Serum insulin and GLP-1 concentrations measured from fasted WT and Sucnr1-deficient (KO) mice
on HFD, measured after intraperitoneal glucose (1.5 g/kg) injection (n = 8 mice per group). G: Representative hematoxylin and eosin–stained
sections of paraffin-embedded pancreas taken from WT and KO mice after 18 weeks on diets (1003 magnification), used to determine islet
area and b-cell number. H: Quantification of islet size measured as a percentage of total pancreas area (n = 6 mice per group).
I: Quantification of b-cell density was assessed by counting the number of nuclei in a 1,740-mm2 islet center area (n = 6 mice per group).
J: Insulin content from acid-extracted pancreata of HFD-fed mice (n = 12 mice per group). K: Representative insulin immunostaining (403
magnification) of pancreas from Sucnr1-deleted mice and WT littermates on HFD (left panels). Immunofluorescence detection (1003
magnification) of insulin (green) and glucagon (red) in islets from Sucnr1-deleted mice and WT littermates on HFD (right panels).
L: Representative Ki67 and caspase-3 staining of pancreas from Sucnr1-deleted mice and WT littermates on HFD. Islets are denoted
by a dotted line, immunopositive cells are marked with an arrowhead. Data represent mean 6 SEM. IPGTT/IPITT analysis was assessed by
repeated-measures ANOVA. *P < 0.05; ***P < 0.001. pAkt, phospho-Akt; prot, protein; S. muscle, skeletal muscle.
mice exhibited a modestly lean phenotype compared with
WT mice, with significantly reduced levels of plasma leptin and reduced WAT mass containing smaller adipocytes
and decreased triglyceride content. This decrease in
adiposity was not associated with ectopic triglyceride storage in liver or plasma (Fig. 5) or skeletal muscle (not
shown) but correlated with a significant increase in glucose tolerance and elevated metabolic activity, including
1166
SUCNR1 Effects on Obesity
increased energy expenditure, which might partly explain
why Sucnr1 KO mice were lean despite hyperphagia. However, this phenotype was seen in the setting of normal
levels of BAT content and tissue expression of UCPs,
which are often elevated in models of increased energy
expenditure (38). Decreased weight gain in Sucnr1 KO
mice was also observed in the HFD study, particularly
during the initial 10 weeks. This finding supports our
observations on SD and strongly suggests that leanness
is a primary outcome of Sucnr1 deletion. Since adipogenesis was not affected by deletion of Sucnr1 and an antilipolytic effect of succinate had been previously described,
this metabolically favorable phenotype might be explained,
in part, by the release from succinate-inhibited lipolysis.
Notably, experiments both in vitro and in vivo verified
that succinate inhibited the breakdown of triglycerides,
although basal and isoproterenol-induced lipolysis was
unchanged in Sucnr1 KO animals. This phenotype, in
part, is consistent with other mouse models of “unrestrained” lipolysis and shows that release from Gi-coupled
inhibition of fat mobilization results in leanness (39–41)
and increased energy expenditure (41). After prolonged
high-fat feeding, Sucnr1 KO mice displayed a rather more
complex phenotype, presumably a result of nutritional
stress induced by the HFD. Under these conditions,
end-of-study body weights of Sucnr1 KO mice were, in
the main, comparable with WT controls, but WAT was
significantly heavier. Furthermore, after 16 weeks on
HFD, Sucnr1 KO mice had significantly greater levels of
blood glucose and failed to secrete insulin in glucose tolerance tests; however, pancreas architecture was not different than in WT mice. Ostensibly, this hyperglycemic
state may have resulted from the failure to buffer serum
glucose, although this inference is tempered by the result
that insulin sensitivity was unchanged in Sucnr1 KO mice.
Still, compensatory mechanisms such as neuronal or
endocrine modulation may account for this discrepancy
between insulin secretion and resistance.
Although our original hypothesis presupposed that
a lean phenotype would result from augmented WAT
lipolysis, our findings of increased metabolic activity,
under basal conditions, in Sucnr1 KO mice points to
a more systemic effect of Sucnr1 deletion. Whether these
phenotypes are triggered directly by differences in adipose
mass in Sucnr1 KO mice or through a more circuitous
route awaits further study. Nevertheless, adiponectin levels, which are typically inversely correlated with obesity,
were decreased in Sucnr1 KO mice and might reflect specific changes in adipocytokine signaling (42), leading us to
hazard that a hyperglycemic phenotype might occur when
fat content reaches a critical mass.
Many mechanisms can account for leanness, but they
all generally result from deregulation of energy balance, as
noted here. We can speculate on several potential
mechanisms that may promote this phenotype in Sucnr1
KO mice (43): hypothalamic signal transduction (44), reduced intestinal absorption of foods leading to decreased
Diabetes Volume 64, April 2015
energy availability (45), enhanced energy metabolism in
peripheral tissues, or a defect in adipose tissue storage
capability. It remains to be determined whether one or
more of these mechanisms are operating in our model.
Finally, it should be noted that an underlying concept
in Sucnr1 signaling is hypoxia-induced modulation of
receptor engagement through local increases in succinate
levels. This is particularly manifest in ischemic retinopathy, where microvascular degeneration results in an
abnormal hypoxia-driven succinate-induced neovascularization through Sucnr1-dependent upregulation of proangiogenic factors (13,46). Interestingly, hyperglycemia can
also result in regional increases in succinate (16,46), most
likely through overactivation of the Krebs cycle (47). Indeed, adipose tissue succinate has been shown to increase
(twofold) in mice under HFD (48), and hypoxia has been
detected during expansion of adipose tissue in obesity
(49,50), doubtless occurring when vascularization is insufficient to maintain oxygen levels. Thus increases in
succinate, hypoxia, and/or hyperglycemia might trigger
Sucnr1-induced signaling at the level of the adipocyte.
Clearly, because Sucnr1 is expressed in organs with high
metabolic demands, tissue-selective KOs will be required
to identify the specific cell type(s) in which Sucnr1 plays
a direct role; nevertheless, our results indicate that activation of Sucnr1 may have therapeutic implications in the
treatment of obesity-related disorders.
Acknowledgments. The authors thank Guadalupe Sabio and Tamas
Rözer for helpful discussions and Roisin Brid Doohan, Ana Belén Ricote, and
Jesús María Ruiz-Cabello for technical assistance (all from CNIC).
Funding. This work was supported in part by a research grant from the
Ministerio de Ciencia e Innovación (SAF2009-07965 to K.J.M. and SAF201015239 to B.G.G.). The CNIC is supported by Ministerio de Economía y Competitividad and the Pro-CNIC Foundation.
Duality of Interest. No potential conflicts of interest relevant to this article
were reported.
Author Contributions. K.J.M. and A.M.C. conceived the project, performed
experiments, interpreted data, and wrote the paper. S.E., B.G.G., M.B., A.d.M., and
P.S. performed experiments and interpreted data. A.M.C. is the guarantor of this
work and, as such, had full access to all the data in the study and takes responsibility
for the integrity of the data and the accuracy of the data analysis.
References
1. Flower DR, Attwood TK. Integrative bioinformatics for functional genome
annotation: trawling for G protein-coupled receptors. Semin Cell Dev Biol 2004;
15:693–701
2. Smith NJ. Low affinity GPCRs for metabolic intermediates: challenges for
pharmacologists. Front Endocrinol (Lausanne) 2012;3:1
3. Blad CC, Tang C, Offermanns S. G protein-coupled receptors for energy
metabolites as new therapeutic targets. Nat Rev Drug Discov 2012;11:603–619
4. Cai TQ, Ren N, Jin L, et al. Role of GPR81 in lactate-mediated reduction of
adipose lipolysis. Biochem Biophys Res Commun 2008;377:987–991
5. Taggart AK, Kero J, Gan X, et al. (D)-beta-Hydroxybutyrate inhibits adipocyte
lipolysis via the nicotinic acid receptor PUMA-G. J Biol Chem 2005;280:26649–
26652
6. Tokonami N, Morla L, Centeno G, et al. a-Ketoglutarate regulates acid-base
balance through an intrarenal paracrine mechanism. J Clin Invest 2013;123:
3166–3171
diabetes.diabetesjournals.org
7. He W, Miao FJ, Lin DC, et al. Citric acid cycle intermediates as ligands for
orphan G-protein-coupled receptors. Nature 2004;429:188–193
8. Krebs HA. Rate control of the tricarboxylic acid cycle. Adv Enzyme Regul
1970;8:335–353
9. Ashrafian H, Czibik G, Bellahcene M, et al. Fumarate is cardioprotective via
activation of the Nrf2 antioxidant pathway. Cell Metab 2012;15:361–371
10. Hohl C, Oestreich R, Rösen P, Wiesner R, Grieshaber M. Evidence for
succinate production by reduction of fumarate during hypoxia in isolated adult rat
heart cells. Arch Biochem Biophys 1987;259:527–535
11. Iles RA, Barnett D, Strunin L, Strunin JM, Simpson BR, Cohen RD. The effect
of hypoxia on succinate metabolism in man and the isolated perfused dog liver.
Clin Sci 1972;42:35–45
12. Correa PR, Kruglov EA, Thompson M, Leite MF, Dranoff JA, Nathanson MH.
Succinate is a paracrine signal for liver damage. J Hepatol 2007;47:262–269
13. Sapieha P, Sirinyan M, Hamel D, et al. The succinate receptor GPR91 in
neurons has a major role in retinal angiogenesis. Nat Med 2008;14:1067–1076
14. Aguiar CJ, Andrade VL, Gomes ER, et al. Succinate modulates Ca(2+)
transient and cardiomyocyte viability through PKA-dependent pathway. Cell
Calcium 2010;47:37–46
15. Sadagopan N, Li W, Roberds SL, et al. Circulating succinate is elevated in
rodent models of hypertension and metabolic disease. Am J Hypertens 2007;20:
1209–1215
16. Toma I, Kang JJ, Sipos A, et al. Succinate receptor GPR91 provides a direct
link between high glucose levels and renin release in murine and rabbit kidney.
J Clin Invest 2008;118:2526–2534
17. Krebs HA. Chemical composition of blood plasma and serum. Annu Rev
Biochem 1950;19:409–430
18. Nordmann J, Nordmann R. Organic acids in blood and urine. Adv Clin Chem
1961;4:53–120
19. Ariza AC, Deen PM, Robben JH. The succinate receptor as a novel therapeutic target for oxidative and metabolic stress-related conditions. Front Endocrinol (Lausanne) 2012;3:22
20. Kaplan NM. The deadly quartet. Upper-body obesity, glucose intolerance,
hypertriglyceridemia, and hypertension. Arch Intern Med 1989;149:1514–1520
21. Wilson PW, D’Agostino RB, Sullivan L, Parise H, Kannel WB. Overweight and
obesity as determinants of cardiovascular risk: the Framingham experience. Arch
Intern Med 2002;162:1867–1872
22. Regard JB, Sato IT, Coughlin SR. Anatomical profiling of G protein-coupled
receptor expression. Cell 2008;135:561–571
23. Skarnes WC, Rosen B, West AP, et al. A conditional knockout resource for
the genome-wide study of mouse gene function. Nature 2011;474:337–342
24. Künnecke B, Verry P, Bénardeau A, von Kienlin M. Quantitative body
composition analysis in awake mice and rats by magnetic resonance relaxometry. Obes Res 2004;12:1604–1615
25. Corbin IR, Furth EE, Pickup S, Siegelman ES, Delikatny EJ. In vivo assessment of hepatic triglycerides in murine non-alcoholic fatty liver disease using
magnetic resonance spectroscopy. Biochim Biophys Acta 2009;1791:757–763
26. Petersen PS, Jin C, Madsen AN, et al. Deficiency of the GPR39 receptor is
associated with obesity and altered adipocyte metabolism. FASEB J 2011;25:
3803–3814
27. Zhang HH, Huang J, Düvel K, et al. Insulin stimulates adipogenesis through
the Akt-TSC2-mTORC1 pathway. PLoS ONE 2009;4:e6189
28. Sabio G, Kennedy NJ, Cavanagh-Kyros J, et al. Role of muscle c-Jun NH2terminal kinase 1 in obesity-induced insulin resistance. Mol Cell Biol 2010;30:
106–115
McCreath and Associates
1167
29. Hinke SA, Navedo MF, Ulman A, et al. Anchored phosphatases modulate
glucose homeostasis. EMBO J 2012;31:3991–4004
30. Testa G, Schaft J, van der Hoeven F, et al. A reliable lacZ expression reporter
cassette for multipurpose, knockout-first alleles. Genesis 2004;38:151–158
31. Adham IM, Khulan J, Held T, et al. Fas-associated factor (FAF1) is required for
the early cleavage-stages of mouse embryo. Mol Hum Reprod 2008;14:207–213
32. Galy B, Ferring D, Benesova M, Benes V, Hentze MW. Targeted mutagenesis
of the murine IRP1 and IRP2 genes reveals context-dependent RNA processing
differences in vivo. RNA 2004;10:1019–1025
33. Zechner R, Zimmermann R, Eichmann TO, et al. FAT SIGNALS—lipases and
lipolysis in lipid metabolism and signaling. Cell Metab 2012;15:279–291
34. Schweiger M, Schreiber R, Haemmerle G, et al. Adipose triglyceride lipase
and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism. J Biol Chem 2006;281:40236–40241
35. Frederich RC, Hamann A, Anderson S, Löllmann B, Lowell BB, Flier JS.
Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action. Nat Med 1995;1:1311–1314
36. Maruhama Y, Ohneda A, Tadaki H, Ohtsuki M, Yanbe A. Hepatic steatosis
and the elevated plasma insulin level in patients with endogenous hypertriglyceridemia. Metabolism 1975;24:653–664
37. Peti-Peterdi J. High glucose and renin release: the role of succinate and
GPR91. Kidney Int 2010;78:1214–1217
38. Silva JE. Thermogenic mechanisms and their hormonal regulation. Physiol
Rev 2006;86:435–464
39. Ahmed K, Tunaru S, Tang C, et al. An autocrine lactate loop mediates insulindependent inhibition of lipolysis through GPR81. Cell Metab 2010;11:311–319
40. Bjursell M, Admyre T, Göransson M, et al. Improved glucose control and
reduced body fat mass in free fatty acid receptor 2-deficient mice fed a high-fat
diet. Am J Physiol Endocrinol Metab 2011;300:E211–E220
41. Jaworski K, Ahmadian M, Duncan RE, et al. AdPLA ablation increases lipolysis and prevents obesity induced by high-fat feeding or leptin deficiency. Nat
Med 2009;15:159–168
42. Patané G, Caporarello N, Marchetti P, et al. Adiponectin increases glucoseinduced insulin secretion through the activation of lipid oxidation. Acta Diabetol
2013;50:851–857
43. Xia Z, Stanhope KL, Digitale E, et al. Acylation-stimulating protein (ASP)/
complement C3adesArg deficiency results in increased energy expenditure in
mice. J Biol Chem 2004;279:4051–4057
44. Nogueiras R, López M, Diéguez C. Regulation of lipid metabolism by energy
availability: a role for the central nervous system. Obes Rev 2010;11:185–201
45. Khalifeh-Soltani A, McKleroy W, Sakuma S, et al. Mfge8 promotes obesity
by mediating the uptake of dietary fats and serum fatty acids. Nat Med 2014;20:
175–183
46. Hu J, Wu Q, Li T, Chen Y, Wang S. Inhibition of high glucose-induced VEGF
release in retinal ganglion cells by RNA interference targeting G protein-coupled
receptor 91. Exp Eye Res 2013;109:31–39
47. Brownlee M. The pathobiology of diabetic complications: a unifying
mechanism. Diabetes 2005;54:1615–1625
48. Cummins TD, Holden CR, Sansbury BE, et al. Metabolic remodeling of white
adipose tissue in obesity. Am J Physiol Endocrinol Metab 2014;307:E262–E277
49. Elias I, Franckhauser S, Ferré T, et al. Adipose tissue overexpression of
vascular endothelial growth factor protects against diet-induced obesity and
insulin resistance. Diabetes 2012;61:1801–1813
50. Regazzetti C, Peraldi P, Grémeaux T, et al. Hypoxia decreases insulin signaling pathways in adipocytes. Diabetes 2009;58:95–103