CTRP3 attenuates diet-induced hepatic steatosis by regulating triglyceride
Jonathan M. Peterson,1,3,4Marcus M. Seldin,1,3Zhikui Wei,1,3Susan Aja,2,3and G. William Wong1,3
1Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland;2Department of
Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland;3Center for Metabolism and Obesity
Research, Johns Hopkins University School of Medicine, Baltimore, Maryland; and4Department of Health Sciences, School
of Public Health, East Tennessee State University, Johnson City, Tennessee
Submitted 29 March 2013; accepted in final form 5 June 2013
Peterson JM, Seldin MM, Wei Z, Aja S, Wong GW. CTRP3
attenuates diet-induced hepatic steatosis by regulating triglyceride me-
tabolism. Am J Physiol Gastrointest Liver Physiol 305: G214–G224,
2013. First published June 6, 2013; doi:10.1152/ajpgi.00102.2013.—
CTRP3 is a secreted plasma protein of the C1q family that helps
regulate hepatic gluconeogenesis and is downregulated in a diet-
induced obese state. However, the role of CTRP3 in regulating lipid
metabolism has not been established. Here, we used a transgenic
mouse model to address the potential function of CTRP3 in amelio-
rating high-fat diet-induced metabolic stress. Both transgenic and
wild-type mice fed a high-fat diet showed similar body weight gain,
food intake, and energy expenditure. Despite similar adiposity to
wild-type mice upon diet-induced obesity (DIO), CTRP3 transgenic
mice were strikingly resistant to the development of hepatic steatosis,
had reduced serum TNF-? levels, and demonstrated a modest im-
provement in systemic insulin sensitivity. Additionally, reduced he-
patic triglyceride levels were due to decreased expression of enzymes
(GPAT, AGPAT, and DGAT) involved in triglyceride synthesis.
Importantly, short-term daily administration of recombinant CTRP3
to DIO mice for 5 days was sufficient to improve the fatty liver
phenotype, evident as reduced hepatic triglyceride content and ex-
pression of triglyceride synthesis genes. Consistent with a direct effect
on liver cells, recombinant CTRP3 treatment reduced fatty acid
synthesis and neutral lipid accumulation in cultured rat H4IIE hepa-
tocytes. Together, these results establish a novel role for CTRP3
hormone in regulating hepatic lipid metabolism and highlight its
protective function and therapeutic potential in attenuating hepatic
adipokine; CTRP; C1q/TNF; fatty liver; hepatic steatosis; NAFLD;
HEPATIC STEATOSIS, or fatty liver, results from an imbalance
between production and removal of hepatic triglycerides
(TAGs) (10). This imbalance can result from excessive alcohol
consumption (alcoholic fatty liver disease) or through other
means (nonalcoholic fatty liver disease, NAFLD). In NAFLD,
elevated hepatic TAG is caused by a combination of excess
dietary lipids and de novo fatty acid synthesis (6, 10, 45). Fat
oxidation and TAG export (in the form of very low-density
lipoprotein, VLDL) aid in removal of hepatic TAGs. NAFLD
is one of the primary causes of abnormal liver function (10)
and is frequently linked to hepatic insulin resistance and
uncontrolled gluconeogenesis in the diabetic state (6, 21, 22,
25, 26, 49). Indeed, up to 70% of clinically obese patients have
NAFLD (31). Furthermore, obese patients with NAFLD are at
a significantly higher risk of developing obesity-associated
comorbidities (e.g., heart disease and Type 2 diabetes) (52).
For reasons still poorly understood, a subset of patients with
NAFLD will go on to develop NASH (nonalcoholic steato-
hepatitis) and cirrhosis (10). Despite the prevalence of NAFLD
in the general population (28, 50), therapeutic options are
As part of an effort to discover novel secreted metabolic
regulators, we recently identified and characterized a family of
15 secreted proteins of the C1q family, designated as C1q/
TNF-related proteins (CTRP1–15) (48, 55–57, 60–62). Sev-
eral of these proteins play important and distinct roles in
regulating insulin sensitivity and energy balance (11, 42, 43,
54–56, 60, 61). We demonstrated that CTRP3 acts on liver to
suppress hepatic glucose output by modulating the expression
of gluconeogenic enzymes (43). A cardioprotective function of
CTRP3 was recently demonstrated in an animal model of
myocardiac infarction (67). In addition, several other functions
attributable to CTRP3, derived from in vitro studies, have been
reported (1–3, 16, 23, 24, 33, 34, 58). In the present study, we
sought to address the role of CTRP3 in regulating lipid me-
tabolism and its protective function in a pathophysiological
context of high-fat feeding. Using a transgenic (Tg) mouse
model, along with short-term recombinant protein supplemen-
tation, we established an important and novel role for CTRP3
in regulating hepatic TAG metabolism and highlighted its
protective function in attenuating diet-induced hepatic steato-
Animals. All animal protocols were approved by the Institutional
Animal Care and Use Committee of The Johns Hopkins University
School of Medicine. CTRP3 Tg mice (on a C57BL/6 genetic back-
ground) and control littermates were housed in polycarbonate cages
on a 12-h light-dark photocycle with ad libitum access to water and
food. Littermates were used throughout the study as wild-type (WT)
controls. Mice were fed a high-fat diet (HFD; 60% kcal derived from
fat, Research Diets; D12492) or the isocaloric-matched low-fat diet
(LFD; 10% kcal derived from fat, Research Diets; D12450B). Diet
was provided for a period of 14 wk, beginning at 4 wk of age.
Metabolic parameters and food intake were measured by using the
Comprehensive Laboratory Animal Monitoring System (CLAMS)
(Columbus Instruments), and body composition was determined by
using a whole-body NMR instrument (EchoMRI) as previously de-
scribed (42). At termination of the study, animals were fasted over-
night and euthanized, when tissues were collected, snap frozen in
liquid nitrogen, and kept at ?80°C until analysis.
Address for reprint requests and other correspondence: G. W. Wong, Dept.
of Physiology and the Center for Metabolism and Obesity Research, Johns
Hopkins Univ. School of Medicine, Baltimore, MD 21205 (e-mail: gwwong
Am J Physiol Gastrointest Liver Physiol 305: G214–G224, 2013.
First published June 6, 2013; doi:10.1152/ajpgi.00102.2013.
0193-1857/13 Copyright © 2013 the American Physiological Societyhttp://www.ajpgi.orgG214
Antibodies and chemicals. Mouse monoclonal anti-FLAG M2
antibody was obtained from Sigma. Antibodies that recognize phos-
pho-Akt (Thr-308), phospho-AMPK? (Thr-172), Akt, and AMPK?
were obtained from Cell Signaling Technology. Antibody that recog-
nizes actin (sc1616) was obtained from Santa Cruz Biotechnology.
Polyclonal rabbit antibody recognizing CTRP3 was obtained from
Novus Biologicals (NBP1-02995).
Generation of CTRP3 transgenic mouse line. Carboxy-terminal
FLAG epitope (DYKDDDDK)-tagged CTRP3 was cloned into the
EcoRI site of pCAGGS vector (40). Expression of Ctrp3 transgene
was driven by the ubiquitous CAG promoter, containing a CMV
enhancer element with a chicken ?-actin promoter. Plasmid construct
was digested with SalI and NotI restriction enzymes, and resulting
DNA fragments (?3.5 and 2.5 kb) were separated on 1% agarose gel.
The ?3.5-kb linear DNA fragment containing the CAG promoter and
enhancer, Ctrp3 transgene, and rabbit ?-globin polyA adenylation
signal was excised from the agarose gel, purified, and verified by
DNA sequencing. Pronuclear injections were performed, and several
founder lines (on a C57BL/6 genetic background) expressing the
Ctrp3 transgene were obtained. One of these mouse lines was main-
tained and expanded for phenotypic analysis. Tg mice are fertile with
no gross abnormality observed.
Mouse serum analysis. Mouse serum samples were collected at
times indicated by using microvette CB 300 (Sarstedt). Glucose
concentrations were determined at time of blood collection with a
glucometer (BD Biosciences). Serum/tissue TAGs (Thermofisher),
nonesterified fatty acids (NEFA; Wako), insulin, tumor necrosis
factor-? (TNF-?), and adiponectin (Millipore) were determined with
commercially available kits. For Western blot analysis, serum samples
were diluted 1:20 in SDS loading buffer (50 mM Tris·HCl, pH 7.4,
2% SDS wt/vol, 6% glycerol wt/vol, 1% 2-mercaptoethanol vol/vol,
and 0.01% bromophenol blue wt/vol).
Intraperitoneal glucose and insulin tolerance tests. Cohorts of
8–10 Tg and WT control littermates were injected with glucose (1
g/kg) or insulin (0.8 units/kg for LFD-fed mice, 1.2 units/kg for
HFD-fed mice). Animals were fasted overnight (16 h) prior to the
glucose tolerance test. For the insulin tolerance test, food was re-
moved 2 h prior to insulin injection. Serum samples were collected at
the indicated time points. Insulin and glucose tolerance tests were
performed when mice were 16 and 17 wk of age, respectively.
Measurement of tissue triglyceride levels. Lipids were extracted as
described by Bligh and Dyer (5). Samples were weighed then homog-
enized in PBS (100 mg/ml) and 1 ml of the sample was added to 3.75
ml of 1:2 (vol/vol) chloroform-methanol. Next, an additional 1.25 ml
chloroform were added; subsequently, 1.25 ml distilled water were
added to the solution. Samples were vortexed for 30 s between each
addition. Samples were then centrifuged at 1,100 g for 10 min at room
temperature to give a two-phase solution (aqueous phase on top and
organic phase below). The lower phase was collected with a glass
Pasteur pipette with gentle positive pressure. This phase was then
washed three times with dH2O, and each time the upper phase was
collected. Samples were then dried under nitrogen gas at 60°C and
dissolved in tert-butyl alcohol-Triton X-100 (3:2). Triglycerides were
then quantified colorimetrically as glycerol by use of a commercial
enzymatic assay (Infinity Triglycerides, Fisher Diagnostics).
Quantitative real-time PCR. Total RNAs from mouse tissues were
isolated with TRIzol (Invitrogen), and 2 ?g of total RNA were reverse
transcribed by use of Superscript III (Invitrogen). Quantitative PCR
analyses were performed on an Applied Biosystems Prism 7500
Sequence Detection System. Samples were analyzed in 25-?l reac-
tions according to the standard protocol provided in the SYBR Green
PCR Master Mix (Applied Biosystems). All expression levels were
normalized to the corresponding 18 S rRNA levels. Primer sequences
can be found in Supplemental Table S1 (Supplemental Material for
this article is available online at the Journal website.).
Quantifying the rate of VLDL-triglyceride secretion. To measure
hepatic TAG production rate, a separate cohort of HFD-fed mice (Tg
and WT littermates) were given an intraperitoneal injection of 1,000
mg/kg poloxamer 407 (Sigma) in saline ?4 h into the light cycle, as
described by Millar et al. (35). Poloxamer 407 is an inhibitor of
β-globin poly A signal
Fig. 1. Generation of CTRP3 Tg mice. A: sche-
matic of Ctrp3 transgenic construct. FLAG-
tagged Ctrp3 transgene is driven by a ubiquitous
CAG promoter. B: semiquantitative RT-PCR
analysis of Ctrp3 transgene expression in mouse
tissues; ?-actin was included as control. C: im-
munoblot analysis for the presence of CTRP3-
FLAG protein in mouse tissues. ?-Actin levels
serve as loading control. WT, wild-type; Tg,
CTRP3 REGULATES HEPATIC TRIGLYCERIDE METABOLISM
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