CTRP3 attenuates diet-induced hepatic steatosis by regulating triglyceride metabolism

Abstract

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 ameliorating 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-α levels, and demonstrated a modest improvement in systemic insulin sensitivity. Additionally, reduced hepatic 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 expression 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 hepatocytes. 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 steatosis.

Keywords: C1q/TNF; CTRP; NAFLD; adipokine; fatty liver; hepatic steatosis; triglyceride synthesis.

Fig. 1.

Generation of CTRP3 Tg mice. A: schematic 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; β-actin was included as control. C: immunoblot analysis for the presence of CTRP3-FLAG protein in mouse tissues. β-Actin levels serve as loading control. WT, wild-type; Tg, Transgenic.

Fig. 2.

Improved insulin tolerance in Tg mice without changes in other metabolic parameters. A: no differences in body weight gain over time between WT and Tg male mice fed a high-fat diet (HFD). B: food intake in Tg and WT mice. C: total body mass, fat mass, and lean mass of HFD-fed WT and Tg mice. D–F: indirect calorimetry analysis of oxygen consumption (D), energy expenditure (E), respiratory exchange ratio (RER = V̇co₂/V̇o₂; F) in HFD-fed Tg and WT mice. G: glucose tolerance test on HFD-fed Tg and WT mice. H: insulin tolerance test on HFD-fed Tg and WT mice. Body weight measurements and glucose and insulin tolerance tests were repeated with multiple cohorts of HFD-fed WT and Tg mice (n = 8–10 per group). Data reported are the results from 1 cohort, with results similar across cohorts. Data are reported as means ± SE of 8–10 mice per group. *P < 0.05 vs. WT. LF, low-fat diet; HF, High-fat diet. V̇o₂, volume of oxygen consumption; V̇co₂, volume of carbon dioxide produced; RER, respiratory exchange ratio.

Fig. 3.

Reduced hepatic triglyceride content and synthesis in CTRP3 Tg mice. A: representative Tg and WT mouse liver sections stained with Oil Red O. B: quantification of hepatic triglyceride content. C: quantification of mRNA expression of gluconeogenic genes in liver, normalized against 18 S rRNA. D: quantification of mRNA expression of representative fatty acid oxidation genes in liver, normalized against 18 S rRNA. E and F: quantitative immunoblot analysis of liver AMPKα (Thr-172) (E) and Akt (Ser-473) (F) phosphorylation in WT and Tg mice. G: quantification of mRNA expression of enzymes involves in triglyceride synthesis. All data are reported as comparisons between WT and Tg mice on an HFD (n = 8–10 per group). Phosphorylated protein levels were normalized to total protein levels. All data are reported as means ± SE. *P < 0.05 vs. WT.

Fig. 4.

Recombinant CTRP3 treatment reduces lipid accumulation in vitro. A: CTRP3 treatment reduces the accumulation of neutral lipids in rat H4IIE hepatocytes treated overnight with 200 μM palmitate and CTRP3 (5 μg/ml), as quantified by Oil Red O staining. B: CTRP3 decreases de novo lipid synthesis in H4IIE hepatocytes, as quantified by [³H]acetate incorporation. C: no change in lipid uptake as measured by [³H]palmitate uptake by H4IIE hepatocytes pretreated with vehicle or CTRP3. Values are mean fold ± SE. *P < 0.05 vs. vehicle.

Fig. 5.

Reduced export of VLDL-triglycerides from the liver of Tg mice. A: triglyceride content was measured in plasma samples taken at 0, 1, 2, 6, and 24 h after poloxamer 407 (lipoprotein lipase inhibitor) administration. B: rate of triglyceride accumulation was calculated for each time frame indicated. *P < 0.05 vs. vehicle (n = 8 mice per group).

Fig. 6.

Short-term administration of recombinant CTRP3 reduces hepatic triglyceride levels in diet-induced obese (DIO) mice. A: time line depicting the daily injection study. After 12 wk on a high-fat diet, wild-type DIO mice were fasted for 8 h before initial blood draw. After 72-h recovery from the initial fast (considered day 0), body weight of DIO mice was determined. CTRP3 (2 μg/g body wt) or vehicle injection was given every 24 h for the next 5 days. After the 5th injection, food was immediately removed, and after an 8-h fast animals were euthanized and liver tissues and sera were harvested. B: daily body weight of vehicle- and CTRP3-injected DIO mice. C: pre- and posttreatment fasting (8 h) blood glucose levels. D: hepatic triglyceride contents in vehicle- and CTRP3-injected DIO mice. E and F: serum triglyceride (E) and ketone (F) levels in vehicle- and CTRP3-injected DIO mice.