CTRP3 deficiency reduces liver size and alters IL-6 and TGFβ levels in obese mice

Abstract

C1q/TNF-related protein 3 (CTRP3) is a secreted metabolic regulator whose circulating levels are reduced in human and rodent models of obesity and diabetes. Previously, we showed that CTRP3 infusion lowers blood glucose by suppressing gluconeogenesis and that transgenic overexpression of CTRP3 protects mice against diet-induced hepatic steatosis. Here, we used a genetic loss-of-function mouse model to further address whether CTRP3 is indeed required for metabolic homeostasis under normal and obese states. Both male and female mice lacking CTRP3 had similar weight gain when fed a control low-fat (LFD) or high-fat diet (HFD). Regardless of diet, no differences were observed in adiposity, food intake, metabolic rate, energy expenditure, or physical activity levels between wild-type (WT) and Ctrp3-knockout (KO) animals of either sex. Contrary to expectations, loss of CTRP3 in LFD- or HFD-fed male and female mice also had minimal or no impact on whole body glucose metabolism, insulin sensitivity, and fasting-induced hepatic gluconeogenesis. Unexpectedly, the liver sizes of HFD-fed Ctrp3-KO male mice were markedly reduced despite a modest increase in triglyceride content. Furthermore, liver expression of fat oxidation genes was upregulated in the Ctrp3-KO mice. Whereas the liver and adipose expression of profibrotic TGFβ1, as well as its serum levels, was suppressed in HFD-fed KO mice, circulating proinflammatory IL-6 levels were markedly increased; these changes, however, were insufficient to affect systemic metabolic outcome. We conclude that, although it is dispensable for physiological control of energy balance, CTRP3 plays a previously unsuspected role in modulating liver size and circulating cytokine levels in response to obesity.

Keywords: C1q/tumor necrosis factor; C1q/tumor necrosis factor-related protein; adipokine; diabetes; fatty liver; obesity.

Fig. 1.

Generation of Clq/TNF-related protein (Ctrp3)-knockout (KO) mice. A: schematic showing the strategy for generating Ctrp3-KO mice by targeted deletion of exon 4/intron 4 of the mouse Ctrp3 gene and replacement with a lacZ reporter and a neomycin resistance cassette. The green circle represents a splice acceptor (SA) site, and the red triangle represents the Flp recombinase target (FRT) site recognized by Flp recombinase. B: genotyping results indicate the successful generation of Ctrp3-wild-type (WT) (+/+), heterozygous (+/−), and homozygous KO (−/−) alleles using the primer set (→ and ←) indicated in A. C: PCR analysis indicates the absence of detectable Ctrp3 mRNA in KO mice. BacVec, bacterial artificial chromosome vector; 5′-UTR, 5′-untranslated region.

Fig. 2.

Body weight and body composition of Ctrp3-WT and -KO mice. A and B: body weight gain over time of WT and KO male and female mice fed a control low-fat diet (LFD). C and D: body weight gain over time of WT and KO male and female mice fed a high-fat diet (HFD). E and F: body composition analyses of fat and lean mass of LFD-fed WT and KO male and female mice. G and H: body composition analyses of fat and lean mass of HFD-fed WT and KO male and female mice. All data are expressed as means ± SE. Sample sizes are indicated.

Fig. 3.

Glucose homeostasis in LFD- or HFD-fed Ctrp3-KO male mice. A and B: fasting blood glucose (A) and serum insulin levels (B) of LFD-fed WT and KO male mice. C: blood glucose levels of LFD-fed WT and KO male mice during the glucose tolerance test (GTT). Glucose was delivered by intraperitoneal injection. D: blood glucose levels of LFD-fed WT and KO male mice during the insulin tolerance test (ITT). E: blood glucose levels of LFD-fed WT and KO male mice during the pyruvate tolerance test (PTT). F and G: fasting blood glucose (F) and serum insulin levels (G) of HFD-fed WT and KO male mice. H: blood glucose levels of HFD-fed WT and KO male mice during the GTT. I: blood glucose levels of HFD-fed WT and KO male mice during the ITT. J: blood glucose levels of HFD-fed WT and KO male mice during the PTT. K: quantitative real-time PCR analysis of gluconeogenic gene (Pck1 and G6Pase) expression in the livers of WT and KO male mice. Expression levels were normalized to 36B4 [also known as ribosomal phosphoprotein P0 (RPLP0)]. All data are expressed as means ± SE. Sample sizes are indicated. **P < 0.01.

Fig. 4.

Glucose homeostasis in LFD- or HFD-fed Ctrp3-KO female mice. A and B: fasting blood glucose (A) and serum insulin levels (B) of LFD-fed WT and KO female mice. C: blood glucose levels of LFD-fed WT and KO female mice during the GTT. Glucose was delivered by intraperitoneal injection. D: blood glucose levels of LFD-fed WT and KO female mice during the ITT. Smaller sample size for ITT was due to hypoglycemia in some mice that had to be removed from the test. E: blood glucose levels of LFD-fed WT and KO female mice during the PTT. F and G: fasting blood glucose (F) and serum insulin levels (G) of HFD-fed WT and KO female mice. H: blood glucose levels of HFD-fed WT and KO female mice during the GTT. I: blood glucose levels of HFD-fed WT and KO female mice during the ITT. J: blood glucose levels of HFD-fed WT and KO female mice during the PTT. All data are expressed as means ± SE. Sample sizes are indicated.

Fig. 5.

Tissue weight, liver lipid content, and serum lipid profiles of LFD- and HFD-fed Ctrp3-WT and -KO mice. A: representative image of visceral (epididymal) fat pads isolated from HFD-fed WT and KO male mice. B: quantification of visceral fat pad weight in WT and KO male and female mice fed HFD and LFD. C: representative image of subcutaneous (SubQ; inguinal) fat pads isolated from HFD-fed WT and KO male mice. D: quantification of SubQ fat pad weight in WT and KO male and female mice fed HFD and LFD. E: representative image of liver isolated from HFD-fed WT and KO male mice. F: quantification of liver weight in WT and KO male and female mice fed a HFD or LFD. G and H: quantification of liver triglyceride (G) and cholesterol (H) levels in HFD-fed WT and KO male mice. I: quantification of serum cholesterol, triglyceride (TG), HDL, and LDL levels in HFD-fed WT and KO male mice. All data are expressed as means ± SE. *P < 0.05. Sample size: for the LFD-fed group, WT (n = 11) and KO (n = 10) male mice and WT (n = 9) and KO (n = 13) female mice were used; for the HFD group, WT (n = 10) and KO (n = 12) male mice and WT (n = 12) and KO (n = 10) female mice were used.

Fig. 6.

Altered hepatic expression of lipid synthesis and fat oxidation genes in HFD-fed Ctrp3-KO mice. Quantitative real-time PCR analysis of triglyceride synthesis (A), de novo lipid synthesis (B), and fat oxidation genes (C) in the livers of HFD-fed WT (n = 10) and KO (n = 10) male mice. Tissues were harvested from overnight-fasted mice. Expression levels were normalized to 36B4 (also known as RPLP0). All data are expressed as means ± SE. **P < 0.01. Agpat, Acyl glycerol phosphate acyltransferase; Gpat, glycerol phosphate acyltransferase; Dgat, diacylglycerol acyltransferase; Acc, acetyl-CoA carboxylase; Fasn, fatty acid synthase; Srebp, sterol responsive element-binding protein; Lcad, long-chain acyl-CoA dehydrogenase; Mcad, medium-chain acyl-CoA dehydrogenase; Acox, acyl-CoA oxidase; Cpt, carnitine palmitoyl transferase.

Fig. 7.

Altered expression and circulating levels of cytokines and adipokines in HFD-fed Ctrp3-KO mice. A–C: quantitative real-time PCR analysis of cytokine gene expression in liver (A), visceral (epididymal) fat depot (B), and SubQ (inguinal) fat depot (C) of WT (n = 8–10) and KO (n = 8–12) male mice. Expression levels were normalized to 36B4 (also known as RPLP0). D–G: quantification of serum transforming growth factor-β1 (TGFβ1; D), IL-6 (E), IL-1β (F), and TNFα (G) levels in WT (n = 8–9) and KO (n = 8–11) male mice. H–J: quantitative real-time PCR analysis of fibrotic gene expression in the liver (H), visceral (epididymal; I), and SubQ (inguinal; J) of WT (n = 10) and KO (n = 10) male mice. K: quantitative real-time PCR analysis of adipokine gene expression in the visceral (epididymal) fat depot of WT (n = 8) and KO (n = 8) male mice. Expression levels were normalized to cyclophilin A. L and M: quantification of serum adiponectin (L) and leptin (M) levels in WT (n = 9) and KO (n = 10) male mice. N and O: quantitative real-time PCR analysis of Ctrp1, Ctrp9, and Ctrp12 gene expression in the visceral (epididymal; N) and SubQ (inguinal; O) fat depot of WT (n = 10) and KO (n = 10) male mice. All data are expressed as means ± SE. *P < 0.05; **P < 0.01. Mcp-1, monocyte chemotactic protein 1; Col, collagen; Mmp12, matrix metalloprotease 12; Adipoq, adiponectin; Lep, leptin; Retn, resistin.

Fig. 8.

Expression of macrophage markers in the fat pads of HFD-fed Ctrp3-KO mice. A and B: quantitative real-time PCR analysis of pan-macrophage marker (F4/80), M1-type macrophage marker (Cd11c), and M2-type macrophage marker (CD206) in the visceral (epididymal; A) and subcutaneous (inguinal; B) fat depots of WT (n = 8) and KO (n = 8) male mice. Expression levels were normalized to β-actin. All data are expressed as means ± SE. *P < 0.05.