CTRP9 transgenic mice are protected from diet-induced obesity and metabolic dysfunction

Abstract

CTRP9 is a secreted multimeric protein of the C1q family and the closest paralog of the insulin-sensitizing adipokine, adiponectin. The metabolic function of this adipose tissue-derived plasma protein remains largely unknown. Here, we show that the circulating levels of CTRP9 are downregulated in diet-induced obese mice and upregulated upon refeeding. Overexpressing CTRP9 resulted in lean mice that dramatically resisted weight gain induced by a high-fat diet, largely through decreased food intake and increased basal metabolism. Enhanced fat oxidation in CTRP9 transgenic mice resulted from increases in skeletal muscle mitochondrial content, expression of enzymes involved in fatty acid oxidation (LCAD and MCAD), and chronic AMPK activation. Hepatic and skeletal muscle triglyceride levels were substantially decreased in transgenic mice. Consequently, CTRP9 transgenic mice had a greatly improved metabolic profile with markedly reduced fasting insulin and glucose levels. The high-fat diet-induced obesity, insulin resistance, and hepatic steatosis observed in wild-type mice were prevented in transgenic mice. Consistent with the in vivo data, recombinant protein significantly enhanced fat oxidation in L6 myotubes via AMPK activation and reduced lipid accumulation in H4IIE hepatocytes. Collectively, these data establish CTRP9 as a novel metabolic regulator and a new component of the metabolic network that links adipose tissue to lipid metabolism in skeletal muscle and liver.

Keywords: AMPK; C1q family; Type 2 diabetes; energy metabolism; fatty acid oxidation; obesity.

Fig. 1.

Diet and metabolic state modulate circulating levels of CTRP9. A: quantitative real-time PCR analyses of Ctrp9 expression in adipose tissue isolated from 12-wk-old chow-fed male mice under fasted or fasted/refed conditions. B: quantitative Western blot analysis of CTRP9 serums levels in 12-wk-old chow-fed male mice under fasted, fasted/refed, or ad libitum conditions. Quantitative real-time PCR (C) and Western blot (D) analysis of CTRP9 mRNA and serum levels in male C57BL/6 mice fed a high-fat diet (HFD) or a low-fat diet (LFD) for 12 wk. Values shown are means ± SE; n = 8–10 mice per group. *P < 0.05. N.S., not significant.

Fig. 2.

Generation of CTRP9 gain-of-function mouse model. A: schematic of CTRP9 transgene construct. HA epitope-tagged CTRP9 transgene is driven by the ubiquitous CAG promoter. B: semiquantitative RT-PCR analysis of CTRP9-HA transgene and β-actin expression in mouse tissues. C: Western blot analysis of CTRP9 in wild-type (WT) and transgenic (Tg) mouse sera. D: Western blot analysis of CTRP9-HA protein in mouse tissues.

Fig. 3.

CTRP9 Tg mice are resistant to HFD-induced obesity. A and B: body weight gain over time between WT and Tg male mice fed a low-fat diet (LFD; A) or a high-fat diet (HFD; B). C: representative images of HFD-fed WT and Tg mice. Percent fat mass (D) and lean mass (E) in WT and Tg mice, as determined by NMR analysis. Data shown are mean ± SE; n = 8–10 mice per group. *P < 0.05 vs. WT.

Fig. 4.

Reduced adiposity and adipocyte size in CTRP9 Tg mice. A: quantification of subcutaneous (inguinal) fat pad mass in WT and Tg mice. B: representative tissue sections of inguinal fat pad of WT and Tg mice. C: quantification of visceral (gonadal) fat pad mass in WT and Tg mice. D: representative tissue sections of gonadal fat pad of WT and Tg mice. Values shown are expressed as means ± SE; n = 8–10 mice per group. *P < 0.05 vs. WT.

Fig. 5.

Indirect calorimetry analysis of CTRP9 Tg mice fed a low-fat diet. A: food intake analysis in WT and Tg mice on a LFD. B: 24-h ambulatory activity of WT and Tg mice on an LFD. Data were binned into 2-h segments. C–F: oxygen consumption (V̇o₂; C), carbon dioxide release (V̇co₂; D), respiratory exchange ratio (RER = V̇co₂/V̇o₂; E), and energy expenditure (F) of WT and Tg mice on an LFD, as determined by indirect calorimetry. Values shown are expressed as means ± SE; n = 6 mice per group.

Fig. 6.

Enhanced fat oxidation and energy expenditure in CTRP9 Tg mice fed a high-fat diet. A: food intake analysis in WT and Tg mice fed a high-fat diet (HFD). B: 24-h ambulatory activity of WT and Tg mice on an HFD. Data were binned into 2-h segments. Oxygen consumption (V̇o₂; C), carbon dioxide release (V̇co₂; D), respiratory exchange ratio (RER = V̇co₂/V̇o₂; E), and energy expenditure (F) of WT and Tg mice on an HFD, as determined by indirect calorimetry. Values shown are means ± SE; n = 8 mice per group. *P < 0.05 vs. WT.

Fig. 7.

Increased mitochondrial content and expression of fat oxidation genes in skeletal muscle of Tg mice. Quantitative real-time analysis of fat oxidation enzyme genes (A) and mitochondrial genes (B) in the skeletal muscles of WT and Tg mice fed an HFD. C: quantitative Western blot analysis of mitochondrion-specific protein COX IV in WT and Tg mice. COX IV levels were normalized to GAPDH protein levels. D: triglyceride content in the skeletal muscles of WT and Tg mice. Values shown are means ± SE; n = 8–10 mice per group. *P < 0.05 vs. WT. LCAD, long-chain acyl-CoA dehydrogenase; MCAD, medium-chain Co-A dehydrogenase; COX II, cytochrome oxidase subunit II; CytoB, mitochondria cytochrome B; COX IV, cytochrome oxidase subunit IV.

Fig. 8.

CTRP9 activates AMPK signaling in vivo and in vitro. A: quantitative Western blot analysis of AMPKα (Thr-172) phosphorylation in the skeletal muscle of WT and Tg mice fed an HFD. Phospho-protein levels were normalized to total AMPKα levels. B: Western blot analysis of AMPKα phosphorylation in rat L6 myotubes stimulated with vehicle control or recombinant CTRP9 (5 μg/ml). C: fatty acid (palmitate) oxidation was measured in L6 myotubes treated with vehicle control or recombinant CTRP9 (5 μg/ml). Values shown are expressed as means ± SE. Representative gels are shown here. *P < 0.05 vs. WT. n = 8–10 mice per group for in vivo studies; n = 6 for in vitro experiments, representing three independent experiments.

Fig. 9.

Reduced hepatic triglyceride accumulation in Tg mice. A: representative images of WT and Tg liver sections stained with oil red O. B: quantification of hepatic triglyceride levels in WT and Tg mice fed an HFD. C: lipid accumulation in rat H4IIE hepatocytes treated overnight with vehicle control or CTRP9 (5 μg/ml) in the presence or absence of 100 μM palmitate. All data shown are expressed as means ± SE; n = 8–10 mice per group for in vivo studies; n = 6 in vitro experiments, representing three independent experiments. *P < 0.05 vs. WT.

Fig. 10.

Improved insulin sensitivity in CTRP9 trangenic mice. Intraperitoneal glucose tolerance test (GTT) for mice fed a low-fat diet (LFD; A) or a high-fat diet (HFD; E). Quantification of the cumulative glucose clearance in GTT by integration of area under the curve (AUC) for LFD-fed (B) and HFD-fed mice (F). Insulin levels during the course of GTT for LFD-fed (C) and HFD-fed mice (G). Quantification of the cumulative insulin release in GTT by integration of AUC for LFD-fed (D) and HFD-fed mice (H). All mice were 13 wk old and had been on LFD or HFD for the previous 9 wk. Values are expressed as means ± SE; n = 6 for LFD group and n = 8–10 mice for HFD group. *P < 0.05 vs. WT.