Growth/differentiation factor 3 signals through ALK7 and regulates accumulation of adipose tissue and diet-induced obesity

Abstract

Growth/differentiation factor 3 (GDF3) is highly expressed in adipose tissue, and previous overexpression experiments in mice have suggested that it may act as an adipogenic factor under conditions of high lipid load. GDF3 has been shown to signal via the activin receptor ALK4 during embryogenesis, but functional receptors in adipose tissue are unknown. In this study, we show that Gdf3(-/-) mutant mice accumulate less adipose tissue than WT animals and show partial resistance to high-fat diet-induced obesity despite similar food intake. We also demonstrate that GDF3 can signal via the ALK4-homolog ALK7 and the coreceptor Cripto, both of which are expressed in adipose tissue. In agreement with a role for ALK7 in GDF3 signaling in vivo, mutant mice lacking ALK7 also showed reduced fat accumulation and partial resistance to diet-induced obesity. We propose that GDF3 regulates adipose-tissue homeostasis and energy balance under nutrient overload in part by signaling through the ALK7 receptor.

Fig. 1.

Gdf3^−/− mutant mice gain less weight during diet-induced obesity despite normal food intake. (A and B) Body weight of 2-month-old WT and Gdf3^−/− mutant mice that were fed on a normal chow diet was monitored during subsequent 14 weeks on either chow (A) or a high-fat diet (HFD) (B). No significant differences were found in chow diet. (C) Weekly food consumption measured from four independent cages per genotype during the first 10 weeks of the period shown in A and B. Results are shown as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student's t test).

Fig. 2.

Reduced accumulation of adipose tissue in mice lacking GDF3. (A) Weight of epididymal fat pads of 5-month-old mice on a chow diet (WT, n = 28; Gdf3^−/−, n = 16), or after 14 weeks of a high-fat diet (WT, n = 50; Gdf3^−/−, n = 29). (B) Histological analysis of paraffin sections from epididymal fat pads of WT and Gdf3^−/− mice by H&E staining. (Scale bar, 100 μm.) (C) Representative MRI images of 5-month old WT and Gdf3^−/− mutant mice fed a chow diet or after 14 weeks on a high-fat diet. (D) Quantification of total fat content by MRI (chow, n = 5 per genotype; high-fat diet, n = 13 per genotype). Results are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01 vs. WT (Student's t test).

Fig. 3.

Alk7 is expressed in adipose tissue and can mediate GDF3 signaling in a Cripto dependent manner. (A) RT-PCR analysis of Gdf3, Alk7, Alk4, ActRII-A, ActRII-B, and Cripto mRNA expression. Gapdh mRNA expression is shown in the bottom-most panel as a loading control. “no input” indicates that no mRNA was used for RT-PCR. (B) Luciferase reporter assay in R4-2 cells transfected with a CAGA-luc reporter and plasmids encoding mature GDF3, ALK7, and Cripto as indicated. Results are expressed in relative luciferase units normalized to control (mean ± SD of triplicate observations). (C) Luciferase reporter assay in HepG2 cells transfected with a BRE-luc reporter and plasmids encoding mature GDF3, Cripto, and chimeric type I receptor ALK7/3 as indicated. BMP4 protein (50 ng/ml) was used as a positive control for reporter activation.

Fig. 4.

Reduced diet-induced weight gain and fat accumulation in mice lacking ALK7. (A) Body weight of 2-month-old WT and Alk7^−/− mutant mice during subsequent 14 weeks on a high-fat diet. (B) Weekly food consumption measured from four independent cages per genotype during the first 10 weeks of the period shown in A. (C) Weight of epididymal fat pads of 5-months-old mice on a chow diet (WT, n = 25; Alk7^−/−, n = 13), or after 14 weeks of a high-fat diet (WT, n = 50; Alk7^−/−, n = 24). (D) Histological analysis of paraffin sections from epididymal fat pads of WT and Alk7^−/− by H&E staining. (Scale bar, 100 μm.) (E) Representative MRI images of 5-month-old WT and Alk7^−/− mutant mice after 14 weeks on a high-fat diet. (F) Quantification of total fat content by MRI (n = 11 for each genotype). Results are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. WT (Student's t test).

Fig. 5.

Mice lacking GDF3, unlike those lacking ALK7, display normal insulinemia and insulin sensitivity. (A) Insulin serum levels after overnight fasting in 5-month-old WT (n = 16), Alk7^−/− (n = 11), and Gdf3^−/− (n = 9) mice. (B) Insulin tolerance test of 5-month-old WT (n = 9), Alk7^−/− (n = 6), and Gdf3^−/− (n = 9) mice maintained on chow diet. (C) Wet liver weight of 5-month-old WT (chow, n = 28; high-fat diet, n = 54), Alk7^−/− (chow, n = 13; high-fat diet, n = 24), and Gdf3^−/− (chow, n = 16; high-fat diet, n = 29) mice after being subjected to chow or high-fat diet. (D) Histological analysis of liver sections from 2-month-old WT, Alk7^−/−, and Gdf3^−/− mice by Oil red-O staining. Results are shown as mean ± SEM. *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001 (Student's t test). (Scale bar, 100 μm.)