Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice

Abstract

Adipocyte complement-related protein (30 kDa) (Acrp30), a secreted protein of unknown function, is exclusively expressed in differentiated adipocytes; its mRNA is decreased in obese humans and mice. Here we describe novel pharmacological properties of the protease-generated globular head domain of Acrp30 (gAcrp30). Acute treatment of mice with gAcrp30 significantly decreased the elevated levels of plasma free fatty acids caused either by administration of a high fat test meal or by i.v. injection of Intralipid. This effect of gAcrp30 was caused, at least in part, by an acute increase in fatty acid oxidation by muscle. As a result, daily administration of a very low dose of gAcrp30 to mice consuming a high-fat/sucrose diet caused profound and sustainable weight reduction without affecting food intake. Thus, gAcrp30 is a novel pharmacological compound that controls energy homeostasis and exerts its effect primarily at the peripheral level.

Figure 1

Characterization of Acrp30, gAcrp30, and a fragment of apm-1 in human plasma. (a) Acrp30 has four domains; the C-terminal globular head region (aa 110–247) makes up the majority. (b) A cleavage product of apm-1, the human homolog of Acrp30, was detected in human plasma with the use of a globular head-specific anti-serum for immunoprecipitation as well as for subsequent Western blotting. The apparent molecular mass of this truncated form is 27kDa, corresponding to about 70% of the complete form of apm-1 (Lane II). This truncated form was not detected when an anti-serum specific for the human nonhomologous region (HDQETTTQGPGVLLPLPKGA) of the protein was used for immunoprecipitation (Lane III), followed by immunoblotting with the globular head-specific anti-serum. Preimmune serum of the same animal did not detect any protein (Lane I). (c) Purification of Acrp30 and gAcrp30 shown by SDS-PAGE. Lane V shows the complete form of purified Acrp30. The apparent molecular mass is 37kDa (including an amino-terminal non-Acrp30 region derived from the plasmid). An incompletely reduced dimer (1) of Acrp30 can also be seen. Lane IV shows the proteolytic cleavage product gAcrp30.

Figure 2

Effect of complete Acrp30 and globular region of Acrp30 (gAcrp30) on plasma FFA, glucose, and triglyceride levels in C57BL/6J mice after a high-fat meal. (a–c) Two groups of animals (▴, control; ⧫, gAcrp30-treated; n = 8, each) were fasted for 3 h before the experiment before a baseline blood sample was taken. A high-fat/sucrose meal was given by gavage (vol. = 1% of body weight). Saline or 25 μg gAcrp30 was injected immediately after the high-fat meal and again at 45 min and at 1 h 45 min. Treatment with gAcrp30 resulted in a significant reduction of plasma FFAs at 1–4 h and of glucose and triglycerides at 2–4 h (P < 0.05). (d–f) A similar experiment was conducted in three groups of animals (▴, control, n = 6; ⧫, 3 × 25 μg; ●, 3 × 50 μg full-length Acrp30 treated, n = 4 each). In contrast to the globular head protein, treatment with Acrp30 showed only very small effects, and a significant reduction of plasma FFAs was only seen at 2 h, and a reduction of glucose was seen at 3 h (P < 0.05). All control animals were injected with saline. Blood samples were immediately put on ice; plasma was prepared and kept at −20°C; and triglycerides, FFAs, and glucose were determined within 24 h.

Figure 3

Treatment with gAcrp30 accelerates the removal of plasma FFAs after Intralipid injection. Two groups of mice (n = 5 each) were injected i.v. with 30 μl of Intralipid-20%. A treated group (◊, gAcrp30-treated) was injected with gAcrp30 (25 μg) at 30 and 60 min before Intralipid was given, and control animals (▴, control) received saline. Plasma was isolated, and FFAs were measured as described in Materials and Methods. Three minutes after Intralipid injection, FFA levels rose from 1.07 ± 0.06 to 1.43 ± 0.2 in control animals and from 1.00 ± 0.08 to 1.62 ± 0.15 in gAcrp30-treated animals. Clearance of FFA from plasma measured at later time points was normalized to FFA levels at 3 min after Intralipid injection (100%). Excursion of plasma FFA between saline- and gAcrp30-treated mice was significantly different at P < 0.05 by repeated measure ANOVA with Fisher's probable least-squares difference for post hoc analysis.

Figure 4

Effect of gAcrp30 treatment on fatty acid metabolism in isolated muscle and in C2C12 cells. (a) EDL and soleus muscles were isolated from both legs of normal C57BL/6J mice (n = 17). One muscle of each pair was incubated in medium with 2.5 μg/ml gAcrp30 (dark gray) and one in medium without gAcrp30 (control, light gray). This experimental design allowed us to compare oleate oxidation in pairs of muscles isolated from the same animal. [1-¹⁴C]Oleate oxidation was determined over 90 min. Incubation with gAcrp30 led to a statistically significant increase in oleate oxidation in both muscles (P = 0.0041, repeated measures ANOVA, univariate tests of hypotheses for within-subject effects). (b) Oleate oxidation was also measured in differentiated skeletal muscle C2C12 cells. The formation of ¹⁴CO₂ was determined in untreated control cells (light gray) and in cells incubated for 90 min in the presence of 2.5 μg/ml gAcrp30 (dark gray). Each experiment was performed in triplicate.

Figure 5

Effects of long-term treatment with gAcrp30 on mice fed a high-fat diet. (a) Ten-week-old male C57BL/6J mice were put on a high-fat/sucrose diet for 19 days. The average body weights at this time were 29.0 ± 2.4, 29.3 ± 2.2, and 32.0 ± 2.6 g for control, Acrp30, and gAcrp30 groups, respectively. The mice were then treated by continuous infusion with either 2.5 μg gAcrp30/day (▵), 5 μg Acrp30/day (■), or physiological saline (◊). The mice were continued on the high-fat diet, and their body weight was recorded. Mice treated with gAcrp30 experienced a significant weight reduction (−3.7%, P = 0.002) during the first 4 days. This reduction in body weight remained significant throughout the study. (b) C57BL/6J mice were fed a high-fat diet for more than 6 months before treatment. The average body weights at this time were 51.2 ± 4.5, 51.9 ± 4.1, and 54.3 ± 3.2 g for control, Acrp30, and gAcrp30 groups, respectively. Three groups of mice (n = 8, each) were injected twice daily with gAcrp30 (▵, 25 μg per injection), Acrp30 (■, 25 μg per injection), or saline (◊). Body weights were recorded at the indicated time points. Treatment with gAcrp30 led to significant (P < 0.05) weight loss at day 3. This effect became even more significant as the study continued. The animals had lost 7.5% of their initial body weight at day 16 (P = 0.001). (c) This effect was paralleled by a drop in plasma FFAs, which reached statistical significance (P < 0.05 vs. saline) at day 3 and persisted throughout the study. Shown is the plasma FFA level at day 16 of the study. The initial FFA plasma concentration was the same in all three groups. (d) Daily food intake averaged over the course of the study was not significantly different in either treatment group when compared with saline-injected animals.