Apelin treatment increases complete Fatty Acid oxidation, mitochondrial oxidative capacity, and biogenesis in muscle of insulin-resistant mice

Abstract

Both acute and chronic apelin treatment have been shown to improve insulin sensitivity in mice. However, the effects of apelin on fatty acid oxidation (FAO) during obesity-related insulin resistance have not yet been addressed. Thus, the aim of the current study was to determine the impact of chronic treatment on lipid use, especially in skeletal muscles. High-fat diet (HFD)-induced obese and insulin-resistant mice treated by an apelin injection (0.1 μmol/kg/day i.p.) during 4 weeks had decreased fat mass, glycemia, and plasma levels of triglycerides and were protected from hyperinsulinemia compared with HFD PBS-treated mice. Indirect calorimetry experiments showed that apelin-treated mice had a better use of lipids. The complete FAO, the oxidative capacity, and mitochondrial biogenesis were increased in soleus of apelin-treated mice. The action of apelin was AMP-activated protein kinase (AMPK) dependent since all the effects studied were abrogated in HFD apelin-treated mice with muscle-specific inactive AMPK. Finally, the apelin-stimulated improvement of oxidative capacity led to decreased levels of acylcarnitines and enhanced insulin-stimulated glucose uptake in soleus. Thus, by promoting complete lipid use in muscle of insulin-resistant mice through mitochondrial biogenesis and tighter matching between FAO and the tricarboxylic acid cycle, apelin treatment could contribute to insulin sensitivity improvement.

FIG. 1.

Chronic apelin treatment in HFD mice improved insulin sensitivity. A: Fasted glucose (left) and insulin blood levels (right) in PBS-treated (control, n = 12) and apelin-treated (n = 12) mice before and after the treatment. Results are means ± SEM. *P ≤ 0.05. B: GTT before and at the end of the apelin treatment in HFD mice (n = 12). C: ITT before and at the end of the apelin treatment in HFD mice (n = 8). Results are means ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

FIG. 2.

Chronic apelin treatment in HFD mice increased lipid oxidation in vivo. A: RER measurement during 24 h in insulin-resistant mice chronically treated with PBS (n = 13) or apelin (n = 12). B: RER during the light and dark periods in PBS- (n = 13) and apelin-treated (n = 12) mice. C: Amount of lipid and glucose oxidized during the light and dark periods calculated as described in research design and methods in PBS- and apelin-treated mice. Results are means ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

FIG. 3.

Effect of chronic apelin treatment on palmitate partitioning in muscle of insulin-resistant mice. A: TG and DAG levels in muscle homogenates of PBS-treated (n = 7) and apelin-treated (n = 8) mice. Results are means ± SEM. B: Measure of [¹⁴C]palmitate incorporation into TG in muscle of PBS-treated (n = 11) and apelin-treated (n = 12) mice. Results are means ± SEM. C: Complete (left) and incomplete (right) FAO measured as described in research design and methods. Results are means ± SEM of PBS-treated (n = 11) and apelin-treated (n = 9) mice. **P ≤ 0.01.

FIG. 4.

Chronic apelin treatment in HFD mice increased mitochondrial oxidative capacities and biogenesis in muscle. A: State 2 and State 3 respiration were measured on fresh permeabilized fibers prepared from soleus skeletal muscle of PBS-treated (n = 7) and apelin-treated (n = 7) mice as described in research design and methods. B: Representative Western blot of the different mitochondrial complexes (left) and quantification (right) in PBS-treated (n = 6) and apelin-treated (n = 7) mice. Results are means ± SEM. *P ≤ 0.05. C: Gene expression in soleus muscle of PBS-treated (n = 5) and apelin-treated (n = 5) mice. Results are means ± SEM. *P ≤ 0.05. D: mtDNA quantity calculated as the ratio of COX1 to cyclophilin A DNA levels determined by real-time PCR in soleus of PBS-treated (n = 4) and apelin-treated (n = 4) mice. **P ≤ 0.01. E: Transmission electron microscopy images at magnification ×6,000 and ×25,000 in SS and IMF mitochondria (left). Quantification of mitochondria number relative to the section area (analysis of three images for each mouse) from soleus of PBS-treated (n = 4) and apelin-treated (n = 5) mice (right).

FIG. 5.

The effects of apelin on FAO and mitochondrial biogenesis in muscle are dependent on AMPK activation. A: Phospho-AMPK and phospho-ACC protein expression after PBS (n = 3) or apelin (n = 4) treatment in muscle of insulin-resistant mice. The graph shows the quantified data (n = 4). B: Malonyl-CoA concentration in soleus muscle of PBS-treated (n = 6) or apelin-treated (n = 6) mice. C: Total FAO measured as described in research design and methods in HFD PBS- and apelin-treated WT and AMPK-DN mice. Results are means ± SEM; n = 4 in each group. D: mtDNA quantity calculated as the ratio of COX1 to cyclophilin A DNA levels determined by real-time PCR in soleus of the different mice; n = 4 in each group. E: Gene expression in soleus muscle of PBS- and apelin-treated WT and AMPK-DN mice. Results are means ± SEM; n = 4 in each group. *P < 0.05. **P ≤ 0.01.

FIG. 6.

Effect of chronic apelin treatment in muscle of insulin-resistant mice on acylcarnitine levels and insulin-stimulated glucose uptake. A: Long-chain species acylcarnitine levels were measured in ND-fed mice (n = 5) and in HFD-fed mice treated with apelin (n = 8) or PBS (n = 7). Results are means ± SEM. *P ≤ 0.05. B: Insulin-induced glucose uptake in soleus muscle of PBS-treated (n = 7) and apelin-treated (n = 6) mice. Results are means ± SEM. *P ≤ 0.05, **P ≤ 0.01. 2-DG, 2-deoxyglucose; Prot, protein.