Apelin is necessary for the maintenance of insulin sensitivity

Abstract

The recently discovered peptide apelin is known to be involved in the maintenance of insulin sensitivity. However, questions persist regarding its precise role in the chronic setting. Fasting glucose, insulin, and adiponectin levels were determined on mice with generalized deficiency of apelin (APKO). Additionally, insulin (ITT) and glucose tolerance tests (GTT) were performed. To assess the impact of exogenously delivered apelin on insulin sensitivity, osmotic pumps containing pyroglutamated apelin-13 or saline were implanted in APKO mice for 4 wk. Following the infusion, ITT/GTTs were repeated and the animals euthanized. Soleus muscles were harvested and homogenized in lysis buffer, and insulin-induced Akt phosphorylation was determined by Western blotting. Apelin-13 infusion and ITTs/GTTs were also performed in obese diabetic db/db mice. To probe the underlying mechanism for apelin's effects, apelin-13 was also delivered to cultured C2C12 myotubes. 2-[3H]deoxyglucose uptake and Akt phosphorylation were assessed in the presence of various inhibitors. APKO mice had diminished insulin sensitivity, were hyperinsulinemic, and had decreased adiponectin levels. Soleus lysates had decreased insulin-induced Akt phosphorylation. Administration of apelin to APKO and db/db mice resulted in improved insulin sensitivity. In C2C12 myotubes, apelin increased glucose uptake and Akt phosphorylation. These events were fully abrogated by pertussis toxin, compound C, and siRNA knockdown of AMPKalpha1 but only partially diminished by LY-294002 and not at all by L-NAME. We conclude that apelin is necessary for the maintenance of insulin sensitivity in vivo. Apelin's effects on glucose uptake and Akt phosphorylation are in part mediated by a G(i) and AMPK-dependent pathway.

Fig. 1.

A and B: insulin (1 U/kg) and glucose (2 g/kg) tolerance tests for generalized apelin-null (APKO) mice; n = 9–14 animals/group. C: representative Western blots of soleus muscle lysates from wild-type and APKO mice at baseline and with insulin stimulation, probing for phosphorylated Akt (p-Akt). Quantification and normalization of band intensity to native Akt is depicted below. *P < 0.05 vs. baseline, †P < 0.05 vs. insulin-treated wild type; n = 3–4 animals/group. D and E: insulin (1.25 U/kg) and glucose (2 g/kg) tolerance tests for APKO mice with diet-induced insulin resistance; n = 6–7 animals/group. F: representative Western blots of soleus lysates from diet-induced insulin-resistant wild-type and APKO mice, probing for p-Akt and normalizing to native Akt. *P < 0.05 vs. baseline; n = 4 animals/group. For all experiments, results are expressed as means ± SE within each group.

Fig. 2.

A and B: insulin (1 U/kg) and glucose (2 g/kg) tolerance tests for generalized APKO mice treated with pyroglutamated apelin-13 or saline for 2 (A) and 4 wk (B). A matched group of wild-type mice was also monitored during the experiment; n = 5–7 animals/group. Results are expressed as means ± SE within each group. C: representative Western blots of soleus muscle lysates from wild-type and APKO mice at baseline and with insulin, probing for p-Akt. Normalization of band intensity to native Akt is depicted at bottom. *P < 0.05 vs. baseline, †P < 0.05 vs. insulin-treated saline mice; n = 3–4 animals/group. For all experiments, results are expressed as means ± SE within each group.

Fig. 3.

Insulin (A; 3 U/kg) and glucose (B; 2 g/kg) tolerance tests for obese C57BL/KLS-lepr^db/lepr^db (db/db) mice treated with pyroglutamated apelin-13 or saline for 2 wk. For all experiments, n = 5–7 animals/group. Results are expressed as means ± SE within each group.

Fig. 4.

A: [2-³H]glucose uptake in cultured C₂C₁₂ myotubes at baseline (left) and with gradually increasing doses of pyroglutamated apelin-13 (right). Samples were incubated for 2 h. †P < 0.05 vs. baseline. B: [2-³H]glucose uptake in C₂C₁₂ myotubes with increasing exposure times to apelin (1 μM). †P < 0.05 vs. 0 min. C: [2-³H]glucose uptake in C₂C₁₂ myotubes treated with apelin (1 μM), insulin (100 nM), LY-294002 (1 mM), pertussis toxin (PTX; 10 ng/ml), compound C (CC; 1 μM), and/or N^ω-nitro-l-arginine methyl ester (l-NAME; 1 mM). †P < 0.01 vs. baseline; *P < 0.05 vs. apelin alone; §P < 0.01 vs. insulin. Results are expressed as means ± SE of 9–13 independent experiments.

Fig. 5.

A and B: p-Akt as determined by Western blotting of C₂C₁₂ lysates exposed to apelin and/or insulin for 30 min (A) and 2 h (B). *P < 0.05 vs. control no insulin; †P < 0.01 vs. control no insulin. C: p-Akt as determined by Western blotting of apelin-treated C₂C₁₂ lysates exposed to LY-294002, PTX, compound C, and l-NAME. *P < 0.05 vs. apelin alone. D: acetyl-CoA carboxylase phosphorylation (p-ACC) as determined by Western blotting of C₂C₁₂ lysates exposed to apelin for 2 h. †P < 0.05 vs. control. E: Akt phosphorylation of lysates derived from C₂C₁₂ cells transfected with siRNAs directed against AMPKα1 and a scrambled sequence. †P < 0.01 vs. scramble. For A, B, C, and E, normalization of band intensity to native Akt is depicted below each blot. For D, normalization to GAPDH is depicted below the blot. All results are expressed as means ± SE of 3–5 independent experiments.