Figure 3. Fgf21 induction in skeletal muscle of Cpt1b^m−/− mice is AMPK- and Akt1-dependent.

(A–B) Gene expression of Fgf21 in mouse primary myotubes treated with an Akt1 inhibitor, A-674563 (30μM) and an AMPK inhibitor, Compound C (CompC, 30μM) for 24h. (A) Results shown are representative of three independent cultures. *P < 0.05 between Cpt1b^m−/− and Cpt1b^fl/fl mice, and ^#P < 0.05 between treatments with vehicle versus inhibitors. (B) Primary muscle cells from Cpt1b^fl/fl mice. Myotubes were treated with BSA-conjugated FA (0.5 mM) in presence of the Cpt1 inhibitor Etomoxir (Et, 100μM) for 24h. Results shown are representative of 3–4 separate experiments*P < 0.05 between control (BSA) versus other treatments, and ^#P < 0.05 due to Akt1 or AMPK inhibitors. (C) Gene expression of FGF21 in human myotubes treated with BSA-conjugated FA (0.5mM) and inhibitors: Etomoxir (Et, 100μM), A-674563 (50μM), and Compound C (CompC, 30μM) for 24h. Human skeletal muscle myoblasts (HSMM) derived from non-diabetic-lean (Normal-Lean) and diabetic-obese subjects were differentiated into myotubes prior to treatments (n=4 per group). Results shown are representative of two independent cultures of HSMM per subject. *P < 0.05 between treatments with BSA-conjugated FA (FA) versus inhibitors, and ^#P < 0.05 due to Akt1 or AMPK inhibitors, and ^&P < 0.05 between myotubes from normal-lean and diabetic-obese subjects. (D) Gene expression of FGF21 in human myotubes treated with BSA-conjugated FA (0.5mM) and Etomoxir (Et, 100μM) and PPAR-inhibitors: GW6471 (for PPARα, 10μM), T0070907 (for PPARγ, 10μM) and GSK3787 (for PPARδ, 10μM) for 24h. *P < 0.05 between treatments with inhibitors versus BSA-control. (E) Activity of mTORC1 and its downstream signaling members P70S6K, IRS1, and basal Akt, as determined by phosphorylation in human myotubes. HSMM originated from normal-lean subjects were differentiated into myotubes prior to treatment with BSA-conjugated FA (0.5mM) and rhFGF21 (200 ng/ml). *P < 0.05 between vehicle (PBS) versus other treatments. All data are presented as means ± s.e.m.,