Lipid-induced insulin resistance is prevented in lean and obese myotubes by AICAR treatment

Abstract

The molecular mechanisms of obesity-associated insulin resistance are becoming increasingly clear, and the effects of various lipid molecules, such as diacylglycerol and ceramide, on the insulin signal are being actively explored. To better understand the divergent response to lipid exposure between lean and obese, we incubated primary human muscle cells from lean [body mass index (BMI) <25 kg/m(2)] and morbidly obese (BMI >40 kg/m(2)) subjects with the saturated fatty acid palmitate. Additionally, given that AMPK-activating drugs are widely prescribed for their insulin-sensitizing effects, we sought to determine whether 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR)-stimulated AMPK activation could prevent or reverse the deleterious effects of lipid on insulin signaling. We found that a 1-h palmitate incubation in lean myotubes reduced (P < 0.05) insulin-stimulated phosphoprotein kinase B (Akt), Akt substrate 160 (AS160), and inhibitory factor kappaBalpha (IkappaBalpha) mass, all of which were prevented with AICAR inclusion. With a longer incubation, we observed that myotubes from morbidly obese individuals appear to be largely resistant to the detrimental effects of 16 h lipid exposure as was evident, in contrast to the lean, by the absence of a reduction in insulin-stimulated insulin receptor substrate (IRS)-1 Tyr phosphorylation, phospho-Akt, and phospho-AS160 (P < 0.05). Furthermore, 16 h lipid exposure significantly reduced IkappaBalpha levels and increased phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and IRS1-Ser(312) in lean myotubes only (P < 0.05). Despite a divergent response to lipid between lean and obese myotubes, AICAR inclusion improved insulin signaling in all myotubes. These findings suggest an important role for regular exercise in addition to offering a potential mechanism of action for oral AMPK-activating agents, such as thiazolidinediones and metformin.

Fig. 1.

The effects of 1 h of 0.45 mM palmitate with and without 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) inclusion on pooled myotubes from lean subjects [body mass index (BMI) <25 m/kg²]. Myotubes were incubated in either control media, 0.45 mM palmitate complexed to 1% BSA in low-glucose DMEM with 1 mM carnitine, or lipid + AICAR, for 1 h, in the presence (filled bars) or absence (open bars) of 10 min insulin (100 nM) and probed for phospho (p)-AMP-activated protein kinase (AMPK) (A), p-acetyl-CoA carboxylase (ACC) (B), p-protein kinase B (Akt) (C), p-Akt substrate 160 (AS160) (D), inhibitory factor κBα (IκBα) (E), and insulin receptor substrate (IRS) 1-pSer³¹² (F). AU, arbitrary units. P < 0.05 for basal vs. insulin stimulation within a given treatment (*), lipid or coincubation (lipid + AICAR) vs. control (#), and coincubation vs. lipid alone (♦).

Fig. 2.

The effects of 16 h of 0.45 mM palmitate exposure on pooled myotubes from lean (BMI <25 m/kg²) and morbidly obese (BMI >40 m/kg²) humans. Myotubes were incubated in control media (open bars) or 0.45 mM palmitate complexed to 1% BSA in low-glucose DMEM with 1 mM carnitine (filled bars) in the presence or absence of 10 min insulin (100 nM) and probed for IRS1-pTyr (A), pAkt (B), pAS160 (C), IκBα (D), p-c-Jun NH₂-terminal kinase (JNK) (E), or IRS1-pSer³¹² (F). P < 0.05 for basal vs. insulin stimulation within a given treatment (*), lipid vs. control (#), and obese vs. lean within a given treatment (♦).

Fig. 3.

The effects of 16 h of 0.45 mM palmitate and AICAR exposure on pooled myotubes from lean (BMI <25 m/kg²) and morbidly obese (BMI >40 m/kg²) humans. Myotubes were incubated in 0.45 mM palmitate complexed to 1% BSA in low-glucose DMEM with 1 mM carnitine (white bars), lipid + 4 h AICAR (gray bars), or lipid + 16 h AICAR (black bars) in the presence or absence of 10 min insulin (100 nM) and probed for pAMPK (A), pACC (B), IRS1-pTyr (C), pAkt (D), pAS160 (E), IκBα (F), pJNK (G), and IRS1-pSer³¹² (H). P < 0.05 for basal vs. insulin stimulation within a given treatment (*), coincubations (4 and 16 h) vs. lipid alone within a given group (#), and obese vs. lean within a given treatment (♦).