AMP kinase activation with AICAR simultaneously increases fatty acid and glucose oxidation in resting rat soleus muscle

Abstract

5-Amino-4-imidazolecarboxamide riboside (AICAR), a pharmacological activator of AMP-activated protein kinase (AMPK), acutely stimulates glucose uptake and fatty acid (FA) oxidation in skeletal muscle. However, it is not fully understood whether AICAR-induced changes in glucose oxidation are secondary to changes in FA oxidation (i.e. glucose fatty acid cycle), or what role AMPK may be playing in the regulation of intramuscular triacylglycerol (TAG) esterification and hydrolysis. We examined the acute (60 min) effects of AICAR (2 mm) on FA metabolism, glucose oxidation and pyruvate dehydrogenase (PDH) activation in isolated resting rat soleus muscle strips exposed to two different FA concentrations (low fatty acid, LFA, 0.2 mm; high fatty acid, HFA, 1 mm). AICAR significantly increased AMPK alpha2 activity (+192%; P<0.05) over 60 min, and simultaneously increased both FA (LFA: +33%, P<0.05; HFA: +36%, P<0.05) and glucose (LFA: +105%, P<0.05; HFA: +170, P<0.001) oxidation regardless of FA availability. While there were no changes in TAG esterification, AICAR did increase the ratio of FA partitioned to oxidation relative to TAG esterification (LFA: +15%, P<0.05; HFA: +49%, P<0.05). AICAR had no effect on endogenous TAG hydrolysis and oxidation in resting soleus. The stimulation of glucose oxidation with AICAR was associated with an increase in PDH activation (+126%; P<0.05) but was without effect on pyruvate, an allosteric activator of the PDH complex, suggesting that AMPK may stimulate PDH directly. In conclusion, AMPK appears to be an important regulator of both FA metabolism and glucose oxidation in resting skeletal muscle.

Figure 1. Incubation protocol for time course experiments for AMP kinase (AMPK), pyruvate dehydrogenase activation (PDHa) and pyruvate (A), fatty acid (FA) metabolism (B), and glucose oxidation (C)

For time course experiments (A) soleus strips were incubated in the presence or absence of AICAR (2 mm) and muscle strips were freezeclamped at the time points indicated for further analysis of AMPK α1 and α2 activities, PDHa and pyruvate concentration. For FA metabolism (B), rat soleus muscle strips were incubated (30°C) in modified Krebs-Henseleit buffer (KHB) through four stages: preincubation (PREINC), pulse ([9,10-³H]palmitate), wash and chase ([1-¹⁴C]palmitate) to monitor endogenous and exogenous FA metabolism. During wash and chase (last 60 min), soleus strips were incubated in the presence or absence of AICAR (2 mm), in KHB with low fatty acid (LFA, 0.2 mm) or high fatty acid (1 mm) concentration. For glucose oxidation (C), soleus strips were incubated in the presence or absence of AICAR (2 mm), in KHB with low fatty acid (LFA, 0.2 mm) or high fatty acid (1 mm) concentration throughout the protocol (60 min) and glucose oxidation was monitored during the last 30 min.

Figure 2. Time course for AMPK α1 (A) and α2 (B) activity in the presence or absence of AICAR in isolated rat soleus muscle

Values are means ± s.e.m., pmol min⁻¹ (mg protein)⁻¹, n = 5–8 per group. *Significantly different from 30 min of same condition (P < 0.05). †Treatment effect of AICAR significantly different from No AICAR (P < 0.05).

Figure 3. Effect of AICAR on fatty acid oxidation and TAG esterification in isolated rat soleus muscle

Effect of AICAR on fatty acid oxidation in low fatty acid (0.2 mm, LFA: A) or high fatty acid (1 mm, HFA: B) modified KHB, on TAG esterification (LFA: C; HFA: D) and on oxidation: TAG esterification ratio (LFA: E; HFA: F). Values are means ± s.e.m., nmol (g wet wt)⁻¹, n = 7 per group. *Significantly different from No AICAR (P < 0.05).

Figure 4. Effect of AICAR on glucose oxidation in isolated rat soleus muscle

Effect of AICAR on glucose oxidation in low fatty acid (0.2 mm: A) or high fatty acid (1 mm: B) modified KHB. Values are means ± s.e.m., nmol (g wet wt)⁻¹, n =6–8 per group. *Significantly different from No AICAR (P < 0.05).

Figure 5. Effect of AICAR on time course for pyruvate dehydrogenase activation and pyruvate content in isolated rat soleus muscle

Time course for pyruvate dehydrogenase activation (PDHa, A) and pyruvate content (B) in the presence or absence of AICAR. Values are means ± s.e.m., n = 6–8 per group. *Significantly different from 30 min of same condition (P < 0.05); **Significantly different from 30 min of different condition (P < 0.05). †Trial effect of AICAR being significantly different from No AICAR (P < 0.05).