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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 1, 2004 as doi:10.1096/fj.03-1067fje. |
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Skeletal Muscle Research Laboratory, School of Medical Sciences, Royal Melbourne Institute of Technology, Bundoora;
* St. Vincent’s Institute of Medical Research, Fitzroy; and
School of Health Sciences, Deakin University, Burwood, Australia
1 Correspondence: Skeletal Muscle Research Laboratory, School of Medical Sciences, RMIT University, P.O. Box 71, Bundoora 3083, Victoria, Australia. E-mail: matthew.watt{at}rmit.edu.au
SPECIFIC AIMS
Intramuscular triacylglycerol (IMTG) is an important metabolic substrate for skeletal muscle during contraction and accumulates with insulin resistance and type 2 diabetes. Hormone-sensitive lipase (HSL) is the rate-limiting enzyme for the degradation of triacylglycerol. Although regulation of HSL has been well studied in adipose tissue, there is a relative paucity of research on its regulation in skeletal muscle. Although hormonal factors such as ß-adrenergic stimulation and circulating insulin are the major factors mediating HSL in adipose tissue, local factors in active skeletal muscle may render the effect of hormones on skeletal muscle HSL as relatively unimportant. In adipose tissue, AMPK phosphorylates the "inhibitory" Ser 565 site, which in turn prevents phosphorylation of Ser 563, indicating that AMPK exerts an antilipolytic effect that may override the stimulation by epinephrine. However, AMPK is expected to be a more important mediator of HSL activity in skeletal muscle than in adipose tissue because of the marked energy turnover in this tissue. In view of the central role of AMPK in fat metabolism and the association between IMTG accumulation and the etiology of insulin resistance, the aim of this study was to investigate whether AMPK is a key regulator of HSL activity capable of overriding ß-adrenergic stimulation in skeletal muscle. We hypothesized that increasing AMPK activity would decrease HSL activity and IMTG degradation during exercise.
PRINCIPAL FINDINGS
1. AMPK decreases HSL activity in human skeletal muscle despite augmented epinephrine in vivo
We first determined the effect of AMPK on HSL activity during muscle contraction in human skeletal muscle in vivo. AMPK activity was manipulated in humans by lowering intramuscular glycogen stores prior to exercise. Male volunteers visited the laboratory on two occasions, exhibiting either normal (CON) or low (LG) resting muscle glycogen content, then cycled for 60 min at 70% of peak pulmonary oxygen uptake. AMPK 
-1 activity was not different between trials and was unaffected by exercise. AMPK -2 activity was not different between trials at rest. Whereas AMPK -2 activity increased 3.7-fold (P<0.05) during exercise in LG, no change was observed in CON. Free AMP was elevated in LG compared with CON. HSL activity was not different between trials at rest. HSL activity increased during exercise in CON; this effect was abolished during LG (Fig. 1
). A strong negative linear relationship (P=0.02) was observed between AMPK -2 activity and HSL activity during exercise. There were no differences in exercise content of phosphorylated ERK1/2, and conventional and atypical PKC between trials, whereas plasma epinephrine was markedly elevated in LG compared with CON during exercise (CON: 1.96±0.29, LG: 4.25±0.60 nM). These results demonstrate that despite increased plasma epinephrine, a known ß-adrenergic stimulus of HSL, the exercise-induced increase in HSL activity is abolished when AMPK activity is elevated. The attenuated HSL response to exercise does not appear to be related to disrupted PKC-ERK signaling.
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2. AMPK completely attenuates ß-adrenergic stimulation of HSL in L6 myotubes
To confirm the inhibitory effect of AMPK on HSL activity, we performed experiments in serum-starved L6 myotubes. The AMPK agonist 5-aminoimidazole-4-carboxamide did not affect HSL activity. HSL activity was elevated 
50% from control in cells treated with epinephrine (10 nM–1 µM); however, coincubation with epinephrine and AICAR (2 mM) completely attenuated the increase observed with epinephrine alone. Therefore, these data show that AMPK is a major regulator of HSL activity that is capable of overriding ß-adrenergic stimulation.
3. IMTG degradation is dissociated from HSL activity
We also determined whether increasing HSL activity above rest was mandatory for IMTG degradation. Muscle samples were analyzed for HSL activity and IMTG content. IMTG content was not different between trials at rest. IMTG content was unchanged after 60 min of exercise in CON but was decreased by 20% in LG. These data demonstrate that in circumstances where AMPK is elevated, IMTG degradation is augmented despite reduced HSL activity.
CONCLUSIONS AND SIGNIFICANCE
Triacylglycerols in skeletal muscle myocytes represent an important energy depot and their accumulation is associated with insulin resistance. The regulation of skeletal muscle HSL is complex and is thought to involve both intrinsic factors related to the free energy charge potential and ß-adrenergic mechanisms. This work represents a significant advance in understanding cellular skeletal muscle triacylglycerol degradation. We have demonstrated for the first time that AMPK is a major regulator of HSL activity that is capable of overriding ß-adrenergic stimulation in skeletal muscle in vivo. In light of our findings, we propose that "triacylglycerol lipase" is a name that more aptly reflects the regulation of this enzyme in muscle, given that hormonal affects are overridden by intrinsic factors in vivo. We found that in circumstances where AMPK is elevated, intramuscular triacylglycerol utilization is augmented despite reduced HSL activity. Thus, the physiological importance of activating HSL for triacylglycerol degradation is questioned and the regulation of this process needs to be elucidated. Moreover, these data support the contention of multiple triacylglycerol lipases in skeletal muscle.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1067fje; doi: 10.1096/fj.03-1067fje
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