In mice and humans, deficiency of ATGL, the rate-limiting TG hydrolase, revealed massive TG accumulation in multiple tissues, including liver and skeletal muscle. Although the expression of ATGL is very low compared to white adipose tissue, ATGL deficiency causes reduced hepatic TG hydrolysis in the liver. Hepatocytes lacking ATGL exhibited an increase of fatty acid (FA) incorporation into TG and reduced release of FA and TG compared to wild-type cells. Conversely, overexpression of ATGL in hepatocytes led to depletion of TG stores. A specific overexpression of ATGL in the liver (liv-ATGL) in vivo resulted in reduced liver TG content. Liv-ATGL mice exhibited decreased VLDL synthesis, indicating that ATGL affects liver TG mobilisation, but showed no changes on mRNA levels of specific genes involved in energy production. Furthermore, ATGL expression and activity is strongly regulated by nutritional state, resulting in a massive upregulation of ATGL protein expression during fasting.Furthermore the influence of ATGL deficiency on energy availability and substrate utilization in working muscle was studied by using a treadmill. Blood energy metabolites and liver glycogen stores were determined. Since ATGL-deficient mice exhibit massive TG accumulation in the heart, we studied ATGL-deficient mice specifically overexpressing ATGL in the cardiac muscle (ATGL-deficient/CM). In contrast to ATGL-deficient mice, these mice did not accumulate TG in the heart. Exercise experiments revealed that ATGL-deficient and ATGL-deficient/CM mice are unable to increase circulating FA levels during exercise. The reduced availability of FA for energy conversion led to rapid depletion of liver glycogen stores and hypoglycemia. These data suggested that the increased energy requirements of the working muscle result in an increased use of carbohydrates for energy conversion. Thus, ATGL activity is required for proper energy supply of the skeletal muscle during exercise.