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Hormone-sensitive lipase – quantitation of enzyme and mRNA in small biopsies of human adipose tissue
Hormone-sensitive lipase (HSL) activity was first identified as an adrenaline-sensitive lipolytic activity in adipose tissue. Hormonal activation of HSL occurs via cAMP-dependent protein kinase, which phosphorylates HSL. HSL is the enzyme responsible for the release of free fatty acids (FFAs) from adipose tissue and, therefore, plays a pivotal role in providing the major source of energy for most tissues. Although its expression is highest in adipose tissue, HSL is also expressed in the adrenal glands, ovaries, testes and, to a lesser extent, in skeletal and cardiac muscle and macrophages. The aim of this article is to describe a modified method of determination of HSL activity and gene expression in 10 mg of frozen human adipose tissue samples obtained by biopsy. The enzymes were extracted from the frozen tissue and activity was tested against a fatty acid-labelled diacylglycerol emulsion. The released fatty acids were extracted using a liquid–liquid partition system. The mean±standard error of the mean (SEM) HSL activity in normal fasting individuals was 2.38±0.75 milliunits (mU) oleate/min/g at 37°C. HSL mRNA levels were assessed in 10 mg of adipose tissue using a competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay and the mean ratio of HSL mRNA molecules to β2-microglobulinmRNA molecules in normal individuals was 0.29±0.04 (mean±SEM). The methods described here are suitable for use in adipose tissue biopsies obtained from normal individuals and provide a means for the examination of the regulation of this enzyme. The importance of this is becoming increasingly apparent, as the inability to suppress lipolysis is associated with conditions of insulin resistance and an increase in propensity to atherosclerosis. This is associated with inappropriate release of FFAs in the postprandial period, resulting in reduced sensitivity of glucose metabolism and increased postprandial lipaemia.