Hormone-sensitive Lipase mRNA Expression Research Articles

Sterol-mediated regulation of hormone-sensitive lipase in 3T3-L1 adipocytes.

We previously reported that intracellular free cholesterol at physiological concentrations regulates the activity of neutral cholesterol esterase (N-CEase) in macrophages. The objective of the present study is to investigate whether the regulation of N-CEase by cholesterol is generally observed in other types of cells such as adipocytes with high activity of hormone-sensitive lipase (HSL), the same gene product as N-CEase. 3T3-L1 adipocytes were cultured with and without cholesterol (1-30 microg/mL) or 25-hydroxycholesterol (0.1-10 microg/mL), and changes in the N-CEase activity, expression of HSL mRNA, and protein were examined. Incubation (24 h) of cells with cholesterol did not change N-CEase activity, but incubation with 25-hydroxycholesterol decreased the activity in a concentration-dependent manner by 24 (24 h) and 54% (36 h). Quantitative reverse transcription-PCR indicated that 25-hydroxycholesterol (10 microg/mL) did not influence expression of HSL mRNA. However, Western blot analysis showed that this sterol reduced HSL protein by 72 (24 h) and by 93% (36 h), respectively. It was concluded that sterol-mediated regulation of HSL/N-CEase occurs not only in macrophages but also in adipocytes, and regulation appears to occur not at a transcriptional level but by a post-transcriptional process. Sterol-mediated proteolysis may be involved in the loss of HSL protein.

Hormone sensitive lipase expression and adipose tissue metabolism show gender difference in obese subjects after weight loss.

The effect of weight reduction on hormone sensitive lipase (HSL) and lipoprotein lipase (LPL) gene expression and their relationship with adipose tissue metabolism were studied in massively obese men and women. Seventeen obese subjects (eight men, nine women) participated in the study (age 44+/-2 y, weight 145+/-8 kg, fat 40+/-2% of body mass, mean+/-s.e.m.), who were going through a gastric-banding operation for weight reduction. HSL and LPL mRNA expressions were analyzed using the reverse transcription competitive polymerase chain reaction. Subcutaneous fat lipolysis was measured in vivo by microdialysis and in vitro in isolated subcutaneous abdominal adipocytes. Measurements were done before and after 1 y of weight reduction. Significant reductions in weight (for men -20.3+/-2.5%, for women -18.3+/-2.1% (mean+/-s.e.m.) and fat mass (for men -27.6+/-7.9%, for women -21.8+/-3.9%) were observed in both genders. In women HSL mRNA expression decreased by 31% (P=0.008) and LPL expression increased slightly, but nonsignificantly (42%, P=0.110). These changes were not observed in men. In men, inhibition of lipolysis with alpha(2)-adrenergic and adenosine agonist was improved (P=0.001) in isolated adipocytes. This study uncovers new differences between genders in adipocyte metabolism along with weight reduction. In women, the observed changes in HSL and LPL gene expression suggest that deposition of lipids into adipose tissue might be favored after weight reduction. In men, the results indicate improved responsiveness to inhibition in adipose tissue metabolism along with weight reduction.

Subcellular localization of insulin receptor substrate family proteins associated with phosphatidylinositol 3-kinase activity and alterations in lipolysis in primary mouse adipocytes from IRS-1 null mice.

To clarify the roles of insulin receptor substrate (IRS) family proteins in phosphatidylinositol (PI) 3-kinase activation and insulin actions in adipocytes, we investigated the intracellular localization of IRS family proteins and PI 3-kinase activation in response to insulin by fractionation of mouse adipocytes from wild-type and IRS-1 null mice. In adipocytes from wild-type mice, tyrosine-phosphorylated IRS-1 and IRS-2, which were found to associate with PI 3-kinase in response to insulin, were detected in the plasma membrane (PM) and low-density microsome (LDM) fractions. By contrast, tyrosine-phosphorylated IRS-3 (pp60), which was found to associate with PI 3-kinase, was predominantly localized in the PM fraction. In adipocytes from IRS-1-null mice, insulin-stimulated PI 3-kinase activity in anti-phosphotyrosine (alphaPY) immunoprecipitates in the LDM fraction was almost exclusively mediated via IRS-2 and was reduced to 25%; however, insulin-stimulated PI 3-kinase activity in the PM fraction was primarily mediated via IRS-3 and was reduced to 60%. To determine the potential functional impact of the distinct subcellular localization of IRSs and associating PI 3-kinase activity on adipocyte-specific metabolic actions, we examined lipolysis in IRS-1 null mice. The level of isoproterenol-induced lipolysis was increased 5.1-fold in adipocytes from IRS-1 null mice as compared with wild-type mice. Moreover, hormone-sensitive lipase (HSL) protein was increased 4.3-fold in adipocytes from IRS-1-null mice compared with wild-type mice, and HSL mRNA expression was also increased. The antilipolytic effect of insulin in IRS-1 null adipocytes, however, was comparable to that in wild-type mice. Thus, discordance between these two insulin actions as well as the transcriptional and translational effect (HSL mRNA and protein regulation) and the PM effect (antilipolysis) of insulin may be explained by distinct roles of both PI 3-kinase activity associated with IRS-1/IRS-2 and PI 3-kinase activity associated with IRS-3 in insulin actions related to their subcellular localization.

Hormone sensitive lipase mRNA in both monocyte and macrophage forms of the human THP-1 cell line

The identity of the neutral cholesteryl ester hydrolase (CEH) in human monocyte/macrophages is uncertain. Prior studies indicate that hormone sensitive lipase (HSL) is a major CEH in mouse macrophages, and that HSL mRNA is present in human THP-1 monocytes. In the present study, HSL mRNA expression was examined in THP-1 cells as a function of differentiation status and cholesterol enrichment. By RT-PCR with primer pairs that span exon boundaries, HSL mRNA was demonstrated in THP-1 monocytes and phorbol-ester differentiated THP-1 macrophages. cDNA identities were confirmed by sequencing. By Northern blotting, with HSL cDNA as probe, THP-1 monocytes were found to contain HSL mRNA of approximately 3 and 3.9 kb. In THP-1 macrophages, the 3 kb mRNA was greatly diminished, while the level of the 3.9 kb mRNA was maintained. mRNA of approximately 3 and 3.9 kb are those expected of the 86-kDa (adipocyte) and 117-kDa (testicular) HSL isoforms, respectively. The presence of the testicular isoform mRNA was confirmed in THP-1 cells by amplification and sequencing of an isoform-specific cDNA. Additionally, Northern-blot comparisons showed that the 3 and 3.9 kb mRNA in THP-1 comigrated with the HSL mRNA in 3T3-L1 adipocytes and rat testis, respectively. The level of the 3.9 kb mRNA did not vary greatly with cholesterol enrichment. Thus, the HSL gene is transcribed in THP-1 cells both before and after differentiation into macrophages; after differentiation, the predominant mRNA is that for the 117-kDa isoform. This isoform is a CEH, and may mediate some CE turnover in THP-1 cells.

Free fatty acids are involved in the inverse relationship between hormone-sensitive lipase (HSL) activity and expression in adipose tissue after high-fat feeding or beta3-adrenergic stimulation.

The hormone-sensitive lipase (HSL) is the rate-limiting enzyme in adipose tissue lipolysis. The aim of this experimental trial was to study the effects of a beta3-adrenergic agonist (Trecadrine) on plasma fatty acids, adipocyte HSL activity, and gene expression in control and cafeteria-induced obese animals. Control and cafeteria-fed rats were treated with a placebo or Trecadrine during 35 days. Plasma fatty acids were measured by an enzymatic method, whereas HSL activity was assessed by using labeled triolein as substrate. Finally, HSL gene expression from white adipose tissue (WAT) was determined using a reverse transcription-polymerase chain reaction method. Trecadrine administration reduced plasma fatty acids and HSL mRNA levels in abdominal WAT, whereas HSL activity was significantly higher in the Trecadrine-treated obese rats than in the obese nontreated rats. Also, abdominal WAT HSL activity significantly increased, whereas WAT HSL gene expression fell in control rats treated with beta3-adrenergic agonist as compared with control untreated animals. In situations of fat accumulation (high-fat feeding) or lipid mobilization (beta3-adrenergic stimulation), changes in HSL activity and HSL gene expression seem to follow a trend related to plasma fatty acids levels, as indicated by the positive correlation (r = 0.39, p < 0.05) between HSL mRNA levels and plasma fatty acids, and the negative correlation (r = -0.38, p < 0.05) between plasma fatty acids and HSL activity. Furthermore, a highly negative correlation (r = -0.59, p < 0.001) between HSL activity and HSL mRNA expression was found, in which plasma-free fatty acids are apparently involved.

Hormone-sensitive lipase expression and activity in relation to lipolysis in human fat cells

Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step in adipocyte lipolysis. The activity of HSL is thought to be primarily regulated by reversible phosphorylation. However, the regulation of HSL activity by pretranslational mechanisms has been poorly studied. The present studies were undertaken to explore the relationship between the levels of HSL protein and mRNA expressions and the lipolytic capacity. The study was performed in human abdominal subcutaneous adipocytes with identical sizes but having either a high (HL) or low (LL) lipolytic capacity (n = 16). Basal and maximal lipolysis induced by catecholamines, an adenylyl cyclase activator forskolin, and a cyclic AMP analogue dibutyryl cAMP were 50% lower in LL- in comparison with HL-fat cells (P < 0.05 or better). No differences in drug sensitivity were found. HSL activity and quantity were about 50% lower in LL- compared with HL-fat cells (P < 0.05). Moreover, the mRNA ratio between HSL and γ-actin was 35% lower in LL-compared with HL-fat cells (P < 0.05). There was a strong linear correlation between the protein and enzymatic HSL measurements (r2 = 0.91). In addition, the maximum lipolytic capacity was significantly correlated with HSL activity (r2 = 0.75) and HSL protein amount (r2 = 0.64).▪ It is concluded that hormone-sensitive lipase (HSL) expression, measured either as total HSL protein by Western blot analysis or as total amount of activatable HSL enzyme, is a major determinant of the maximum lipolytic capacity of human fat cells. In addition, HSL protein expression is at least, in part, determined by HSL mRNA expression.—Large, V., P. Arner, S. Reynisdottir, J. Grober, V. Van Harmelen, C. Holm, and D. Langin. Hormone-sensitive lipase expression and activity in relation to lipolysis in human fat cells.

Evidence for hormone-sensitive lipase mRNA expression in human monocyte/macrophages.

The role of hormone-sensitive lipase (HSL) in the hydrolysis of adipose tissue triacylglycerol to provide free fatty acids for energy requirements has been well established. However, the role of HSL in other tissues, including macrophages, is not well understood. The demonstration that HSL is capable of hydrolyzing cholesteryl esters at approximately the same rate as triacylglycerol raised the possibility that HSL activity in macrophages may influence the accumulation of cholesteryl esters in foam cells of atherosclerotic lesions. We and others have previously demonstrated that HSL mRNA is expressed in murine peritoneal macrophages and macrophage cell lines; however, it was previously reported that HSL mRNA is absent in human monocyte-derived macrophages, suggesting that a species difference may exist. To clarify this point, we have further examined the issue of HSL mRNA expression in human macrophages. In the current study, we demonstrate that HSL mRNA is detectable in human monocyte-derived macrophages and in the THP-1 human monocyte cell line using reverse transcription coupled to polymerase chain reaction (RT-PCR). Specific amplification of cDNA derived from mRNA was ensured by using primers that span an intron within the human HSL gene, and the identity of PCR products as HSL was confirmed by hybridization to HSL cDNA and by DNA sequencing. Using a semiquantitative PCR assay, we establish that HSL mRNA levels in monocyte/macrophages are approximately 1/40 the levels in human adipose tissue. These results indicate that further studies addressing the role of HSL in macrophage metabolism and its potential role in development of foam cells in human atherosclerotic lesions are warranted.

Expression of hormone-sensitive lipase mRNA in macrophages.

Macrophages contain a neutral cholesteryl ester hydrolase that can be activated by cAMP-dependent protein kinase. Immunological studies strongly suggest that hormone-sensitive lipase (HSL) is probably responsible for the cholesteryl ester hydrolase activity in macrophages; however, due to the very low level of expression in macrophages, it has been difficult to determine whether the macrophage cholesteryl ester hydrolase and adipose HSL are, in fact, products of the same gene. We have used the sensitive polymerase chain reaction (PCR) technique to demonstrate expression of HSL mRNA in resident and thioglycollate-elicited mouse peritoneal macrophages, as well as in the P388D1 mouse macrophage cell line. PCR was performed using oligonucleotide primer sequences present on adjacent exons of the mouse HSL gene to allow discrimination between products derived from HSL mRNA or genomic DNA sequences; specificity of the PCR was demonstrated by the absence of a product in liver, which does not express HSL mRNA. Northern blot analysis of poly (A)+ RNA from peritoneal macrophages with a mouse adipose HSL cDNA probe demonstrated a low abundance of mRNA of 3.2 kb, identical in size to HSL mRNA in adipose tissue. These findings, together with the results of previous studies demonstrating similarities between HSL and macrophage neutral cholesteryl ester hydrolase, strongly support the conclusion that both are products of a single gene. The development of a PCR assay for HSL mRNA may allow further study of the regulation of neutral cholesteryl ester hydrolase expression in macrophages and foam cells, and its potential role in atherogenesis.

Increased expression of the mRNA for hormone-sensitive lipase in adipose tissue of cancer patients

The expression of genes coding for regulatory enzymes involved in the uptake, synthesis and mobilisation of lipid was measured in adipose tissue of cancer patients. Total RNA was isolated from subcutaneous adipose tissue of control and cancer patients and the various mRNAs measured by Northern blot analysis. The total lipoprotein lipase enzymic activity and the relative levels of the mRNAs for lipoprotein lipase and for fatty acid synthase were not significantly different between cancer patients and control patients. However, there was a significant two-fold increase in the relative level of mRNA for hormone-sensitive lipase (HSL) in adipose tissue of cancer patients compared with control patients. The cancer patients also exhibited a two-fold elevation in serum triacylglycerol levels and serum free fatty acid levels. There was a significant correlation between the serum free fatty acid level and expression of HSL mRNA in the adipose tissue. The serum levels of insulin and tumour necrosis factor-α were not different between cancer and control patients. The results suggest that at least one of the mechanisms for depletion of lipid from adipose tissue in cancer patients operates at the level of increased expression of mRNA of the lipolytic regulatory enzyme, hormone-sensitive lipase.