Hans Tornqvist

Insulin and Cortisol Promote Leptin Production in Cultured Human Fat Cells

The aim of this study was to investigate the regulation of leptin expression and production in cultured human adipocytes using the model of in vitro differentiated human adipocytes. Freshly isolated human preadipocytes did not exhibit significant leptin mRNA and protein levels as assessed by reverse transcriptase (RT)-polymerase chain reaction (PCR) and radioimmunoassay (RIA). However, during differentiation induced by a defined adipogenic serum-free medium, cellular leptin mRNA and leptin protein released into the medium increased considerably in accordance with the cellular lipid accumulation. In fully differentiated human fat cells, insulin provoked a dose-dependent rise in leptin protein. Cortisol at a near physiological concentration of 10−8 mol/l was found to potentiate this insulin effect by almost threefold. Removal of insulin and cortisol, respectively, was followed by a rapid decrease in leptin expression, which was reversible after readdition of the hormones. These results clearly indicate that both insulin and cortisol are potent and possibly physiological regulators of leptin expression in human adipose tissue.

Adipose Triglyceride Lipase and Hormone-sensitive Lipase Are the Major Enzymes in Adipose Tissue Triacylglycerol Catabolism

The mobilization of free fatty acids from adipose triacylglycerol (TG) stores requires the activities of triacylglycerol lipases. In this study, we demonstrate that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major enzymes contributing to TG breakdown in in vitro assays and in organ cultures of murine white adipose tissue (WAT). To differentiate between ATGL- and HSL-specific activities in cytosolic preparations of WAT and to determine the relative contribution of these TG hydrolases to the lipolytic catabolism of fat, mutant mouse models lacking ATGL or HSL and a mono-specific, small molecule inhibitor for HSL (76-0079) were used. We show that 76-0079 had no effect on TG catabolism in HSL-deficient WAT but, in contrast, essentially abolished free fatty acid mobilization in ATGL-deficient fat. CGI-58, a recently identified coactivator of ATGL, stimulates TG hydrolase activity in wild-type and HSL-deficient WAT but not in ATGL-deficient WAT, suggesting that ATGL is the sole target for CGI-58-mediated activation of adipose lipolysis. Together, ATGL and HSL are responsible for more than 95% of the TG hydrolase activity present in murine WAT. Additional known or unknown lipases appear to play only a quantitatively minor role in fat cell lipolysis.

Hormone-sensitive lipase and monoacylglycerol lipase are both required for complete degradation of adipocyte triacylglycerol

The respective roles of monoacylglycerol lipase and hormone-sensitive lipase in the sequential hydrolysis of adipose tissue triacylglycerols have been examined. An adipose tissue preparation, containing both lipases in approximately the same proportion as in the intact tissue, hydrolyzed emulsified tri- or dioleoylglycerol to fatty acids and glycerol, with little accumulation of di- or monooleoylglycerol. Selective removal of the monoacylglycerol lipase by immunoprecipitation markedly reduced the glycerol release. Isolated hormone-sensitive lipase hydrolyzed acylglycerols with a marked accumulation of monoacylglycerol in accordance with the positional specificity of this enzyme (Fredrikson, G. and Belfrage, P. (1983) J. Biol. Chem. 258, 14253-14256). Addition of increasing amounts of isolated monoacylglycerol lipase led to a corresponding increase in glycerol release, due to hydrolysis of the monoacylglycerols formed. The reaction proceeded to completion when the relative proportion of the two lipases was similar to that in the intact tissue. These findings indicate that hormone-sensitive lipase catalyzes the hydrolysis of triacylglycerol in the rate-limiting step of adipose tissues lipolysis, and of the resulting diacylglycerol, whereas the action of monoacylglycerol lipase is required in the final hydrolysis of the 2-monoacylglycerols produced.

Alterations of lipid metabolism in healthy volunteers during long-term ethanol intake

Abstract. Nine young, healthy male volunteers were given ethanol (75 g/day) for 5 weeks. The ethanol was divided into five daily doses and taken so that blood ethanol levels never exceeded 0.04% (w/v).

Human SGBS Cells - a Unique Tool for Studies of Human Fat Cell Biology

The human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell strain provides a unique and useful tool for studies of human adipocyte biology. The cells originate from an adipose tissue specimen of a patient with SGBS. They are neither transformed nor immortalized, and provide an almost unlimited source due to their ability to proliferate for up to 50 generations with retained capacity for adipogenic differentiation. So far, the cells have been used for a number of studies on adipose differentiation, adipocyte glucose uptake, lipolysis, apoptosis, regulation of expression of adipokines, and protein translocation. The cells are efficiently differentiated in the presence of PPARγagonists and in the absence of serum and albumin. SGBS adipocytes respond to insulin stimulation by increasing glucose uptake several-fold (EC50 approximately 100 pmol/l), and by very effectively inhibiting (IC50 approximately 10 pmol/l) catecholamine-stimulated lipolysis.

Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes. Studies using the selective inhibitor wortmannin.

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3‐kinase (PI 3‐kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC50≈ 100 nM), the insulin‐induced activation and phosphorylation of cGMP‐inhibited cAMP phosphodiesterase (cGI‐PDE) as well as the activation of the insulin‐stimulated cGI‐PDE kinase (IC50≈ 10–30 nM). No direct effects of the inhibitor on the insulin‐stimulated cGI‐PDE kinase, the cGI‐PDE and the hormone‐sensitive lipase were observed. These data suggest that activation of PI 3‐kinase upstream of the insulin‐stimulated cGI‐PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin‐induced cGI‐PDE activation/ phosphorylation and anti‐lipolysis.

Characterization of the transcriptional and functional effects of fibroblast growth factor-1 on human preadipocyte differentiation

We recently established that fibroblast growth factor (FGF)‐1 promotes adipogenesis of primary human preadipocytes (phPA). In the current report, we have characterized the adipogenic effects of FGF‐1 in phPA and also in a human PA strain derived from an individual with Simpson‐Golabi‐Behmel syndrome (SGBS PA), which exhibit an intrinsic capacity to differentiate with high efficiency. In further studies, we compared these models with the well‐characterized murine 3T3‐L1 preadipocyte cell line (3T3‐L1 PA). FGF‐1 up‐regulated the adipogenic program in phPA, with increased expression of peroxisome proliferator‐activated receptor‐γ in confluent PA prior to induction of differentiation and increased expression of adipocyte markers during differentiation. Moreover, phPA differentiated in the presence of FGF‐1 were more insulin responsive and secreted increased levels of adiponectin. FGF‐1 treatment of SGBS PA further enhanced differentiation. For the most part, the adipogenic program in phPA paralleled that observed in 3T3‐L1 PA; however, we found no evidence of mitotic clonal expansion in the phPA. Finally, we investigated a role for extracellular regulated kinase 1/2 (ERK 1/2) in adipogenesis of phPA. FGF‐1 induced robust phosphorylation of ERK1/2 in early differentiation and inhibition of ERK1/2 activity significantly reduced phPA differentiation. These data suggest that FGF‐1 treated phPA represent a valuable in vitro model for the study of adipogenesis and insulin action and indicate that ERK1/2 activation is necessary for human adipogenesis in the absence of mitotic clonal expansion.—Newell, F. S., Su, H., Tornqvist, H., Whitehead, J. P., Prins, J. B., Hutley, L. J. Characterization of the transcriptional and functional effects of fibroblast growth factor‐1 on human preadipocyte differentiation FASEB J. 20, E2133–E2145 (2006)

Hormone-sensitive lipase of rat adipose tissue: Identification and some properties of the enzyme protein

The cyclic AMP-dependent stimulation of hormonesensitive lipase activity in adipose tissue preparations, originally described by Bizack [ 1 ] and confirmed by others [2,3] has been shown to be mediated via a protein kinase-catalyzed phosphorylation [4,5]. However, direct evidence for the molecular events taking place upon stimulation is lacking since the enzyme protein has not been isolated. We have recently purified monoacylglycerol lipase from rat adipose tissue by solubilization and fractionation in non-ionic detergent [6]. Using the same technique we have been able to separate solubilized hormonesensitive lipase from other lipolytic enzymes of rat adipose tissue, to identify the enzyme protein and to demonstrate some of its properties, notably a protein kinase-catalyzed phosphorylation.

Enzymes catalyzing the hydrolysis of long-chain monoacyglycerols in rat adipose tissue

Acetone-ether preparations of epididymal fat pads from fasted or fed rats contained two enzymes catalyzing the hydrolysis of long-chain monoacylglycerols. The enzymes were identified as monoacylglycerol lipase (Tornqvist, H. and Belfrage, P., (1976) J. Biol Chem. 251, 813--819) and lipoprotein lipase by their apparent pI values after electrofocusing in non-ionic detergent, selective inhibition properties, substrate specificity and positional specificity. It was estimated that monoacylglycerol lipase accounted for about 90% of the total monoacylglycerol-hydrolyzing activity in acetone-ether preparations from fasted and 70% from fed rats. Its enzyme activity did not change with the nutritional state in contrast to that of lipoprotein lipase. The latter enzyme hydrolyzed 2-monoacylglycerols at a much lower rate than the 1(3)-isomers. Monoacylglycerol lipase was located almost entirely in the adipocytes, thus most of the enzyme activity towards monoacylglycerols in the adipose tissue was found in this site. Fractionated sucrose homogenates of rat epididymal fat pads also contained a third enzyme with monoacylglycerol-hydrolyzing activity, identified as hormone-sensitive lipase by its pI, selective inhibition properties and substrate specificity. It was estimated that hormone-sensitive lipase accounted for less than 20% of the total activity against monoacylglycerols in these tissue preparations from fasted rats. Over-all quantitative estimations emphasized the dominant role of monoacylglycerol lipase over the other two enzymes in the hydrolysis of monoacylglycerols.

Expression of the transcription factor 7-like 2 gene (TCF7L2) in human adipocytes is down regulated by insulin.

Variants in the TCF7L2 gene (transcription factor 7-like 2) have shown strong association with type 2 diabetes with two defined risk haplotypes, HapA and HapB(T2D). TCF7L2 may play a role in both glucose homeostasis and adipogenesis. Our aim was to characterize the TCF7L2 mRNA expression and regulation in human adipose tissue. We quantified TCF7L2 mRNA levels in cultured human adipocytes and in biopsies from visceral (VAT) and subcutaneous (SAT) adipose tissue from 38 obese non-diabetic subjects, using real-time PCR. The influence of haplotype and clinical traits on TCF7L2 mRNA levels were investigated. In vitro, insulin decreased TCF7L2 mRNA expression. This effect was attenuated in cells incubated with the free fatty acids palmitate or oleate. In vivo, we found significantly higher expression in SAT from more insulin resistant subjects. No correlations between TCF7L2 mRNA expression and obesity measures were observed. TCF7L2 expression was higher in VAT than in SAT and when stratifying for haplotype, this difference was seen in HapA carriers but not in non-HapA carriers. In conclusion, TCF7L2 mRNA levels in adipocytes are decreased by insulin and seem to increase in insulin resistant subjects and in HapA carriers.