The lipolytic stimulation of 3T3-L1 adipocytes promotes the translocation of hormone-sensitive lipase to the surfaces of lipid storage droplets

Hormone-sensitive lipase catalyzes the rate-limiting step in the release of fatty acids from triacylglycerol-rich lipid storage droplets of adipocytes, which contain the body's major energy reserves. Hormonal stimulation of cAMP formation and the activation of cAMP-dependent protein kinase leads to the phosphorylation of hormone-sensitive lipase and a large increase in lipolysis in adipocytes. By contrast, phosphorylation of hormone-sensitive lipase by the kinase in vitro results in a comparatively minor increase in catalytic activity. In this study, we investigate the basis for this discrepancy by using immunofluorescence microscopy to locate hormone-sensitive lipase in lipolytically stimulated and unstimulated 3T3-L1 adipocytes. In unstimulated cells, hormone-sensitive lipase is diffusely distributed throughout the cytosol. Upon stimulation of cells with the beta-adrenergic receptor agonist, isoproterenol, hormone-sensitive lipase translocates from the cytosol to the surfaces of intracellular lipid droplets concomitant with the onset of lipolysis, as measured by the release of glycerol to the culture medium. Both hormone-sensitive lipase translocation and lipolysis are reversed by the incubation of cells with the beta-adrenergic receptor antagonist, propranolol. The treatment of cells with cycloheximide fails to inhibit lipase translocation or lipolysis, indicating that the synthesis of nascent proteins is not required. Cytochalasin D and nocodazole used singly and in combination also failed to have a major effect, thus suggesting that the polymerization of microfilaments and microtubules and the formation of intermediate filament networks is unnecessary. Hormone-sensitive lipase translocation and lipolysis were inhibited by N-ethylmaleimide and a combination of deoxyglucose and sodium azide. We propose that the major consequence of the phosphorylation of hormone-sensitive lipase following the lipolytic stimulation of adipocytes is the translocation of the lipase from the cytosol to the surfaces of lipid storage droplets.     

Development of lipoprotein lipase in cultured 3T3-L1 cells

The 3T3-L1 mouse fibroblast resembles an adipocyte after reaching a confluent stage of growth. Lipoprotein lipase activity was released with heparin and was present in acetone-ether extracts of these cells. During the early post-confluent period both activities increased rapidly. A wide variation in enzyme activities was noted in subclones suggesting that spontaneous heritable change continues to take place in these cells. Since lipoprotein lipase activity was measurable before triglyceride accumulation, it may be the earliest marker of adipocyte expression in this line. This system appears to offer a unique opportunity to study the processes of cellular differentiation and fat metabolism $\text{in vitro}$.     

Insulin regulation of lipoprotein lipase in cultured 3T3-L1 cells

Lipoprotein lipase activity is produced by the 3T3-L1 cell an established mouse fibroblast line which resembles an adipocyte after reaching a confluent stage of growth. Since insulin has been shown to be an important regulator of lipoprotein lipase in other mammalian systems, a two hour incubation period was utilized to determine if insulin could enhance an acute response of enzyme activity. Over the range of concentrations tested (0.4, 4.0 and 40 ng/ml), insulin increased lipoprotein lipase activity in acetone ether powders of cells (intracellular enzyme) and the activity secreted into the culture medium. A simultaneous decrease in lipoprotein lipase activity releasable with heparin in a subsequent incubation (membrane bound activity) indicates two distinct effects of insulin on the enzyme in this system.     

Lipopolysaccharide-treated RAW 264.7 cells produce a mediator that inhibits lipoprotein lipase in 3T3-L1 cells

RAW 264.7 cells upon stimulation with lipopolysaccharide secrete a protein mediator(s) that suppresses lipoprotein lipase activity in differentiated 3T3-L1 cells. The mediator(s), which is absent from unstimulated culture supernatants, is nondialyzable and thermolabile. Preliminary characterization suggests that this mediator(s) may be the same as that previously found in medium from lipopolysaccharide-treated thioglycollate-elicited mouse peritoneal macrophage cultures.     

Synthesis and secretion of lipoprotein lipase in 3T3-L1 adipocytes. Demonstration of inactive forms of lipase in cells

3T3-L1 adipocytes in culture incorporated [35S]methionine into a protein which could be immunoprecipitated with chicken antiserum to bovine lipoprotein lipase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed this protein had an Mr of 55,000, similar to that of bovine lipoprotein lipase, and accounted for 0.1-0.5% of total protein synthesis in the adipocytes. Lipoprotein lipase protein was present in small amounts in confluent 3T3-L1 fibroblasts, and the amount increased many-fold as the cells differentiated into adipocytes. This increase was accompanied by parallel increases in cellular lipase activity and secretion. When cells were grown with [35S]methionine, the amount of label incorporated into lipoprotein lipase increased for 2 h and then leveled off. Pulse-chase experiments showed that half-life of newly synthesized lipase was about 1 h. Turnover of lipoprotein lipase in control cells involved both release to the medium and intracellular degradation. When N-linked glycosylation was blocked by tunicamycin, the cells synthesized a form of lipase that had a smaller Mr (48,000), was catalytically inactive, and was not released to the medium. Radioimmunoassay demonstrated that 3T3-L1 adipocytes contained an unexpectedly large amount of lipoprotein lipase protein. 55% of the enzyme protein in acetone/ether powder of the cells was insoluble in 50 mM NH3/NH4Cl at pH 8.1, a solution commonly used to extract lipoprotein lipase; 27% of the lipase protein was soluble but did not bind to heparin-Sepharose and had very low lipase activity; and the remaining 13% was soluble, bound to heparin-Sepharose, and had high lipolytic activity. About one-half of the lipase released spontaneously to the medium was inactive, and lipase inactivation proceeded in the medium with little loss of enzyme protein. Lipoprotein lipase released heparin, in contrast, was fully active and more stable. When protein synthesis was blocked by cycloheximide, the level of lipoprotein lipase activity in adipocytes decreased more rapidly than the amount of lipase protein in the cells. Most of the inactive lipoprotein lipase in adipocytes probably results from dissociation of active dimeric lipase, but some could be a precursor of active enzyme.     

Human recombinant TNF suppresses lipoprotein lipase activity and stimulates lipolysis in 3T3-L1 cells

Tumor necrosis factor (TNF) has been reported to be identical to "cachectin," a monokine which we have previously proposed as a mediator of the enhanced catabolism observed in patients or animals responding to various invasive stimuli such as infections. Detailed quantitative studies were conducted on the effects of TNF on fatty acid metabolism in 3T3-L1 cells in order to explore the extent of the catabolic effects exerted by TNF compared with those by the crude cachectin. 3T3-L1 adipocytes responded to recombinant human TNF, showing a decrease in LPL activity and an increase in intracellular lipolysis. When TNF in the crude cachectin preparation was completely neutralized with anti-TNF antibody, about 75% of LPL suppression activity in the crude cachectin was absorbed, indicating that most of the mediator responsible for LPL suppression in the crude preparation is TNF. In contrast to the above effect on LPL, TNF markedly increased the lipolysis of stored fat in the cells. The effect on LPL was observed as early as 2 h after the addition of TNF, but enhancement of lipolysis required a time lag of at least 3 h before any increase of glycerol release became apparent. The effective concentrations of TNF for the stimulation of lipolysis were much higher (2.5 to 49 nM) than those for LPL suppression (50 pM to 50 nM), but both were in the same range as the concentration required for tumoricidal effect. These results demonstrate that cachectin is synonymous with TNF and that it plays an important role in the pathophysiology of deranged lipid metabolism through both suppression of LPL and enhancement of lipolysis in patients coping with invasive conditions such as infections.     

Regulation of lipoprotein lipase mRNA content in 3T3-L1 cells by tumour necrosis factor.

U-937 cells differentiated by exposure to dibutyryl cyclic AMP respond to complement fragment C5a with a marked increase in cytoskeletal F-actin, which can be detected by fluorescence-activated cell sorting (f.a.c.s.) analysis of their rhodamine phalloidin-stained cytoskeletons. The C5a-induced increase in F-actin content can be prevented by prior exposure of the cells to cytochalasin B and pertussis toxin. It is insensitive to removal of extra cellular Ca2+, to cholera toxin or to neomycin. Phorbol myristate acetate (PMA), an activator of protein kinase C, does not induce actin polymerization in the differentiated cells. Both C5a and PMA stimulate superoxide production. The action of C5a on superoxide formation is also inhibited by neomycin, a phospholipase inhibitor. These results suggest that the cytoskeletal response to C5a requires activation of a G protein, but probably does not involve phospholipase C and protein kinase C, and is not highly dependent on the availability of Ca2+. Phospholipase C and kinase C may, however, be components of the pathway leading from C5a binding to superoxide production.     

Adenovirus transduction of 3T3-L1 cells

3T3-L1 cells offer an excellent model system for studies of differentiation and biochemistry of fat cells. However, these cells are limited in their utility by the low efficiency with which DNA can be introduced by transfection. Gene delivery by viral vectors, particularly adenovirus, has proven a powerful means for introduction of genes into certain cell types. Furthermore, adenovirus transduction has been used to study mechanisms involved in the differentiation of 3T3-L1 cells into mature fat cells. We show in this study that 3T3-L1 cells are inefficiently transduced by adenovirus. The potential advantages offered by adenovirus transduction led us to examine methods designed to enhance transduction of 3T3-L1 cells by adenovirus. Of these methods, polylysine-mediated enhancement demonstrates considerable promise because it permits up to 100% of cells to be transduced and because it does not inhibit differentiation of 3T3-L1 cells. -- Orlicky D. J., and J. Schaack. Adenovirus transduction of 3T3-L1 cells. J. Lipid Res. 2001. 42: 460--466.     

LMO4 modulates proliferation and differentiation of 3T3-L1 preadipocytes

Previous microarray analyses revealed that LMO4 is expressed in 3T3-L1 preadipocytes, however, its roles in adipogenesis are unknown. In the present study, using RT-PCR sequencing and quantitative real-time RT-PCR, we confirmed that LMO4 gene is expressed in 3T3-L1 preadipocytes and its expression peaks at the early stage of 3T3-L1 preadipocyte differentiation. Further analyses showed that LMO4 knockdown decreased the proliferation of 3T3-L1 preadipocytes, and attenuated the differentiation of 3T3-L1 preadipocytes, as evidenced by reduced lipid accumulation and down-regulation of PPARγ gene expression. Collectively, our findings indicate that LMO4 is a novel modulator of adipogenesis.     

Multiple biotin-containing proteins in 3T3-L1 cells.

Extracts of 3T3-L1 cells prepared after labelling the monolayer cultures with [3H]biotin contained numerous protein bands that were detected by fluorography of dried SDS/polyacrylamide electrophoresis gels. All labelled proteins in the extracts could be removed by avidin affinity chromatography. The biotin-containing subunits of acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, with molecular masses of approx. 220, 120, 75 and 72 kDa respectively, were detected together with minor bands at 100, 85 and 37 kDa that did not appear to be partial degradation products. Additional labelled bands increased in amount during incubation of cell extracts or did not occur in extracts prepared with trichloroacetic acid, 9.5 M-urea or proteolytic inhibitors, and were tentatively classified as partial degradation products. The unknown bands were not removed by incubation of cell monolayers for 24 h, a treatment that gave degradation rate constants of 0.47 day-1 for acetyl-CoA carboxylase and 0.28 day-1 for pyruvate carboxylase. Upon two-dimensional electrophoresis, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase had isoelectric points of 6.4, 7.2 and 6.4 respectively. Several additional discrete spots with isoelectric points below 6.2 were also present. All the unknown biotin-containing proteins banded with intact mitochondria during density-gradient centrifugation. We conclude that several unknown biotin-containing proteins are present in the mitochondria of 3T3-L1 cells, whereas others are partial breakdown products of mitochondrial proteolysis.     

Neuropeptide Y potentiates beta-adrenergic stimulation of lipolysis in 3T3-L1 adipocytes

Recently, we have shown that neuropeptide Y (NPY) is produced and upregulated in visceral adipose tissue of an early-life programmed rat model of central obesity. Moreover, we have demonstrated that NPY promotes proliferation of adipocyte precursor cells and contributes to the pathogenesis of obesity. However, the role of NPY in regulating adipocyte metabolism is poorly understood. The present study was designed to examine the effects of NPY on adipocyte metabolic function using 3T3-L1 adipocytes as an in vitro cell model system. We found that although it did not affect basal lipolysis, NPY potentiated isoproterenol (a β-adrenergic receptor agonist) stimulated lipolysis. Furthermore, this potentiation occurred upstream of adenylyl cyclase, since NPY did not enhance forskolin (an activator of adenylyl cyclase) stimulated lipolysis. In addition, NPY also augmented isoproterenol-stimulated phosphorylation of hormone sensitive lipase. In contrast, NPY did not alter the expression of several key lipolytic and lipogenic enzymes/proteins. Taken together, our results revealed a novel cross talk between the NPY and β-adrenergic signaling pathways in regulating lipolysis. Thus, the present findings add a new dimension to the dynamic role NPY plays in regulating energy balance.     

Porcine phosphotyrosine interaction domain containing 1 modulates 3T3-L1 preadipocyte proliferation and differentiation

The phosphotyrosine interaction domain containing 1 (PID1) gene was firstly isolated from obese subjects and involved in obesity-associated insulin resistance. In the present study, Duroc×Landrace×Yorkshire (DLY) pig PID1 cDNA was cloned. The entire open reading frame of the cloned porcine PID1 is 654 bp. The predicted protein is composed of 217 amino acids residues with a molecular mass of 24,774 Da. Over-expression of porcine PID1 significantly accelerated the proliferation of 3T3-L1 preadipocyte, but inhibited preadipocyte differentiation by decreasing the numerous fat droplets appeared and down-regulating the mRNA expression levels of peroxisome proliferators-activated receptor-γ, CCAAT/enhancer binding protein α, fat acid synthase and lipoprotein lipase. Together, these results suggest that porcine PID1 plays a role in regulating adipose development.     

Regulation of lipoprotein lipase synthesis and 3T3-L1 adipocyte metabolism by recombinant interleukin 1

When fully differentiated 3T3-L1 adipocytes were exposed to purified, recombinant murine interleukin 1 (rIL-1), a dose-dependent suppression of lipoprotein lipase activity was observed. The loss of activity reached a maximum of 60-70% of control and appeared to be due to an effect on the synthesis of the enzyme as judged by a suppression of the ability to incorporate [35S]methionine into immunoprecipitable lipoprotein lipase. There was no general effect on protein synthesis as determined by radiolabel incorporation into acid precipitable protein; however, after a 17 h exposure of the 3T3-L1 cells to recombinant interleukin 1, the synthesis of two proteins (molecular weights, 19,400 and 165,000 daltons) was enhanced several-fold. When the effect of Il-1 on the major metabolic pathways of the adipocyte was investigated, lipolysis as measured by glycerol release from the cells was markedly enhanced after a 17 h incubation with the hormone, while no effect was observed on de novo fatty acid synthesis. These effects on the metabolism of the adipocytes occur at concentration on a basis of molecules per cell, similar (only a 3-fold difference) to those required for stimulation of [3H]thymidine incorporation into mouse thymocyte DNA, suggesting that IL-1 may be a physiologically significant effector of adipocyte metabolism.     

SNARE expression and distribution during 3T3-L1 adipocyte differentiation

Differentiation of 3T3-L1 cells into adipocytes presupposes the expression of the glucose transporter isoform GLUT4 and the acquisition of insulin-dependent GLUT4 translocation from intracellular storage vesicles to plasma membrane. This ability to translocate GLUT4 depends on the presence of a set of proteins of the SNARE category that are essential in the fusion step. The expression and levels of some of these SNARE proteins are altered during 3T3-L1 differentiation. Levels of the v-SNARE protein cellubrevin and of the t-SNARE protein syntaxin 4 were increased in this process in parallel to GLUT4. However, the levels of SNAP-23, another t-SNARE, were maintained during differentiation. Immunofluorescence images of SNAP-23 showed the initial distribution of this protein in a perinuclear region before differentiation and its redistribution towards plasma membrane in the adipocyte form. These results suggest a capital role in the expression levels and cellular distribution, during 3T3-L1 differentiation, of SNARE proteins involved in the late steps of GLUT4 translocation.