1Neuroendocrine and Obesity Biology Unit, Liverpool Centre for Nutritional Genomics, School of Clinical Sciences, University of Liverpool, Liverpool L69 3GA; and 2The Henry Wellcome Laboratory of Molecular and Cellular Gastroenterology, School of Clinical Sciences, University of Liverpool, Liverpool L69 3BX, United Kingdom
Submitted 18 February 2004 ; accepted in final form 19 April 2004
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ABSTRACT |
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nerve growth factor; neurotropin; white adipose tissue; sympathetic nervous system; tumor necrosis factor-
WAT is innervated by the sympathetic nervous system (SNS), although less extensively than brown adipose tissue (2, 3), and a recent report suggests that there may also be a parasympathetic innervation (27). The sympathetic system is considered to be the major physiological regulator of lipolysis in WAT (2, 3, 17), and it has also been strongly implicated in the regulation of cell number (2, 3) and the synthesis of several adipokines, particularly leptin (38, 42, 43). In a number of tissues, the development and survival of sympathetic neurons are dependent on the presence of target-derived neurotropins, the best characterized of which is nerve growth factor (NGF) (40, 44). NGF exists as a 130,000-molecular weight pentameric protein complex, with the -subunit responsible for all biological activity. Two receptors for NGF have been identified, trkA and p75 (40, 44). The neurotropic activity of NGF is mediated only by trkA, and this receptor is both specific for NGF and of high affinity (13, 44).
Previous studies have demonstrated that NGF is synthesized and released by brown adipose tissue, with NGF expression in brown adipocytes being inhibited by prolonged (but not short-term) cold exposure and by norepinephrine, i.e., by sympathetic stimulation (35, 36). However, whether the major WAT depots also produce NGF and whether this target-derived neurotropin is an important factor in the development and maintenance of the sympathetic innervation in white fat have not been established. There is, nevertheless, evidence for NGF synthesis in white adipocytes locally associated with cutaneous wound healing (18) and also in subepicardial adipose tissue (surrounding coronary arteries) linked with atherosclerotic lesions (4, 5).
In the present study, we demonstrate that the NGF gene is expressed in the major WAT depots of mice, as well as in human white fat, and that NGF itself is secreted from 3T3-L1 adipocytes in culture. We also demonstrate that the cytokine TNF- is a major factor in the stimulation of NGF expression and secretion, consistent with the role of the neurotropin in inflammatory responses. In addition, we show that the genes encoding the low-affinity p75 and the high-affinity trkA NGF receptors are expressed in WAT.
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MATERIALS AND METHODS |
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A portion of freshly dissected subcutaneous adipose tissue was digested with collagenase (Sigma, Poole, UK) to separate mature adipocytes from the stromal vascular (S-V) cells, as described previously (33). These fractions were then stored at 80°C until use.
Human tissue. Subcutaneous fat and omental white fat were obtained from four subjects (3 male and 1 female; body mass index >45, age 3748 yr) undergoing gastroplasty; the subjects did not exhibit any ongoing disease (e.g., infection or cancer). After removal, the fat samples were frozen in liquid nitrogen and stored at 80°C until analysis. Ethical permission was obtained through the Sefton Ethics Committee; the subjects gave informed consent for the removal of fat samples.
Cell culture. 3T3-L1 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured at 37°C in a humidified atmosphere of 5% CO2-95% air. The cells were maintained in culture medium containing the following constituents: Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Paisley, UK) with 25 mM glucose, 1.0 mM pyruvate, 4.02 mM L-alanyl-L-glutamine, and 10% (vol/vol) fetal calf serum (FCS, Sigma). Differentiation of the cells was initiated 24 h after confluence by incubation for 2 days in culture medium containing 0.25 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine and 5 µg/ml insulin (Sigma). This was followed by maintenance in feeding medium (renewed every 2 or 3 days) consisting of culture medium containing 5 µg/ml insulin.
Between three and six individual wells in cell culture plates were treated with each agent or used as controls. The cells were preincubated 11 days after differentiation for 24 h with feeding medium (containing insulin and FCS), insulin-free feeding medium (for the study on the effects of insulin), or FCS-free feeding medium (for the studies on the effects of TNF-, IL-6, and LPS). Cells were then treated with medium containing each of the specific agents (treatment medium with TNF-
, Il-6, or LPS was FCS free); the control cells for each study had their preincubation medium renewed. Cells were collected 24 h later in 700 µl of TRI Reagent (Sigma). Medium was also collected and centrifuged at 1,000 rpm for 10 min, the supernatant being taken and stored together with the collected cells at 20°C until use.
To investigate the time course of NGF secretion during adipocyte differentiation, medium was collected every 2 days. In studies on the regulation of NGF synthesis, 3T3-L1 adipocytes were used at day 12 after the induction of differentiation and incubated for 24 h with the following agents: BRL-37344 (Tocris, Avonmouth, UK), norepinephrine (Fluka, Buchs, Switzerland), isoprenaline, insulin, dexamethasone, TNF-, IL-6, LPS (Sigma), neuropeptide Y (Bachem, St. Helens, UK), and rosiglitazone (GlaxoSmithKline, Uxbridge, UK). The dose-dependent effect of TNF-
on NGF expression was studied by treating cells with increasing concentrations of TNF-
(01,000 ng/ml) for 24 h.
Cell cultures were usually performed at least twice to allow confirmation of the reproducibility of the findings, but results are presented for one representative cell culture experiment. Group numbers in the legends for Figs. 26 refer to the number of wells from which independent samples were collected and processed.
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RT-PCR.
One microgram of total RNA was reverse-transcribed to cDNA in a 20-µl reaction volume with anchored oligo(dT) primer using Reverse-iT 1st Strand Synthesis Kit (ABgene, Epsom, UK). One microliter of the RT product was then used as template for PCR using 1.1x ReddyMix PCR Master Mix (ABgene). RT and PCR were performed according to the manufacturer's protocols on PCR Express thermal cyclers (Hybaid, Ashford, UK). Primers were designed using Primer Premier 5 software (Biosoft International, Palo Alto, CA) and synthesized commercially (MWG Biotech, Ebersberg, Germany). Mouse -actin was utilized as the control gene. The primers used for amplification, together with their specific optimum cycling conditions, were as follows.
Mouse -actin (463-bp product): sense 5'-TGC TGT CCC TGT ATG CCT CT-3'; antisense 5'-AGG TCT TTA CGG ATG TCA ACG-3'; annealing temperature (TA) 60°C; 20 cycles.
Mouse NGF (412-bp product): sense 5'-TGT GCC TCA AGC CAG TGA AAT T-3'; antisense 5'-TCC ACA GTG ATG TTG CGG GTC T-3'; TA 55°C; 35 cycles.
Mouse resistin (266-bp product): sense 5'-GAA GAA CCT TTC ATT TCC CCT CC-3'; antisense 5'-CTT CAC GAA TGT CCC ACG AGG C-3'; TA 60°C; 30 cycles.
Mouse p75 (464-bp product): sense 5'-CCT ATG GCT ACT ACC AGG ACG AG-3'; antisense 5'-CAC AAC CAC AGC AGC CAA GAT-3'; TA 56°C; 35 cycles.
Mouse trkA (324-bp product): sense 5'-AAT GCT CGG CAG GAC TTT CAG-3'; antisense 5'-ACC CAC CAG ACA GTT GCG TGT-3'; TA 56°C; 35 cycles.
Human -actin (281-bp product): sense 5'-GTG GCA TCC ACG AAA CTA CCT T-3'; antisense 5'-GGA CTC GTC ATA CTC CTG CTT G-3'; TA 56.2°C; 28 cycles.
Human NGF (401-bp product): sense 5'-GCA GTC CAA GGG GCT GGA TG-3'; antisense 5'-CGG GGA GGC TGG GTG CTA AA-3'; TA 58.5°C; 37 cycles.
The amplification parameters for PCR were an initial 15-min denaturation step followed by cycles consisting of denaturation at 94°C for 20 s, annealing at the specified optimum temperature for 30 s, and extension at 72°C for 45 s. The specified optimum total number of cycles was followed by a 10-min extension at 72°C. PCR products were separated by electrophoresis on a 1% agarose gel stained with ethidium bromide and photographed under UV light. The products were sequenced commercially to confirm their identity (MWG Biotech).
Real-time PCR.
Mouse NGF gene expression was analyzed by relative quantitation with the 2Ct method (31) using real-time PCR with an ABI Prism 7700 instrument (Applied Biosystems, Foster City, CA). All samples were normalized to values of
-actin, and results expressed as fold changes of threshold cycle (Ct) value relative to controls. Primer and Taqman probe sequences were designed using Primer Express software (Applied Biosystems). Primers and probes were synthesized commercially (Sigma-Genosys, Haverhill, UK, and Eurogentec, Romsey, UK, respectively), and the sequences were as follows.
Mouse NGF (78-bp product): sense 5'-GCC AAG GAC GCA GCT TTC TAT-3'; antisense 5'-AGT GAT CAG AGT GTA GAA CAA CAT GGA-3'; Taqman probe 5'-FAM-CTG GCC GCA GTG AGG TGC ATA GC-TAMRA-3'.
Mouse -actin (71-bp product): sense 5'-ACG GCC AGG TCA TCA CTA TTG-3'; antisense 5'-CAA GAA GGA AGG CTG GAA AAG-3'; Taqman probe 5'-FAM-ACG AGC GGT TCC GAT GCC CTG-TAMRA-3'.
RT was performed as described above. Real-time PCR was performed in 96-well plates using a qPCR Core Kit (Eurogentec) according to the supplier's instructions, with 900 nM forward and reverse primers, 225 nM probe, and 1 µl of cDNA in a 26-µl final reaction volume. Each sample was run in triplicate with the NGF primers and probe and in duplicate with the -actin primers and probe. Amplifications were performed commencing with a 2-min activation stage at 50°C and then a 10-min denaturation stage followed by 40 cycles consisting of a denaturation step of 15 s at 95°C and a combined primer annealing and extension step for 60 s at 60°C. Data were collected and analyzed with Sequence Detector software (Applied Biosystems).
ELISA. NGF was measured in culture medium using the NGF Emax Immunoassay System (Promega, Southampton, UK), a specific and highly sensitive ELISA kit with a stated intra-assay coefficient of variation of 4.2%. The kit displays cross-reactivity across several species, including mouse and human. ELISA assays were performed according to the manufacturer's instructions in Nunc MaxiSorp 96-well microplates (Fisher Scientific, Loughborough, UK). Absorbance was measured at 450 nm with a Benchmark Plus microplate spectrophotometer (Bio-Rad, Hemel Hempstead, UK). Data were collected and analyzed with Microplate Manager software (Bio-Rad).
Statistical analysis. The statistical significance of differences between groups of treated 3T3-L1 cells was assessed by Student's unpaired t-test, and between mouse WAT depots by one-way ANOVA with post hoc multiple comparisons using a Bonferroni correction. Differences were considered to be significant when P < 0.05.
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RESULTS |
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In subsequent experiments, NGF mRNA levels were quantitated using real-time PCR. Although RT-PCR indicated that the NGF gene is expressed in all WAT sites (Fig. 1B), the signal intensity appeared to vary between depots. Comparison by real-time PCR shows clear differences in NGF mRNA levels between depots, with the level in the epididymal and perirenal sites being significantly higher than in subcutaneous and omental fat (Fig. 1C).
NGF gene expression and secretion in cell culture. In the next studies, 3T3-L1 cells in culture were utilized to assess whether NGF protein is secreted from adipocytes. Differentiation of the 3T3-L1 cells was evident from the accumulation of intracellular lipid droplets, and for all experiments the differentiation rate was >90%. NGF gene expression was first examined in 3T3-L1 cells by RT-PCR, before and after the induction of differentiation into adipocytes. NGF mRNA was readily detected both before and after differentiation (Fig. 2A). Resistin mRNA was also examined in the same samples for comparison. In contrast to NGF mRNA, resistin mRNA was not detected before or shortly after the induction of differentiation; a signal was, however, present at day 2 postinduction and thereafter (Fig. 2A), as in previous work (19).
A specific ELISA was used to determine whether immunoreactive NGF was secreted by 3T3-L1 adipocytes. NGF was readily detected in the cell culture medium both before and after the induction of differentiation (Fig. 2B). The rate of release into the medium fell markedly, however, after induction, being lowest at days 2 and 4 postinduction. The concentration of NGF in the medium then rose to approximately one-half the preinduction level and was essentially stable from day 10 (Fig. 2B). In view of this time course, subsequent studies on the control of NGF production were conducted at day 12 postinduction.
Regulation of NGF gene expression and secretion in 3T3-L1 adipocytes.
The first experiments on the regulation of NGF gene expression and secretion examined the effects of sympathetic mediators. Treatment of 3T3-L1 adipocytes with norepinephrine resulted in a small, but statistically significant, reduction in NGF mRNA level as measured by real-time PCR (Fig. 3A). With the higher dose of norepinephrine, the NGF mRNA level was decreased by just 18% compared with the controls. There was also a small decrease in NGF concentration in the medium with norepinephrine, but this was statistically significant only at the high dose where the level of NGF was reduced by 36%. The addition of the -adrenoceptor agonist isoprenaline had no effect on the NGF mRNA level at either low or high doses. There was, however, a reduction in NGF concentration in the medium; this was statistically significant with the high dose, which caused a 43% fall in NGF protein concentration (Fig. 3B).
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The next experiments investigated the effects of insulin, dexamethasone, and rosiglitazone. Insulin had no effect on NGF secretion into the medium, but there was a significant reduction in NGF mRNA level (35% with both low and high doses) (Fig. 4A). However, a substantial decrease in both NGF mRNA and protein occurred when the adipocytes were treated with dexamethasone (Fig. 4B). With the lower dose of the synthetic glucocorticoid, there was a fourfold decrease in NGF mRNA level in the cells and in NGF protein in the medium. At the higher dose, the NGF mRNA level fell 11-fold, whereas the amount of NGF in the medium decreased sevenfold, relative to the controls.
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The subsequent experiments examined the effects of three inflammatory agents: LPS, TNF-, and IL-6. LPS induced a statistically significant increase in NGF mRNA and protein, although in the case of the mRNA the effect was no greater with the high than with the low dose. The concentration of NGF in the medium was increased more than threefold relative to the control with the higher dose of LPS (Fig. 5A).
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In view of the substantial effects of TNF- on NGF production at the two concentrations employed, an extended dose-response study was undertaken, doses of between 0.01 and 1,000 ng/ml of TNF-
being used. The level of NGF mRNA in 3T3-L1 adipocytes was significantly increased with a concentration of TNF-
of 3.3 ng/ml and above (Fig. 6A); at the highest doses of the cytokine, NGF mRNA level was increased ninefold. The amount of NGF in the medium was significantly raised at the lower concentration of TNF-
of 0.33 ng/ml. At the highest doses of TNF-
, the amount of NGF in the medium was elevated as much as 30- to 40-fold relative to the controls (Fig. 6B).
NGF gene expression in human WAT. The studies on NGF gene expression and secretion reported in the previous sections involved mouse tissue and a murine cell line. RT-PCR was used to investigate whether the NGF gene is also expressed in human WAT. Figure 7 shows that NGF mRNA was present in both human depots examined, namely subcutaneous and omental fat; the signal was rather stronger, however, in the omental tissue.
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DISCUSSION |
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A number of cell types have been shown to secrete NGF, including fibroblasts (30), airway smooth muscle cells (14), cardiac myocytes (32), and vascular endothelial cells (16). The S-V fraction of adipose tissue contains a variety of other cell types, such as fibroblasts, vascular endothelial cells, and immune cells (including mast cells and macrophages), in addition to preadipocytes (20). In the present study, the NGF mRNA level appeared higher in mature adipocytes than in the S-V fraction, but in contrast was lower in 3T3-L1 cells after differentiation into adipocytes than before differentiation. The explanation for this difference may be that, although some of the other cell types in the S-V fraction can also express NGF (16, 30), this may be to a lesser extent than the preadipocytes and so dilute the overall level of NGF mRNA within the S-V fraction relative to what might be expected from the preadipocytes alone. The possibility that 3T3-L1 adipocytes, as a clonal cell line, may not fully mirror quantitatively what is happening in mature adipocytes within WAT cannot, of course, be excluded.
Differences were clearly evident between the levels of NGF mRNA in the different WAT depots of mice, as measured by real-time PCR, and there are several possible explanations for this observation. NGF gene expression within the S-V fraction appears to be lower than in mature adipocytes, as noted above; thus, if the proportion of mature adipocytes to S-V cells were to be significantly different between the depots, then this might contribute to the overall differences in NGF mRNA levels between the various sites. Differences in the degree of sympathetic innervation, or sympathetic activity, might also account for the variability between depots in NGF mRNA levels. Additionally, it is possible to speculate that variations in the extent to which depots may be involved in inflammatory responses could also influence the site-specific expression of NGF.
The function originally attributed to NGF was as a key signal for the development and survival of the sympathetic innervation to a tissue (40). NGF is, however, now recognized to have a far wider role than simply that of a neurotropic signal, being involved in both immune and inflammatory responses in particular (28, 44). NGF has been previously identified in the other form of adipose tissue, namely brown adipose tissue, where its synthesis is suppressed by norepinephrine (36). Similarly, chronic cold exposure also inhibits NGF production in brown fat (36). Thus, in brown adipose tissue, the sympathetic system downregulates a key signal responsible for its own development and survival.
Previous studies have indicated that NGF can be produced by white adipocytes, although this relates to the specific circumstances of fat cells associated with cutaneous wound healing (18) and those adipocytes surrounding coronary arteries in association with atherosclerotic lesions (4, 5). The neurotropin appears to be an important general signal in wound healing and in tissue repair (26). Whether white adipocytes in the major WAT depots produce NGF has not previously been established.
It is well recognized that the SNS plays a central role in the stimulation of lipolysis in WAT, despite the relatively sparse innervation of the tissue (2, 3, 17). Similarly, the sympathetic system has been shown to be a regulatory factor in cell proliferation (2, 3) and in the production of several adipokines, including adiponectin (10) and haptoglobin (37), but particularly of leptin (38, 42, 43). In the case of leptin, it has been suggested that not only does the SNS provide a negative feedback loop to the adipocyte in the control of leptin production but that sympathetic tone is the pivotal regulator in the synthesis of this hormone (38, 42). Leptin also stimulates the sympathetic innervation in several tissues, including brown adipose tissue and the kidneys (22, 23). There is, therefore, a two-way interaction between leptin and the SNS.
The results reported here utilizing 3T3-L1 cells indicate that a number of hormones and other factors affect the production of NGF by white adipocytes, both at the level of gene expression and in the overall release of the protein. A reduction in NGF gene expression and secretion occurred with norepinephrine, isoprenaline, and a selective 3-adrenoceptor agonist, but the effects were, at most, small. The modest sympathetic inhibition that was observed appears to be mediated via
3-adrenoceptors, given the effects of BRL-37344 (a selective
3-adrenoceptor agonist), and this receptor is the key
-subtype in rodent adipose tissues (1). Neuropeptide Y, a cotransmitter released from sympathetic neurons, appears to play no regulatory role. Thus, as neither of the main sympathetic transmitters has a substantial effect on the expression and secretion of NGF by white adipocytes, it would appear that the sympathetic system is not a major factor in the regulation of NGF production in WAT, in contrast to the situation in brown fat (36). This may reflect the fact that, unlike brown fat, WAT is not a highly innervated tissue, so that many aspects of its overall function may be more subject to regulation by factors other than sympathetic activity.
Insulin appears to have little effect on NGF synthesis by white adipocytes. In marked contrast, a strong inhibition of NGF gene expression and protein secretion occurred with dexamethasone, indicating that glucocorticoids are important in the regulation of NGF production by adipocytes, and this is similar to other cell types (12, 30). The production of several adipokines is strongly influenced by glucocorticoids, with the expression of leptin, resistin, and haptoglobin, for example, being stimulated by dexamethasone (8, 19, 37). The peroxisome proliferator-activated receptor- (PPAR
) nuclear receptor may also be involved in the control of NGF production by adipocytes, in view of the inhibition of NGF gene expression and secretion that was observed with rosiglitazone. As with glucocorticoids, the PPAR
receptor has been implicated in the synthesis of several other adipokines, including leptin and resistin (7, 19, 41). The inhibition of NGF production by both dexamethasone and rosiglitazone may relate to their anti-inflammatory effect, given the role of the neurotropin in inflammatory and immune responses (28, 44). Although these agents may act directly to inhibit NGF expression, it is also possible that this inhibition is indirect, via downregulation of proinflammatory cytokines.
The factors discussed so far are either inhibitory, or have little or no effect, on NGF production by white adipocytes. Examination of the effects of inflammatory agents showed that LPS increases NGF mRNA levels and NGF secretion; this presumably occurs via the Toll-like receptors (TLR)-4, which are located in adipocytes (29). A very substantial increase in NGF gene expression and NGF secretion occurred with TNF-, and the response to this proinflammatory cytokine was of a much greater magnitude than with any of the other factors examined. Similarly, we have recently reported that TNF-
has a larger effect on haptoglobin gene expression in 3T3-L1 adipocytes than other hormones and cytokines (37).
The strong stimulation of NGF expression and secretion by TNF- may partly relate to the recognized neuroprotective function of the neurotropin, TNF-
having an apoptotic effect on neurons that is inhibited by NGF (21, 39). NGF may also be associated directly with the inflammatory response of adipocytes. Such a proposition is consistent with the general role that is now recognized for NGF in immunity and inflammation (28, 44). NGF would seem to link with factors such as IL-1
, IL-6, and haptoglobin, as well as TNF-
itself, as inflammation-related products of adipocytes (15, 34, 37, 42).
Despite the scale of the response to TNF-, the regulation of NGF production by proinflammatory cytokines is complex. IL-6 had the opposite effect to TNF-
, although to a much less dramatic extent, inhibiting both NGF gene expression and the amount of the protein secreted from 3T3-L1 adipocytes. IL-6 is considered to be released from sympathetic nerve endings, so that the inhibition of NGF by this cytokine may be equivalent to the effect of catecholamines in terms of a sympathetically induced downregulation. Both IL-6 and TNF-
synthesis in WAT are increased in obesity (24, 25, 45), but whether one or the other has a dominant effect in terms of NGF production in the obese state is unclear. In an initial study, however, we did observe a modest increase in the level of NGF mRNA in epididymal WAT of obese (ob/ob) mice compared with lean controls (unpublished results).
In conclusion, the present study clearly demonstrates that the NGF gene is expressed in the main WAT depots of mice, and that NGF is secreted from adipocytes. By analogy with other tissues, NGF is likely to play a key role as a target-derived neurotropin in the development and survival of sympathetic neurons within white fat. In addition, in view of the profound effect of TNF- on NGF gene expression and secretion, NGF would appear to be linked with the inflammatory response of WAT. Obesity and diabetes are now considered to represent states of chronic mild inflammation (6, 9, 11), and adipocyte-derived NGF may be part of that inflammatory response, along with well-recognized markers such as IL-6, TNF-
, and haptoglobin. Finally, NGF is a member of the rapidly expanding family of proteins secreted by white adipocytes, the adipokines.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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