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,
,1,,
,,
* Children’s Health Research Institute; and
Department of Obstetrics and Gynecology,
Department of Physiology and Pharmacology, and
Department of Medicine, Lawson Health Research Institute; University of Western Ontario, London, Ontario, Canada
1Correspondence: Children’s Health Research Institute, Rm. A5–132, Victoria Research Laboratories,Westminster Campus, 800 Commissioners Rd. East, London, Ontario, Canada N6A 4G5. E-mail: kyang{at}uwo.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: obesity • Zucker rat • adipogenesis • 3T3-L1 cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. There is strong evidence for a genetic component to human obesity (2)
. Multiple systems regulate energy homeostasis (3
, 4)
, and a number of genes associated with human obesity have been identified (5)
; yet, the genetic component of this condition cannot explain the dramatic increase in the prevalence of obesity in recent years.
A large number of epidemiological studies have revealed a robust association between poor fetal growth and the subsequent development of type 2 diabetes, hypertension, and obesity, visceral obesity in particular (6)
. These observations were made initially by Osmond and Barker (7)
in England but have now been reproduced in a diverse range of populations worldwide. These findings have led to the "fetal origins" hypothesis, which states that an adverse intrauterine environment programs or imprints the development of fetal tissues, permanently determining physiological responses and ultimately producing dysfunction and disease later in life (8)
. However, the molecular mechanisms that underpin this relationship remain elusive.
Since worldwide maternal malnutrition is the most common cause for poor early growth and amino acids play a critical role in fetal growth (9)
, the maternal protein restriction (MPR) rat model has become one of the most extensively studied models of early-life origins of adult diseases (10)
. In this model, rat dams are subjected to a low-protein diet (8% protein) instead of control diet (20% protein) throughout pregnancy and lactation. As a consequence, the resulting offspring exhibit low birth weight and become diabetic, insulin resistant, and hypertensive (11
, 12)
. It has been reported that MPR permanently programs the structure and function of certain organs, such as pancreas (13
14
15
16
17)
, liver (18
, 19)
, and muscle (20)
. In addition, MPR also has programming effects on adipose tissue growth and development (21)
. However, given the compelling evidence of a causal link between visceral obesity and metabolic disorders (22)
, the molecular mechanisms underlying increased visceral adiposity are unknown but represent a crucial area of investigation.
Recently, we established an MPR rat model in which to study the metabolic programming of adult diseases (15)
. We made the original observation that MPR led to fetal growth restriction and development of increased visceral adiposity in adult male rat offspring (23)
. We also obtained evidence suggesting that increased visceral adiposity in our rat model was characterized by adipocyte hyperplasia and distinct patterns of visceral adipose tissue gene expression profiles. Indeed, adipocyte precursors derived from MPR offspring exhibited an accelerated rate of proliferation, even a few days after removal from their in vivo environment (24)
. This suggested that MPR permanently altered adipocyte development but that the factors and molecular mechanisms that are responsible for programming this aberrant phenotype remain to be determined.
As a first step in identifying the causal factors involved, we used a candidate gene approach by capitalizing on our previously published visceral adipose tissue gene expression profiling database generated with our rat model of increased visceral adiposity. Candidate genes were selected on the basis that 1) they are known to stimulate nonadipose cell proliferation, and 2) their expression is up-regulated in our rat model. One such candidate is the gene encoding neuropeptide Y (NPY), the expression of which is augmented >4-fold. NPY is produced in the brain, where it functions as the most potent orexigenic factor (25
26
27)
. In addition, NPY stimulates proliferation in a wide variety of nonadipose cells (28
29
30
31
32)
. It also acts on adipocytes to inhibit lipolysis and modulate gene expression (33
34
35
36
37)
. Importantly, during the preparation of this article, two studies were published, one of which (38)
reported that NPY mRNA was expressed in the mouse subcutaneous abdominal adipose tissue and its expression was up-regulated only in mice that were subjected to a combination of cold stress and high-fat diet but not in those exposed to either conditions individually. The other study (39)
showed that NPY protein was secreted by the isolated adipocytes from human abdominal subcutaneous fat and its secretion was enhanced by insulin. One important question remains to be addressed, which is whether NPY is synthesized by visceral adipose tissue and, if so, whether its expression is increased in obesity, because of the established link between increased visceral adiposity and a cluster of metabolic and cardiovascular disorders (22)
.
In the present study, we first confirmed and extended our previous DNA microarray findings of an up-regulated NPY expression in visceral adipose tissue of our rat model with real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and enzyme immunoassay (EIA). We also determined if this novel finding could be extended to the obese Zucker rat, a well-characterized rodent model of obesity. Using both rat primary preadipocytes and the murine preadipocyte cell line 3T3-L1 cells as in vitro model systems, we then studied the effects of NPY on preadipocyte proliferation and differentiation and also examined the NPY receptor subtype as well as the signal transduction pathway involved. In addition, we determined whether increased visceral adiposity in our rat model was glucocorticoid dependent, because of a recently established link between elevated adipose tissue glucocorticoid activity and visceral obesity (40)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MPR rats
The early-life programmed rat model of visceral adiposity, abbreviated as MPR rat model, was established as described previously (23)
. In brief, virgin female Wistar rats (Charles River Laboratories, Wilmington, MA, USA) weighing 240–260 g were housed individually and maintained at 22°C on a 12:12-h light-dark (7 AM–7 PM) cycle. They were mated, and day 0 of gestation was set as the day on which vaginal plugs were expelled. The pregnant dams were fed either a diet containing 20% protein (control diet) or an isocaloric diet containing 8% protein (low-protein diet) throughout pregnancy and lactation. Calories were equalized between diets by the addition of carbohydrate to the low-protein diet. At 3 days of age, litters were randomly reduced to eight pups, thus ensuring a standard litter size per mother. At 21 days of age, all offspring were weaned onto a 20% protein diet. At 130 days of age, male offspring were euthanized and their visceral fat pads (composed of mesenteric, omental, and retroperitoneal fat masses for all fat tissue collections) were isolated. The dissected fat tissues were flash frozen in liquid nitrogen and stored at –80°C. For simplicity, the two groups of offspring will be termed control and MPR rats.
Zucker rats
Lean and obese male Zucker rats at 6 wk of age were purchased from Charles River Laboratories. They were housed at 22°C on a 12:12-h light-dark (7 AM–7 PM) cycle and allowed free access to standard rat chow and drinking water for 3–4 days before treatment. Lean and obese rats were randomly assigned to one of the three treatment groups (control, glargine, and dexamethasone treated), respectively. All animals were weighed daily before treatment, and the dosage of drugs was adjusted daily based on their body weight. The long-acting insulin analog glargine (5 IU/kg body wt) and the synthetic glucocorticoid dexamethasone (120 µg/kg body wt) were injected subcutaneously daily at 10 AM. The reason for using glargine instead of insulin is that administration of this analog in humans results in a constant level of insulin in circulation, thereby eliminating the need to adjust blood glucose levels by a glucose clamp (41)
. The dosage of glargine (5 IU/kg body wt) and dexamethasone (120 µg/kg body wt) represented the therapeutic dosage given to type 2 diabetic patients (41)
and stress levels of glucocorticoid (42)
, respectively. Drugs were diluted in 0.9% saline solution for injection, and a matched volume of the saline solution was injected daily to the control groups. On the eighth day of treatment, animals were euthanized, and visceral adipose tissues were collected, flash frozen in liquid nitrogen, and stored at –80°C.
Wistar rats
Visceral fat pads were also collected from male Wistar rats at 8 wk of age and used immediately for isolation of preadipocytes as described below.
Determination of NPY protein levels
Protein was extracted from visceral adipose tissue samples according to the protocol, as described previously (43)
. Levels of NPY protein in tissue extracts were determined with the NPY EIA kit (Phoenix Pharmaceuticals, Burlingame, CA, USA), following the manufacturer’s instructions. NPY levels were expressed as nanograms of peptide per gram of issue. To eliminate interassay variations, all samples (6 control and 6 MPR) were analyzed in triplicate in one assay, and the intra-assay coefficient of variation was <5%.
Determination of visceral adipose tissue NPY cDNA sequence
Total RNA was extracted from the brain and visceral adipose tissues of a male MPR rat at 130 days of age, as described previously (23)
. A standard RT-PCR was performed using primers that were designed to amplify the entire coding region of the published rat brain NPY cDNA (Table 1
). The PCR products were sequenced from both orientations by a standard automated sequencing protocol at the London Regional Genomics Centre (London, ON, Canada). The sequences generated from both rat brain and visceral adipose tissues were aligned with the published rat NPY cDNA sequence (GenBank accession no. NM_012614).
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Determination of corticosterone levels
Levels of corticosterone in visceral adipose tissue samples were determined using an established ELISA, as described previously (44)
. The level of corticosterone was expressed as nanograms per gram of tissue. To eliminate interassay variations, all samples (6 control and 6 MPR) were analyzed in triplicate in one assay, and the intra-assay coefficient of variation was <5%.
Isolation and culture of rat preadipocytes
Preadipocytes were isolated, and their purity was verified by >95% conversion to adipocytes as determined by Oil Red O staining following an established differentiation protocol, as described previously (24)
. Briefly, the visceral fat pads were dissected from visible blood vessels and connective tissue, weighed, finely minced, and digested in digestion buffer (3 ml/g tissue) consisting of Dulbecco modified Eagle medium (DMEM; Invitrogen Life Technologies, Burlington, ON, Canada), 0.5 mg/ml collagenase class IV (Sigma, Oakville, ON, Canada), and 1.5% bovine serum albumin (Sigma) for 45 min at 37°C under mild controlled agitation. The resultant digest material was filtered through 250 µm nylon mesh and centrifuged at 600 g for 5 min to separate the floating adipocytes. The cell pellet was resuspended, washed with Dulbecco phosphate-buffered saline (DPBS) containing 10% newborn calf serum (Invitrogen), filtered through 25 µm nylon mesh, and then centrifuged. The pelleted preadipocytes were either used immediately for total RNA isolation or resuspended in standard culture medium (DMEM/F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Sigma), 50 U/ml penicillin, and 50 µg/ml streptomycin (Invitrogen). Preadipocytes were cultured on 24-well plates in a humidified incubator at 37°C in the presence of 5% CO2.
Culture of 3T3-L1 preadipocytes
The murine 3T3-L1 preadipocyte cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in growth medium, consisting of DMEM (Sigma) and 10% FBS (Sigma). Cultures were maintained in a humidified incubator at 5% CO2 and 37°C. Medium was replaced every 2–3 days.
Proliferation assay: [3H]thymidine incorporation
Proliferation capacity of 3T3-L1 preadipocytes and rat primary preadipocytes was assessed by measuring [3H]thymidine incorporation, as described previously (24)
. Briefly, 3T3-L1 cells and rat preadipocytes were plated on 24-well plates and cultured in growth medium until 40–50% confluence. Cells were growth arrested in serum-free growth medium for 24 h and were then treated in the serum-free medium with NPY for 24 h or as indicated otherwise. During the last 4 h of treatment, cells were pulsed labeled with [3H]thymidine (0.5 µCi/well) (75.2 Ci/mmol, PerkinElmer Life and Analytical Sciences, Woodbridge, ON, Canada). Cells were washed twice with ice-cold PBS, once with 5% trichloroacetic acid, and twice with 95% ethanol. Cells were then solubilized by the addition of 200 µl of 0.5 M NaOH. The solubilized cell lysate (100 µl) was added to 4 ml of scintillation fluid, and the incorporation of [3H]thymidine into DNA was determined by scintillation counting. Protein concentrations in the cellular lysates were determined by the Bradford technique, and [3H]thymidine incorporation was normalized by protein content. Results are expressed as a percentage of control.
Mitogen-activated protein kinase activation: Western blot analysis
3T3-L1 preadipocytes were plated on 24-well plates and cultured under standard condition until they reach 30–40% confluency. Cells were starved for 24 h and then treated with 10 nM of NPY in serum-free medium. At discrete times thereafter (0–20 min), cells were lysed in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol, and 0.01% wt/vol bromphenol blue) and stored at –80°C.
Levels of extracellular related kinase (ERK) 1/2 proteins in cell lysates were determined by standard Western blot analysis (45)
. In brief, equal fractions of the cell lysates were subjected to a standard 12% SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose using a Bio-Rad mini transfer apparatus. Both total ERK1/2 and phosphorylated ERK1/2 proteins were detected on the nitrocellulose filter using the PhosphoPlus p44/42 mitogen-activated protein kinase (MAPK; Thr202/Tyr204) antibody kit (Cell Signaling) and an enhanced chemiluminescence (ECL) Western blotting analysis system (Pharmacia, Baie D’Urte, QC, Canada) following the manufacturers’ instructions. Briefly, the nitrocellulose filter was blocked for 2 h at 4°C with 10% Blotto in TTBS (0.1% Tween-20 in TBS) and incubated with primary antibodies in TTBS overnight at 4°C. The primary antibodies were polyclonal rabbit anti-ERK1/2 antibody (1:1000) and polyclonal rabbit anti-pERK1/2 antibody (1:1000). After three 5 min washes with TTBS, the filter was incubated with horseradish peroxidase-labeled second antibody (1:5000 dilution) and developed in ECL detection reagents. The filter was then exposed to X-ray film (Eastman Kodak, Rochester, NY, USA) for 10 s to 5 min.
Adipocyte differentiation
3T3-L1 preadipocytes were cultured and differentiated into adipocytes as described previously (46)
. Briefly, cells were grown in growth medium and allowed to reach confluence. At 2 days postconfluence (referred as day 0), cells were induced to differentiate by the addition of a differentiation cocktail containing 0.5 mM of 3-isobutyl-1-methylxanthine (IBMX; Sigma), 0.25 µM of dexamethasone (Alpharma, Boucherville, QC, Canada), and 1 µg/ml of insulin (Eli Lilly Canada, Toronto, ON, Canada). After 48 h (day 2), the medium was replaced with growth medium supplemented with 1 µg/ml of insulin. Subsequently, the medium was changed at days 4 and 6 with fresh growth medium. By day 8, >90% of cells had acquired adipocyte phenotype (i.e., containing lipid droplets). To study the effects of NPY on adipocyte differentiation, 3T3-L1 preadipocytes were subjected to the same differentiation protocol except that insulin was replaced by NPY. Negative controls in the absence of insulin were also included. At day 8, differentiation was assessed by Oil Red O staining of lipid droplets and by determining levels of mRNAs encoding the key adipogenic transcription factor peroxisome proliferator-activated receptor 
(PPAR) and the adipocyte marker adipocyte fatty acid binding protein (aP2) as described below.
Oil Red O staining
Oil Red O staining was performed as described previously (24)
. Briefly, differentiated adipocyte monolayers were washed with DPBS, fixed for 1 h with 4% paraformaldehyde at room temperature, and incubated in 60% isopropanol for 5 min. Oil Red O (3 g/L; Sigma) in 99% isopropanol was diluted with water, filtered, and added to the fixed cell monolayers for 5 min, and then the nuclei were stained with hematoxylin for 30 s. Cell monolayers were then washed with water, and the stained triglyceride droplets were visualized and photographed. The extent of adipocyte differentiation was quantitated by determining the amount of extracted dye, as measured by the optimal absorbance at 510 nM after elution of Oil Red O with isopropanol (47)
.
Analysis of NPY receptor expression: RT-PCR
Expression of Y1, Y2, and Y5 receptors was analyzed by standard RT-PCR. Briefly, total RNA was isolated from cultured rat preadipocytes and 3T3-L1 cells treated with and without 10 nM of NPY as well as mouse and rat brains using TRIZol reagent (Invitrogen) and was subsequently purified by RNeasy mini kit (Qiagen, Mississauga, ON, Canada) coupled with on-column DNase digestion with the RNase-Free DNase set (Qiagen) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed in a volume of 20 µl with the high capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA), following the manufacturer’s instructions. For every RT reaction, one RNA sample was set up without the RT enzyme to provide a negative control against possible genomic DNA contamination. PCR reactions were carried out in a total volume of 50 µl containing 1 µl of RT and 1.5 U of Platium TaqDNA polymerase (Invitrogen). The primers specific for mouse and rat GAPDH, Y1, Y2, and Y5 receptors as well as their expected product sizes are shown in Table 1
. PCR reactions were performed for 35 cycles (32 cycles for the positive controls and 26 cycles for GAPDH) with denaturing at 95°C, annealing at 55°C, and extension at 72°C. PCR products were confirmed with standard restriction enzyme digestions and sequencing analysis.
Analysis of Y1 receptor protein expression: Western blotting
Expression of Y1 receptor protein was analyzed by standard Western blot analysis, as described previously (45)
. Briefly, rat primary preadipocytes and 3T3-L1 cells as well as rat and mouse brain tissues were lysed or homogenized in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol, and 0.01% wt/vol bromphenol blue). Cell lysates and tissue homogenates were then subjected to a standard 12% SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose using a Bio-Rad mini transfer apparatus. The Y1 receptor and GAPDH (to serve as a control) proteins were detected on the nitrocellulose filter using an ECL Western blotting analysis system (Pharmacia) following the manufacturer’s instructions. Briefly, the nitrocellulose filter was blocked for 2 h at 4°C with 10% Blotto in TTBS and incubated with primary antibodies in 3% Blotto overnight at 4°C. The primary antibodies were polyclonal rabbit anti-mouse Y1 receptor antibody (ADI Inc., San Antonio, TX, USA; NPY1R11-A; 3 µg/ml) and polyclonal rabbit anti-human GAPDH antibody (Imgenex Corp., San Diego, CA, USA; IMG-5567; 1:4000). After three 5 min washes with TTBS, the filter was incubated with horseradish peroxidase-labeled second antibody (1:5000 dilution) and developed in ECL detection reagents. The filter was then exposed to X-ray film (Eastman Kodak).
Real-time qRT-PCR
Expression of NPY, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), glucocorticoid receptor (GR), and the key adipocyte markers aP2 and PPAR was analyzed by a two-step real-time qRT-PCR, as described previously (23)
. Briefly, 1 µg of total RNA was reverse transcribed in a volume of 20 µl with the high capacity cDNA archive kit (Applied Biosystems), following the manufacturer’s instructions. For every RT reaction, one RNA sample was set up without the RT enzyme to provide a negative control against possible genomic DNA contamination. Gene-specific primers were designed by using Primer Express software (Applied Biosystems), and the optimal concentrations for each gene were determined empirically. The primers used are listed in Table 2
. The SYBR Green I assay was performed with the SYBP Green PCR master mix (Applied Biosystems) and a modified universal thermal cycling condition (2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 10 s each at 95, 60, and 72°C) with the standard disassociation/melting parameters (15 s each at 95, 60, and 95°C) on the ABI Prism 7900HT sequence detection system (Applied Biosystems). The specificity of the SYBR Green I assay was verified by performing a melting curve analysis and by subsequent sequencing of the PCR products.
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Levels of 28S rRNA (housekeeping gene) and target mRNAs in each RNA sample were quantified by the relative standard curve method (Applied Biosystems). Briefly, standard curves for 28S rRNA and each target gene were generated by performing a dilution series of a mixed cDNA pool. For each RNA sample, the amount of target mRNA relative to that of 28S rRNA was obtained. For each target gene, fold changes in the treatment groups compared with the control were then calculated and expressed as mean ± SE.
Statistical analyses
Results are presented as mean ± SE of four to six independent experiments or individual rats, as indicated. Data were analyzed using Student’s t test and one-way or two-way ANOVA followed by Tukey’s post hoc test, as indicated in the figure legends. Significance was set at P < 0.05. Calculations were performed using Prism 3.0 software (GraphPad, San Diego, CA, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, we mined our published DNA microarray data as deposited in the National Center for Biotechnology Information database (accession no. GSE1813). We found that expression of the gene encoding NPY was up-regulated 4-fold in visceral adipose tissues of MPR offspring when compared with controls. To verify this, we determined the abundance of NPY mRNA in the same total RNA samples as used in the previously published study (23)
with qRT-PCR. As shown in Fig. 1
A, MPR increased the level of NPY mRNA in visceral adipose tissues by 6-fold. To determine whether this increase was carried through to a corresponding rise in NPY protein, we measured levels of NPY protein in tissue extracts with EIA. We showed that NPY protein levels were 4.6x higher in visceral adipose tissues from MPR offspring than those from controls (Fig. 1B
).
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We then determined whether NPY produced in the visceral adipose tissue is the same as the one produced by the brain. To do so, we designed primers that encompass the entire coding region of the published rat NPY cDNA and performed RT-PCR using total RNA samples extracted from both rat brain and visceral adipose tissues as templates. Sequence analysis of the RT-PCR products revealed that the 443 bp NPY cDNA generated from both rat brain and visceral adipose tissues displayed 100% sequence identity to the published rat NPY cDNA (data not shown).
NPY expression in visceral adipose tissue of obese Zucker rats
To determine if the increased expression of NPY could be observed in other rodent models of obesity, we assessed NPY mRNA abundance in visceral adipose tissue from lean and obese male Zucker rats. We found that adipose tissue levels of NPY mRNA were >2x higher in obese than in lean rats (Fig. 2
; P<0.01), suggesting that the increased local NPY expression within visceral adipose tissue may be a common feature of increased adiposity.
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Regulation of NPY expression in visceral adipose tissue by insulin and glucocorticoid in Zucker rats
Having established an elevated NPY expression in visceral adipose tissue of obese Zucker rats, we then determined the causal factors involved. Given that circulating levels of both insulin (48)
and corticosterone (49)
are elevated in obese Zucker rats, coupled with the recent findings that insulin increased NPY secretion from isolated human adipocytes (39)
, we hypothesized that insulin and/or glucocorticoid might stimulate NPY expression in Zucker rats. To test this hypothesis, we studied the effects of the long-acting insulin analog glargine and the synthetic glucocorticoid dexamethasone on adipose tissue NPY expression in Zucker rats. As shown in Fig. 2
, treatment of lean Zucker rats for 7 days with either glargine or dexamethasone augmented NPY mRNA expression in visceral adipose tissue to levels seen in nontreated obese Zucker rats. In contrast, neither of the two treatments was effective in obese Zucker rats.
Expression of NPY receptors in preadipocytes
As a first step in determining if visceral adipose tissue-derived NPY functions locally to regulate preadipocyte proliferation and/or differentiation, we examined the expression of Y1, Y2, and Y5 receptors in cultured rat preadipocytes and a murine preadipocyte cell line, 3T3-L1 cells, treated with and without NPY using standard RT-PCR. We showed that the Y1 receptor mRNA was abundantly expressed in both rat preadipocytes and 3T3-L1 cells and that treatment of these cells with NPY did not alter the mRNA abundance (Fig. 3
A). In contrast, mRNAs encoding the Y2 and Y5 receptors were undetectable in both cell types irrespective of NPY treatment. As expected, mRNA coding for all three NPY receptor subtypes was present in rat and mouse brain tissues (Fig. 3A
).
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To provide further evidence for the expression of Y1 receptors in these cells, we determined Y1 receptor protein by Western blotting. We showed that Y1 receptor protein was present in both rat preadipocytes and 3T3-L1 cells. Consistent with the mRNA data, NPY had no effect on Y1 receptor protein expression (Fig. 3B
).
Effects of NPY on preadipocyte proliferation
Given that accelerated adipogenesis (i.e., preadipocyte proliferation and differentiation) is a hallmark of increased adiposity (50)
and NPY is potent mitogen in a variety of nonadipose cells (28
29
30
31
32)
, we first determined if NPY stimulated preadipocyte proliferation. We showed that in both 3T3-L1 and rat primary preadipocytes NPY increased [3H]thymidine incorporation in a concentration-dependent manner, being effective at 1 nM (P<0.05) and reaching a maximal effect at 10 nM (>200% increase; Fig. 4
). This confirms and extends a recent study (38)
in which a single unspecified concentration of NPY increased [3H]thymidine incorporation in 3T3-L1 preadipocytes by 
190%.
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Involvement of the Y1 receptor and ERK1/2 in mediating the effect of NPY on preadipocyte proliferation
Since data presented in Fig. 3
indicate that the Y1 receptor was the primary NPY receptor isoform expressed in rodent preadipocytes, we determined if this receptor mediated the proliferative effect of NPY in 3T3-L1 cells. To do so, we first studied the effects of several NPY analogues (with various degrees of specificity for Y1, Y2, and Y5 receptors) on preadipocyte proliferation. As shown in Fig. 5
A, [Leu31,Pro34]-NPY, which binds to both Y1 and Y5 receptors, was as effective as NPY in stimulating [3H]thymidine incorporation. In contrast, both the Y2 receptor-specific agonist NPY13–36 and the Y2/Y5 receptor agonist NPY3–36 were ineffective (Fig. 5B, C
).
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To confirm the involvement of the Y1 receptor in mediating the stimulatory effects of NPY on preadipocyte proliferation, we treated 3T3-L1 preadipocytes with NPY in the presence and absence of the Y1 receptor antagonist BIBP3226 (Bachem, Torrance, CA, USA) or the Y5 receptor antagonist L-152,804 (Tocris, Ellisville, MO, USA). BIBP3226, but not L-152,804, blocked the NPY-induced increases in [3H]thymidine incorporation (Fig. 6
A).
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Given that the mitogenic effects of NPY in nonadipose cells are mediated through the activation of ERK1/2 (28
, 29
, 31)
, we explored the involvement of this MAPK in mediating the proliferative effect of NPY on 3T3-L1 preadipocytes using U0126, a specific pharmacological inhibitor of ERK1/2. As shown in Fig. 6A
, U0126 prevented NPY-induced increases in [3H]thymidine incorporation, suggesting that the stimulatory effects of NPY on preadipocyte proliferation are mediated through the ERK1/2 signaling pathway. Indeed, NPY induced rapid activation of ERK1/2, as evidenced by elevated levels of phosphorylated ERK1/2 (Fig. 6B
).
Effects of NPY on adipocyte differentiation
Having established a role for NPY in preadipocyte proliferation, we then studied its effects on adipocyte differentiation. To do so, 3T3-L1 cells were exposed to various concentrations of NPY (known to stimulate preadipocyte proliferation as shown in Fig. 4
) and 1 µg/ml of insulin (served as a positive control), respectively, in the presence of IBMX and dexamethasone. NPY and insulin were added to the medium from the start of differentiation (day 0) to day 4. At day 8, differentiation was assessed using both morphological (i.e., Oil Red O staining) and biochemical (i.e., expression of key adipocyte markers) methods. As shown in Fig. 7
A, B, NPY did not influence lipid accumulation. This was consistent with the lack of an effect of NPY on levels of mRNAs encoding the key adipocyte marker proteins aP2 and PPAR (Fig. 7C, D
). As expected, insulin enhanced both lipid accumulation (Fig. 7A, B
) and expression of adipocyte markers (Fig. 7C, D
).
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Effects of MPR on key components of glucocorticoid signaling in visceral adipose tissue
Given that an enhanced activity of glucocorticoid in visceral adipose tissue is linked to visceral obesity and metabolic syndrome (40)
, we sought changes in the three key components of the glucocorticoid signaling in visceral adipose tissue of our rat model. As shown in Fig. 8
, visceral adipose tissue levels of corticosterone, GR mRNA, and 11β-HSD1 mRNA were not different between control and MPR rats, suggesting that increased visceral adiposity in our rat model is glucocorticoid independent.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We first confirmed our previous DNA microarray data regarding the up-regulation of NPY expression in visceral adipose tissue of MPR offspring with qRT-PCR and demonstrated that the magnitude of the increase in NPY mRNA was similar between the two methodologies. Importantly, we revealed a corresponding rise in NPY protein, indicating that visceral adipose tissue is a bona fide site of NPY biosynthesis. Furthermore, we presented evidence that visceral adipose tissue-derived NPY was identical to that produced by the brain. We also showed that NPY expression was elevated in visceral adipose tissue of obese Zucker rats. These novel observations not only suggest that aberrant expression of NPY in visceral adipose tissue may be a common feature of increased visceral adiposity but also underscore the potential role of this locally derived NPY in the pathogenesis of visceral obesity.
Although there is robust evidence that white adipose tissue is innervated and NPY can be released from nerve endings (36)
, it is not until very recently that the potential of white adipose tissue to synthesize NPY has been revealed. During the preparation of this article, Kuo et al. (38)
reported that 2 wk of cold stress together with a high-fat diet led to increased visceral adiposity and up-regulation of NPY mRNA specifically in the subcutaneous abdominal fat pads in mice. They also reported that human white adipose tissue contained NPY protein as revealed by immunohistochemistry, which could result from local production and/or be taken up from circulation. A more recent study (39)
provided evidence that NPY was secreted, and by inference produced, by isolated human adipocytes. Thus, our present findings corroborated and extended these two recent publications by providing the first evidence that NPY was not only expressed in rat visceral adipose tissue but that the level of its expression was up-regulated in the two distinct models of increased adiposity.
Given that circulating levels of both insulin (48)
and corticosterone (49)
are elevated in obese Zucker rats, coupled with the previous findings that glucocorticoid stimulated NPY expression in rat blood cells in vivo (51)
as well as in neuronal cell lines in vitro (52)
and insulin increased NPY secretion in isolated human adipocytes in vitro (39)
, we hypothesized that the elevated level of insulin and corticosterone might be responsible for the increased adipose tissue NPY expression in obese Zucker rats. To test our hypothesis, we treated both lean and obese Zucker rats for 7 days with either the long-acting insulin analog glargine or the synthetic glucocorticoid dexamethasone. We demonstrated that both glargine and dexamethasone increased adipose tissue levels of NPY mRNA in lean but not obese Zucker rats. Thus, our results not only provide the first in vivo evidence that NPY expression in visceral adipose tissue was regulated by insulin and glucocorticoid but also suggest that the elevated levels of these two hormones, either singly or together, might be responsible for the increased expression of NPY in obesity. If our present findings could be extrapolated to humans, they would have significant and far-reaching clinical implications because insulin levels are elevated in insulin resistance/type 2 diabetes and the intra-adipose tissue level of glucocorticoid is increase in obese humans, either one of which could lead to elevated levels of peripheral NPY expression within adipose tissue. Obviously, this contention requires future scrutiny.
The identification of visceral adipose tissue as a new peripheral site of NPY biosynthesis begs the question of whether NPY may function locally in an autocrine and/or paracrine fashion to regulate adipogenesis. Given that NPY is a potent mitogen in a variety of nonadipose cells (28
29
30
31
32)
and that adipocyte hyperplasia is a hallmark of increased adiposity (50)
, we hypothesized that NPY stimulates preadipocyte proliferation. As a first step in examining this hypothesis, we determined NPY receptor expression in the murine preadipocyte cell line 3T3-L1 and cultured rat preadipocytes with RT-PCR. Our results revealed that the Y1 receptor was expressed in both cell types, suggesting that 3T3-L1 preadipocytes are a suitable in vitro model system in which to study the proliferative effect of NPY. Importantly, we showed that NPY, at physiologically relevant concentrations, increased thymidine incorporation, a well-known marker of proliferation, not only in 3T3-L1 cells but also in cultured rat preadipocytes.
We further addressed the functional involvement of the Y1 receptor in mediating the proliferative effect of NPY in 3T3-L1 preadipocytes using a variety of NPY analogues with varying degrees of selectivity for the three primary subtypes of NPY receptors as well as specific and commercially available NPY receptor antagonists. We showed that the Y1/Y5 agonist [Leu31,Pro34]-NPY was as effective as NPY in stimulating preadipocyte proliferation, while both the Y2 agonist NPY13–36 and the Y2/Y5 agonist NPY3–36 were ineffective. Furthermore, the Y1 receptor antagonist BIBP3226, but not the Y5 receptor antagonist L-152,804, blocked the mitogenic effect of NPY. Taken together, our results provided compelling evidence that the stimulatory effect of NPY on preadipocyte proliferation was mediated by the Y1 receptor. Although our present findings corroborate in principle those of Kuo et al. (38)
, there is one discrepancy in that the Y2 receptor was implicated in the previous study. There are no apparent reasons for this discrepancy, especially considering that the same preadipocyte cell line and the same proliferation assay were used. However, it is noteworthy that both studies showed that the mRNA encoding the Y2 receptor was undetectable in 3T3-L1 preadipocytes under nonstimulated conditions. Importantly, the Y2 receptor antagonist BIIE0246 did not completely block NPY-induced increases in thymidine incorporation in the previous study (38)
, while the Y1 receptor antagonist BIBP3226 did so in the present study.
Another novel aspect of our present study was that we examined the signal transduction pathway involved in mediating the proliferative effect of NPY on adipocyte precursors. We showed that NPY activated the ERK-signaling pathway, as evidenced by a rapid rise in levels of phosphorylated ERK1/2 proteins. We also demonstrated that the pharmacological inhibitor of ERK1/2 U0126 prevented NPY-induced increases in thymidine incorporation. Our present findings are consistent with the literature, which showed that NPY-elicited proliferation was mediated primarily by the ERK1/2 signaling pathway in a variety of nonadipose cells, including neurons (29)
, cardiomyocytes (28)
, pancreatic β-cells (31)
, smooth muscle cells (53)
, and tumor cells (32
, 54)
. These proliferative effects appeared to engage either Y1 or Y5 receptors.
Adipogenesis involves both proliferation and differentiation of adipocyte precursor cells. The fact that NPY stimulates preadipocyte proliferation raises the question of whether NPY influences adipocyte differentiation. To address this question, we treated 3T3-L1 preadipocytes with various concentrations of NPY that were known to be effective in promoting preadipocyte proliferation and established that NPY had no effect on lipid accumulation. This was corroborated by the lack of an effect of NPY on the expression of the two key adipocyte marker genes aP2 and PPAR. Our present findings are consistent with our recently published study (24)
in which we identified an aberrant phenotype of adipocyte precursor cells derived from MPR rats. These precursors exhibited an accelerated rate of proliferation with an unremarkable differentiation profile. However, our present data are in contrast with those of Kuo et al. demonstrating that NPY mimicked the effects of insulin by increasing lipid filling of the new adipocytes, as revealed by Oil Red O staining. There are no apparent reasons for this discrepancy, and direct comparisons could not be made between the two studies, because no details were provided about the differentiation and treatment protocols in the previous study. Clearly, these discrepancies, including the one described above, await resolution by future independent studies.
At periphery, NPY is colocalized with norepinephrine (NE) and released from sympathetic nerve terminals on sympathetic stimulation (55)
, and it is known that white adipose tissue is innervated by the sympathetic nervous system (56
, 57)
. Interestingly, sympathetic denervation of white adipose tissue leads to increased adiposity and fat cell numbers in hamsters (58
59
60)
. This was likely due to the removal of an inhibitory effect of NE on adipocyte proliferation (59)
. Given the stimulatory effect of NPY on preadipocyte proliferation, we can only speculate that the effect of NE is dominant over that of NPY within the sympathetic nerve system of white adipose tissue. Our present findings and those of Kuo et al. (38)
revealed a new source of NPY, in addition to that released from sympathetic nerve endings, within white adipose tissue. However, the relative contribution of these two sources to the total NPY pool in white adipose tissue remains to be determined. It is also unclear as to which cell type is responsible for NPY biosynthesis in the adipose tissue, although one recent study (39)
showed that isolated human adipocytes were capable of releasing NPY into the culture medium.
The discovery of NPY production by visceral adipose tissue also raises the question of whether this peripheral source of NPY is the same as that produced by the brain. To address this question, we sequenced the NPY cDNAs generated by RT-PCR from both rat brain and visceral adipose tissue and compared these sequences to the published rat NPY cDNA sequence. Our results revealed that the adipose tissue-derived NPY cDNA sequence was identical to both the brain-derived and the published NPY cDNA sequences, indicating that NPY produced by rat visceral adipose tissue is the same as the one produced by the brain. Based on our present results of NPY stimulation of preadipocyte proliferation in vitro, we speculate that this novel peripheral source of NPY may function locally to promote proliferation of adipocyte precursor cells in vivo, thereby contributing to the pathogenesis of obesity. It is also possible that adipose tissue-derived NPY may enter the circulation and even the brain where it may complement the role of centrally derived NPY in regulating appetite. Obviously, the relative role of the brain- and adipose tissue-derived NPY in mediating obesity requires future study.
Given the recently established link between an enhanced intra-adipose tissue glucocorticoid activity and visceral obesity (40)
, we sought changes in the three key components of glucocorticoid signaling in our rat model of increased visceral adiposity. We found that visceral adipose tissue levels of cortcosterone, 11β-HSD1 mRNA, and GR mRNA were similar between control and MPR offspring, suggesting that the increased visceral adiposity in our rat model is glucocorticoid independent. The present data contrast with those in the literature showing an up-regulation of at least one of these three key components in the local glucocorticoid signaling in a variety of rodent models of obesity (61)
, including the recently reported mouse model of stress and high-fat diet-induced obesity in which both corticosterone concentration and 11β-HSD1 mRNA abundance were elevated in the abdominal adipose tissue (38)
. Conflicting results have been reported in humans, with some showing an increase in white adipose tissue levels of 11β-HSD1 mRNA and activity (62
63
64
65)
, while others report no change (66)
. These discrepancies probably reflect complex and distinct etiologies of obesity in humans and different animal models of obesity.
In conclusion, the present study identifies rat visceral adipose tissue as a new site of NPY biosynthesis, and reveals that NPY expression within the adipose tissue is up-regulated in our early-life programmed rat model of increased visceral adiposity and obese Zucker rats. Importantly, our study provides the first in vivo evidence that adipose tissue NPY expression is subject to regulation by insulin and glucocorticoid, the two most important hormones involved in the pathogenesis of obesity and its associated metabolic disorders. We also show that NPY promotes preadipocyte proliferation in vitro. Thus, our data corroborate and extend those reported recently by Kuo et al. (38)
in a mouse model of stress and high-fat diet-induced obesity. Together, the two studies provide a strong rationale for exploiting the local NPY system within white adipose tissue as a potential therapeutic target for combating visceral obesity and its associated metabolic disorders.
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ACKNOWLEDGMENTS |
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Received for publication October 28, 2007. Accepted for publication February 14, 2008.
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