Fat depot-related differences in gene expression, adiponectin secretion, and insulin action and signalling in human adipocytes differentiated in vitro from precursor stromal cells
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- Perrini, S., Laviola, L., Cignarelli, A. et al. Diabetologia (2008) 51: 155. doi:10.1007/s00125-007-0841-7
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The distinct metabolic properties of visceral and subcutaneous adipocytes may be due to inherent characteristics of the cells that are resident in each fat depot. To test this hypothesis, human adipocytes were differentiated in vitro from precursor stromal cells obtained from visceral and subcutaneous fat depots and analysed for genetic, biochemical and metabolic endpoints.
Stromal cells were isolated from adipose tissue depots of nondiabetic individuals. mRNA levels of adipocyte-specific proteins were determined by real-time RT-PCR. Insulin signalling was evaluated by immunoblotting with specific antibodies. Glucose transport was measured by a 2-deoxy-glucose uptake assay. Adiponectin secretion in the adipocyte-conditioned medium was determined by a specific RIA.
With cell differentiation, mRNA levels of PPARG, C/EBPα (also known as CEBPA), AP2 (also known as GTF3A), GLUT4 (also known as SLC2A4) were markedly upregulated, whereas GLUT1 (also known as SLC2A1) mRNA did not change. However, expression of C/EBPα, AP2 and adiponectin was higher in subcutaneous than in visceral adipocytes. By contrast, adiponectin was secreted at threefold higher rates by visceral than by subcutaneous adipocytes while visceral adipocytes also showed two- to threefold higher insulin-stimulated glucose uptake. Insulin-induced phosphorylation of the insulin receptor, IRS proteins, Akt and extracellular signal-regulated kinase-1/2 was more rapid and tended to decrease at earlier time-points in visceral than in subcutaneous adipocytes.
Subcutaneous and visceral adipocytes, also when differentiated in vitro from precursor stromal cells, retain differences in gene expression, adiponectin secretion, and insulin action and signalling. Thus, the precursor cells that reside in the visceral and subcutaneous fat depots may already possess inherent and specific metabolic characteristics that will be expressed upon completion of the differentiation programme.
KeywordsAdiponectinAktErkExtracellular signal-regulated kinaseGlucose uptakeInsulin signallingSubcutaneous fatVisceral fat
CCAAT/enhancer binding protein (C/EBP), alpha
extracellular signal-regulated kinase
phosphatase and tensin homologue (mutated in multiple advanced cancers 1)
protein tyrosine phosphatase 1B
SH2 containing inositol phosphatase
By regulating triacylglycerol metabolism and secreting a variety of cytokines and hormones with pleiotropic effects on multiple tissues, adipose tissue has an enormous capacity to regulate fuel utilisation, energy homeostasis and cardiovascular function. Dysfunctional adipose tissue, clinically evident as excessive fat mass (obesity) or abnormally reduced fat (lipodystrophy), leads to impaired glucose and lipid metabolism, insulin resistance and type 2 diabetes, as well as to increased risk of cardiovascular disease. Indeed, the distribution of body fat appears to be more important than the total amount of fat. Abdominal, and in particular visceral, adiposity shows the closest association with metabolic and cardiovascular diseases [1, 2]. An excess of visceral fat may promote decreased insulin effects on glucose uptake, NEFA re-esterification and inhibition of lipolysis . Conversely, surgical elimination of visceral fat resulted in dramatic improvements in insulin sensitivity in rodents  and humans , while removal of subcutaneous fat did not prevent insulin resistance and metabolic abnormalities [6, 7].
The adverse metabolic impact of visceral fat has been attributed to distinct biological properties of adipocytes in this particular fat depot compared with those in other sites, including differences in metabolic responses, gene expression, adipokine secretion and insulin action [8, 9]. For example, the lipolytic effect of catecholamines was shown to be more pronounced and the anti-lipolytic effect of insulin weaker in omental than in subcutaneous adipocytes . Differences in the secretion of cytokines, such as adiponectin, plasminogen activator inhibitor, type I (PAI-1), IL-6 and leptin, between the two abdominal fat compartments have also been extensively described [9, 11]. Recently, we investigated insulin signalling in different fat depots in humans in vivo and demonstrated higher, more rapid and transient activation of the insulin signalling cascade in visceral than in subcutaneous fat . These observations are in line with the reportedly greater effect of insulin in stimulation of glucose uptake in visceral fat, both in humans in vivo  and in isolated human adipocytes in vitro .
It should be recognised, however, that information on the metabolic properties of adipose tissue largely derives from studies in whole fat tissue in vivo, adipose tissue fragments ex vivo or isolated mature adipocytes in vitro, thus probably reflecting the influence of environmental factors in the tissue of origin. These experimental conditions cannot clarify whether the depot-related specificity of adipose cells is due to their innate characteristics or, alternatively, to extrinsic factors, such as tissue microenvironment, local circulation, local innervation and/or heterogeneity in cellularity. In this study, we used adipocytes differentiated in vitro from visceral and subcutaneous precursor stromal cells to investigate whether these cells retain the depot-related differences in gene expression, adiponectin secretion, and insulin action and signalling that are seen in intact adipose tissue and freshly isolated adipocytes.
Individuals and adipose tissue biopsies
Paired abdominal subcutaneous and omental fat biopsies were obtained from 13 non-obese individuals with normal glucose tolerance (seven men, six women; aged 45 ± 1 years; weight 68.4 ± 3.4 kg; height, 167 ± 3 cm), who underwent elective open abdominal surgery for nonmalignant diseases (i.e. cholecystectomy, inguinal hernia, hysterectomy). They were all otherwise healthy and not taking any regular medication. All participants gave their informed consent before the surgical procedure. The study protocol was approved by the Independent Ethical Committee at the Azienda Ospedaliero-Universitaria Policlinico Consorziale, University of Bari School of Medicine. Anthropometric and metabolic characterisation, including both height and weight measurements and fasting blood chemistry, were performed 2 to 3 days before the surgical procedure; see Electronic supplementary material (ESM) for additional details.
Isolation and differentiation of adipocyte precursor cells
Human preadipocytes were isolated according to the method of Rodbell , with minor modifications, and differentiated for about 30 days in DMEM/F-12 medium supplemented with insulin, dexamethasone, triiodothyronine and rosiglitazone, in line with recently published methods for differentiation of human preadipocytes ; for further details on preadipocyte isolation and differentiation, see ESM. Differentiation of cells into adipocytes was assessed through morphological analysis and Oil-Red-O staining. Cells in three wells were fixed with 3.7% (vol./vol.) formaldehyde in PBS, and their triacylglycerol content was stained with 0.3% (wt/vol.) Oil-Red-O in 60% (vol./vol.) isopropanol. After repeatedly washing with water, the differentiated adipocytes were estimated by direct counting under light microscope. Cells were defined as differentiated when their cytoplasm was completely filled with multiple fat droplets. All experimental analyses on adipocytes were carried out following culture for 2 days in washout DMEM/F-12 medium without insulin, dexamethasone, triiodothyronine and rosiglitazone.
RNA extraction and real-time PCR
Total RNA was isolated from preadipocytes, mature adipocytes and whole adipose tissue using kits (RNeasy Mini and RNeasy Lipid Tissue Mini, respectively; Qiagen, Hilden, Germany). The mRNA levels of multiple proteins were analysed by quantitative real-time RT-PCR using a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA), as previously described  (see ESM for primer sequences). The mRNA level of each target gene was normalised using 18S as internal control.
Measurement of adiponectin secretion and glucose uptake
Adipocytes were starved for 12 h in serum-free DMEM/F10 medium containing 0.2% (wt/vol.) BSA and then incubated for 2 h in fresh medium. A fraction of the culture medium was removed for subsequent adiponectin evaluation by RIA using a specific assay kit (Linco Research, St Charles, MO, USA). The kit intra-assay and inter-assay CVs were 6.21 and 6.90%, respectively. Adiponectin secretion is reported as ng/ml of culture medium per μg of cell protein. Glucose transport measurements in human adipocytes were performed as previously described  (see ESM).
Analysis of insulin signalling
Subcutaneous and omental adipocytes were incubated for 3 h in serum-free medium containing 0.5% (wt/vol.) BSA and then stimulated with 10 nmol/l insulin for the indicated times or left untreated. Then the cells were rapidly processed and analysed by immunoblotting, as described previously [12, 18] (see ESM).
Differences between the two groups were analysed by the Student’s t test for independent samples. For the evaluation of changes in the phosphorylation profiles of the proteins investigated, one-way ANOVA and Tukey’s post hoc test were used. All data are expressed as mean ± SE. p values <0.05 were considered to represent statistical significance.
mRNA levels of adipocyte transcription factors, adiponectin and glucose transporters
To evaluate the effects of insulin on glucose transport, subcutaneous and omental mature adipocytes were incubated with 10 nmol/l insulin for various times, and glucose transport was measured by determining the rates of 2-deoxy-d-[3H]glucose uptake. Basal glucose uptake was not different in subcutaneous and omental adipocytes (p = 0.850) (Fig. 3b). In subcutaneous adipocytes, a statistically significant increase in insulin-stimulated glucose uptake was evident after 30 min (p = 0.017 vs basal), with a maximum effect after 60 min (Fig. 3b,c). In omental adipocytes, a significant increase in insulin-induced glucose transport was detected at all time points evaluated (p = 0.0001 vs basal), with a maximum effect after 60 min of hormone stimulation. In addition, at the 15, 30 and 60 min time-points, the magnitude of glucose transport stimulation by insulin was significantly and markedly greater in omental than in subcutaneous adipocytes (sixfold vs twofold, respectively, p < 0.05) (Fig. 3b,c). Next, glucose transport was determined in subcutaneous and omental adipocytes following incubation with various concentrations of insulin for 60 min. In both cell types, insulin elicited an increase in glucose transport rates with statistically significant effects at 0.1 to 100 nmol/l insulin (p = 0.02 and p = 0.001 vs basal, for subcutaneous and omental cells, respectively) (Fig. 3d,e). All insulin doses were found to raise glucose transport to greater levels in omental than in subcutaneous adipocytes (five- to sixfold vs approximately twofold, respectively, p < 0.05) (Fig. 3e). Furthermore, omental adipocytes showed a greater sensitivity to insulin stimulation, since an initial significant effect was evident at 0.1 nmol/l insulin, whereas in subcutaneous adipose cells glucose transport increased significantly only with 1 nmol/l insulin (Fig. 3d,e).
Insulin receptor and IRS proteins
IRS tyrosine phosphorylation, collectively evaluated by direct anti-phosphotyrosine immunoblotting, was not statistically different in subcutaneous and omental adipocytes in the basal state (p = 0.95) (Fig. 4c). Insulin markedly stimulated IRS tyrosine phosphorylation in the subcutaneous adipocytes, in which a peak was observed at 15 min; then, IRS tyrosine phosphorylation showed a gradual decrease, even though it remained above basal levels for up to 120 min (p = 0.017 vs basal) (Fig. 4c,d). In omental adipocytes, by contrast, insulin-induced IRS tyrosine phosphorylation exhibited a maximum increase after 5 min (p = 0.008 vs basal), returning to basal levels after 120 min (Fig. 4c,d). Therefore, IRS tyrosine phosphorylation was significantly lower in omental than in subcutaneous adipocytes after 120 min of insulin stimulation (p = 0.017 vs subcutaneous cells) (Fig. 4d). IRS-1 protein levels were not significantly different in the two experimental cell cultures (p = 0.45) (Fig. 4c; and data not shown). IRS-2 protein levels could not be measured, because they were below the detection threshold of the immunoblotting analysis in human adipocytes (data not shown).
To assess whether the depot-specific phosphorylation kinetics of the IR and IRS proteins could be associated with different cellular levels of tyrosine phosphatases, the total protein content of protein tyrosine phosphatase 1B (PTP-1B), SH2 containing inositol phosphatase (SHIP-2) and phosphatase and tensin homologue (mutated in multiple advanced cancers 1) (PTEN) was determined. However, PTP-1B, SHIP-2 and PTEN protein levels were found to be similar in subcutaneous and omental adipocytes (p = 0.67, p = 0.72 and p = 0.81, respectively) (ESM Fig. 1).
Akt and extracellular signal-regulated kinase
The biological diversity of adipose tissue depots has become a fundamental issue in recent years, in light of its potential impact on human health . It is known that visceral adipose tissue is morphologically and functionally different from subcutaneous adipose tissue . Depot-related variations have been described for a variety of biological endpoints, such as activation of the insulin signalling pathway , glucose uptake responses [13, 14] and adiponectin secretion . However, the precise mechanisms underlying the observed differences remain unclear. It has been proposed that extrinsic factors, including depot-specific blood flow, cell density, cell heterogeneity and/or innervation  could contribute to distinct gene expression patterns and metabolic profiles in adipocytes of different anatomical regions. In this study, we demonstrate that depot-related differences in gene expression and adiponectin secretion are still evident when adipocyte precursor cells are isolated from bioptic adipose tissue fragments, differentiated in vitro and studied several weeks after removal from their original tissue environment. In addition, after insulin stimulation, adipocytes differentiated from omental stromal cells show earlier and more transient kinetics of activation of multiple signalling intermediates, including the IR, IRS proteins, Akt and Erk-1/2, as well as significantly greater glucose transport rates than adipocytes derived from subcutaneous stromal precursors. Interestingly, the results on insulin signalling and glucose uptake responses in this in vitro experimental system resemble previous observations in whole fat tissues in vivo [12, 13]. These findings suggest that the biological differences of the two fat depots depend, at least in part, on innate characteristics of subcutaneous and omental adipose cells. This concept is supported by the recent demonstration that preadipocytes from subcutaneous and omental fat tissues retain fat depot-specific dynamic characteristics and gene expression patterns even after 40 cell doublings .
Adipogenesis involves the sequential induction of multiple transcription factors, acquisition of specific metabolic competences and ability to synthesise and secrete fat-specific proteins. In our experimental system, both subcutaneous and visceral adipocytes exhibited accumulation of cytoplasmic triacylglycerol droplets and markedly increased expression of PPARγ, C/EBPα, AP2, adiponectin and GLUT4 mRNAs, as compared with precursor stromal cells, in line with previous reports . Altogether, these findings indicate that the subcutaneous and visceral cells completed the differentiation process into mature adipocytes.
C/EBPα, AP2 and PPARγ cross-regulate each other’s expression and govern the entire adipogenic programme, by coordinating the expression of lipogenic enzymes and other adipocyte-specific genes [21, 22], including glycerophosphate dehydrogenase, adiponectin and GLUT4 . In contrast to C/EBPβ (also known as CEBPB) and C/EBPδ (also known as CEBPD), whose mRNA levels increase transiently at the onset of adipocyte differentiation, C/EBPα is induced at a subsequent phase and remains expressed at high levels in mature adipocytes . Adiponectin mRNA levels rise immediately after the increase of C/EBPα, remaining at high levels in mature adipocytes. In this study, mRNA levels of C/EBPα and AP2 were expressed at higher levels in subcutaneous adipocytes than in omental adipose cells, in line with a previous report ; adiponectin mRNA levels were also higher in subcutaneous adipocytes and in subcutaneous fat biopsies (Fig. 2). This result is in contrast with the results of others , who found no differences in adiponectin gene expression in freshly isolated subcutaneous and omental human adipocytes. However, it should be noted that subjects in that study had a mean BMI of 46.4 ± 2.0 kg/m2 (range, 32–58.4 kg/m2). Since CCAAT/enhancer binding protein (C/EBP), alpha (C/EBPα) is an important transcription factor for the human adiponectin gene , the differences in adiponectin gene expression in subcutaneous compared with omental adipocytes may be related to the different levels of C/EBPα mRNA.
Using RT-PCR primers that do not discriminate between the two isoforms of peroxisome proliferator-activated receptor gamma (PPARγ), i.e. γ1 and γ2, PPARγ mRNA levels were found to be similar in subcutaneous and omental adipocytes, in line with results obtained in both whole fat tissue specimens and isolated adipocytes in this study and elsewhere [26–28]. This finding is apparently in contrast with the reportedly higher levels of PPARγ found in subcutaneous compared with visceral adipocytes differentiated in vitro in the study by Tchkonia et al. . However, in that previous study adipocytes were analysed 15 days after induction of differentiation, whereas in this study they were studied after a longer time.
Similarly to PPARγ, mRNA expression levels of GLUT1 and GLUT4 did not vary significantly between omental and subcutaneous adipocytes. However, GLUT4 mRNA was found to be slightly higher in subcutaneous than in omental adipocytes, even though this difference was not statistically significant, a finding which is analogous to previous observations in adipose tissue fragments isolated from subcutaneous and omental fat depots . On the other hand, GLUT1 was already expressed at high levels in preadipocytes and did not show prominent changes with differentiation, in line with similar observations in 3T3-L1 cells .
In previous studies on isolated fat cells, visceral adipocytes exhibited a higher adiponectin release than subcutaneous adipocytes, both in the basal state (although not significantly) and following insulin or thiazolidinedione incubation . In this study, the rates of adiponectin secretion were approximately threefold higher in the adipocytes differentiated from omental than from subcutaneous precursors. The apparent discrepancy between changes in adiponectin mRNA expression, which was found to be higher in subcutaneous-derived adipocytes and in subcutaneous fat biopsies (Fig. 2), and changes in adiponectin protein release in the culture medium, which was higher in omental cells (Fig. 3a), suggests that adiponectin gene expression and protein secretion may be regulated differently in specific fat depots. Indeed, adiponectin gene expression is under the transcriptional control of C/EBPα , which was found to be expressed at higher levels in subcutaneous than in omental adipocytes (Fig. 2a), whereas adiponectin secretion can be regulated by other factors, including insulin, as shown in 3T3-L1 adipocytes [31, 32]. Whether more rapid insulin signalling responses, as observed in omental compared with subcutaneous adipocytes, may result in faster adiponectin secretion rates will be the focus of future studies. Additionally, it has been recently demonstrated that the intracellular deacetylase sirtuin (silent mating type information regulation 2 homologue) 1 (S. cerevisiae) (SIRT1) stimulates adiponectin gene transcription by cooperating with forkhead box O1 (FOXO1) and C/EBPα , while it suppresses adiponectin secretion . Thus, it will also be interesting to investigate whether SIRT1 may be differently expressed and/or activated in visceral than in subcutaneous fat depots.
Insulin-stimulated glucose transport was also found to be higher, by approximately two- to threefold, in omental than in subcutaneous adipocytes. This finding is again in accordance with earlier studies in whole fat tissues in humans in vivo  and isolated human adipocytes in vitro , which demonstrated twofold greater rates of insulin-stimulated glucose uptake in the visceral fat depot. Thus, regional variations between visceral and subcutaneous adipocytes, including differences in adiponectin release and glucose metabolism, appear to be preserved in cultures of stromal cells differentiated into mature adipocytes in vitro, suggesting the existence of intrinsic traits of subcutaneous- and visceral-oriented fat cells.
In omental adipocytes, insulin induced a more rapid and transient activation of IR, IRS proteins, Akt and Erk-1/2 compared with subcutaneous cells, in which activation of these proteins was more gradual and somewhat sustained. These results strikingly recall the distinct temporal profile of activation of the same insulin signalling proteins observed in human omental and subcutaneous fat depots in response to insulin administration intravenously in vivo . In that previous study, omental fat showed maximum increases in IR, Akt and Erk phosphorylation 6 min after insulin injection, with subsequent decreases at 30 min, whereas in subcutaneous fat activation of insulin signalling reached its maximum 30 min after insulin stimulation . The phosphorylation levels of specific signalling proteins, such as the IR and Erk-1/2, were found to be higher in omental than in subcutaneous fat in the in vivo study , whereas in this study the omental and subcutaneous adipocytes differentiated in vitro exhibited different temporal profiles but similar magnitude of activation of all signalling reactions. It should be noted, however, that omental fat tissue as a whole is characterised by higher protein content of IR and Erk-1/2 than that found in subcutaneous fat and that the differences in the extent of phosphorylation between the two fat depots are minimised when they are normalised per amount of protein . The more rapid and less persistent phosphorylation profile of the IR and downstream signalling molecules in response to insulin stimulation in omental adipocytes could be potentially explained by higher levels of specific phosphotyrosine phosphatases. However, we found comparable protein content of PTP-1B, SHIP-2 or PTEN in subcutaneous vs omental adipocytes (see ESM Fig. 1). It is possible that differences in specific phosphatase activities and/or the involvement of other, as yet unknown phosphatases, may contribute to the different signalling kinetics in the two fat cell types.
In conclusion, even when differentiated in vitro, subcutaneous and omental adipose cells retain fat depot-specific characteristics in gene expression, adiponectin release, and insulin signalling and action. It is tempting to speculate that there may be an early commitment of fat precursor stromal cells towards a depot-oriented differentiation, which may precede and be independent of extrinsic influences from the tissue environment. Additional studies are needed to elucidate how extrinsic and environmental factors interact with the innate properties of adipocytes under normal conditions and in disease states.
This work was supported by grants from the Ministero dell’Università e Ricerca (Italy), the Cofinlab 2000—Centro di Eccellenza ‘Genomica comparata: geni coinvolti in processi fisiopatologici in campo biomedico e agrario’ (Italy) and grants from Pfizer Italia (ARADO Programme) and Novo Nordisk (LIBRA Programme) to F. Giorgino.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.