Glucocorticoids (GCs) have several effects on adipose tissue (AT) biology: among others, they regulate terminal adipocyte differentiation, AT endocrine function and lipogenic-lipolysis balance [1
]. Some of these effects are more predominant on visceral (VAT) rather than on subcutaneous (SCAT) AT function [4
]. One clear example is the Cushing’s syndrome (CS) phenotype, mainly characterized by high GC levels in blood and increased VAT rather than SCAT mass [5
]. Other features of CS, some of them shared by the human metabolic syndrome (MS) phenotype, are the presence of VAT hypertrophic adipocytes, altered lipid metabolism and impaired adipokine secretion [6
]. The existence of these alterations suggests that GCs have a pivotal role in the pathogenesis of central obesity and the associated alterations seen in the CS phenotype.
Neonatal i.p. administration of the neurotoxin monosodium l
-glutamate (MSG) in rodents causes neuronal loss, mainly at the hypothalamic arcuate nucleus level [7
], triggering a failure in hypothalamus-pituitary-adrenal axis regulation, which results in high plasma GC levels [8
]. Because of this reason, the MSG treatment has been widely used to generate animal models resembling that of the human CS phenotype. We have extensively used the MSG rat model, which is characterized by increased VAT mass with hypertrophic adipocytes and an altered peripheral pattern of adipokines [8
It is generally accepted that the ability of an AT depot to expand its mass depends on two processes: the generation of new adipocytes (adipogenesis) and the hypertrophy of mature AT cells. It has been proposed [10
] that at the AT level, hyperplasia contributes to maintaining a pool of small and functional adipocytes, thus preventing the development of metabolic alterations associated with hypertrophic obesity [11
]. Indeed, adipocyte hypertrophy is associated with cell dysfunction, such as insulin resistance [12
] and changes in the adipokine secretion pattern [13
During the adipogenic process, adipose precursor cells (APCs) differentiate into mature adipocytes in two sequential steps: commitment of mesenchymal stem cells (MSCs) to APCs, acquiring their adipogenic potential and restricting them to the adipocyte linage, followed by terminal adipocyte differentiation [15
]. CD34 is a cell surface antigen that distinguishes between adipogenic and non-adipogenic cell subpopulations in the stromal vascular fraction (SVF) of the AT [16
]. This CD34+
subpopulation expresses almost exclusively the transcriptional factor Zinc finger protein 423 (Zfp423) [17
], which in turn activates the basal expression of cell peroxisome proliferator-activated receptor (PPAR)-γ2, a key pro-adipogenic signal that assures APCs conversion into adipocytes [18
]. The differential expression of both transcriptional factors determines the competency of APCs, that is, the cell’s ability to differentiate into adipocytes after the addition of defined stimuli [15
GCs are one of the most important cell differentiation inducers through their inhibitory effect on Preadipocyte Factor 1 (Pref-1) and Wingless-type MMTV integration site family member 10b (Wnt-10b) expression [19
]. Both, Pref-1 and Wnt-10b, are highly expressed in preadipocytes and absent in mature adipocytes [22
]. In vitro experiments with the preadipocyte cell line 3T3-L1 have shown that Pref-1 is an early target for dexamethasone (DXM) action and that its expression decreases with high DXM concentrations, at the same time that adipocyte differentiation increases [19
]. Similarly, in vivo and in vitro studies demonstrated that the inhibition of the Wnt/b-catenin signaling pathway by methylprednisolone or DXM, respectively, can promote adipocyte differentiation [21
]. Several studies show that GCs can affect adipogenesis through their binding to mineralocorticoid (MR) and/or glucocorticoid (GR) receptors [25
], although the contribution of MR and GR in mediating GCs’ adipogenic effect has not been fully understood.
While the activity of GCs on adipocyte terminal differentiation has been extensively studied, the effect of chronic GC exposure of SVF cells remains almost unexplored. Using adult male MSG rats, it has been previously demonstrated that adipogenesis is inhibited as a consequence of long-term GC exposure of SVF cells, thus contributing to hypertrophic retroperitoneal AT (RPAT) mass expansion [28
]. The present study focuses on the APCs’ adipogenic potential during in vivo RPAT mass expansion under an earlier chronic GCs circulating excess.
Enhanced VAT is associated with an increased risk of the development of several pathologies, such as diabetes mellitus type 2 and cardiovascular disease. Conversely, SCAT function is considered as protective against them [35
]. This difference has been attributed, at least in part, to a higher adipogenic capacity of SCAT [37
]. However, a system has been recently developed for the inducible and permanent labelling of mature adipocytes in vivo, which indicates that hyperplastic expansion of the AT occurs mainly at the epididymal, but not at the SCAT depot [38
]. The present work showed that high chronic GC plasma levels are associated with early activation of adipogenesis (30-day-old MSG rats), supporting the concept that RPAT could expand through the generation of new adipocytes. In contrast, our previous studies revealed the inhibition of the RPAT adipogenic process at advanced age (60 day-old MSG rats) [28
]. This age-/time-dependent shift in the adipogenic potential could be a consequence of changes in the expression levels of APCs’ competency factors.
GCs have diverse effects on both AT functions [39
] and distribution [41
], favoring an increase in VAT, but not in SCAT mass. MSG rats share several features of the human CS phenotype, which is characterized by chronic high circulating levels of GC, enhanced AT mass and adipocyte size and an altered pattern of adipokine secretion, among others. In the MSG rat phenotype, restoring to normal the peripheral levels of GC reverses most of such dysfunctions [43
]. Our present data indicate that the MSG animal phenotype is already established at 30 days of age, and some characteristic alterations get differentially worse over time.
Regarding the terminal stage of adipogenesis, an increase in all differentiation parameters was observed in MSG cells from 30-day-old rats. According to these findings, the expression of anti-adipogenic factors, such as Pref-1 and Wnt-10b decreased in undifferentiated MSG cells. In fact, the pro-adipogenic action of GCs is due to its inhibitory effect on Pref-1 and Wnt/b-catenin signaling pathway expression [21
]. As mentioned above, these results show evidence of a dual behavior of the adipogenic process by itself in a GC-rich endogenous environment, characterized by adipogenesis activation at an initial step and the subsequent inhibition of the process, as shown before [28
Cristancho et al. [15
] distinguished between two functional terms: cell competency and cell commitment, where adipogenic competency refers to the cell’s ability to differentiate into adipocyte upon the addition of defined stimuli, and adipogenic commitment indicates the multipotent cell type fate to undergo its conversion into adipocyte. Within this context, the adipogenic potential of an AT pad depends, besides any adipogenic stimulus environment, on the number and competency of APCs present in the local SVF. GCs are known to inhibit the proliferation capacity of APCs and to play an important role driving APCs to differentiate into adipocytes.
Over the last few years, great effort has been made to find cell surface markers that unequivocally identify APCs applying diverse criteria. Most of them agree that the CD34 antigen can distinguish between cells that are able or not to differentiate into mature adipocytes [44
]. Interestingly, the expansion of different AT pads, both in mouse and human obese phenotypes, has been proposed to depend on changes in progenitor cell number [10
]. To our knowledge, the effect of chronic exposure to high GC levels on APCs number in VAT pad has not been yet explored. In fact, our study is the first to show that APCs number in the SVF of RPAT from MSG rats is not altered at 30 or 60 days of age, and consequently, this parameter does not seem to be responsible for the changes observed in their adipogenic potential.
Zfp423 has been described as a marker of APCs competency, because it regulates PPARγ2 expression throughout the amplification of SMAD protein activity [17
]. Thus, since PPARγ2 plays a key role within the overall adipogenic process, we are persuaded that PPARγ2 should be considered as a competency factor by itself. In SVF cells from 30 day-old MSG rats, the levels of PPARγ2 and Zfp423 mRNA were higher than in CTR SVF cells, which indicates high APC competency. These data are opposite to those found in 60 day-old MSG rat cells where both PPARγ2 and Zfp423 mRNA levels were low. Our results demonstrate that changes in APCs competency are responsible for the different adipogenic capacities of SVF cells from MSG rats of 30 and 60 days of age.
In obese phenotypes, a recruitment of immune cells occurs in VAT, including macrophages, lymphocytes and neutrophils, thus contributing to the development of a chronic inflammatory state [48
]. Furthermore, there is a macrophage polarization toward the M1 type, which secretes pro-inflammatory cytokines (e.g., TNFα, IL1β and MCP-1) to the detriment of the anti-inflammatory M2 type (IL-10) [52
]. These pro-inflammatory macrophage-secreted factors have been involved in an antiadipogenic activity, by decreasing the expression levels of adipogenic markers, such as aP2 and PPARγ2 in human and 3T3-L1 preadipocytes [54
]. The role of GCs mediating the inflammatory response in AT depends on the MR or GR activation, which will determine a pro- or anti-inflammatory response, respectively, due to the decrease or increase of pro-inflammatory cytokines’ secretion [26
]. Nevertheless, CS patients display increased adiposity, but the establishment of a chronic inflammatory state is controversial [56
]. We have previously shown that RPAT expression of TNFα, IL-6, MCP-1 and F4/80 does not increase in 60-day-old MSG rats, probably due to the anti-inflammatory effect of GCs exerted through GR activation [28
]. In this regard, further studies are needed to evaluate the AT inflammatory response in MSG rats at 30 days of age.
Pro-adipogenic activity of GCs has been clearly recognized; then, the co-existence of high GC levels and a reduced adipogenic cell capacity could be indicative of changes in the RPAT sensitivity to GCs. This could explain why high levels of a potent adipogenic factor do not stimulate the process continuously, preventing hypertrophy of VAT. It is known that aldosterone is able to promote differentiation of APCs in both 3T3-L1 [58
] and human preadipocytes. Notably, expression silencing or activity blocking of MR in both cell types prevent adipogenesis, but this is not the same for GR inactivation [25
]. We found that MR mRNA levels did not change in 30-day-old MSG rats, though they decreased in 60-day-old rats, coinciding with the low adipogenic capacity found in them [28
]. This could suggest that MR is involved in the development of a GC-resistant state, although the participation of MR or GR in the biological actions of GCs upon the AT is still being debated [25
]. However, the contribution of GR to the lack of GC effect cannot be ruled out, despite not finding any change in cell GR expression.
The distribution of adipocyte size has been previously used to determine the presence of AT hypertrophy and/or hyperplasia [10
]. Analysis of the adipocyte size distribution in RPAT from MSG animals at both ages showed interesting data. Indeed, MSG rats at 30 days of age displayed one hypertrophic adipocyte population, while MSG rats at 60 days of age presented two adipocyte size populations, one smaller and another larger than the only adipocyte size population found in CTR pads. The additional presence of small adipocytes in 60-day-old MSG rats suggests that increased APC competency (seen in 30-day-old MSG rats) leads to adipogenesis stimulation between Days 30 and 60 of age.