The steps involved in adipocyte formation have remained unresolved for the past two decades. To gain insight into this process, we used a reductionist approach with two related aims: first, to test the hypothesis that individual components of the DIM cocktail regulate discrete commitment states in the progression from preadipocyte to adipocyte; second, to assess at the molecular level the contributions of the individual components to adipogenesis. Through this approach we determined conditions for differentiation of 3T3-L1 preadipocytes that are independent of insulin addition, as previously demonstrated (Schmidt et al., 1990
; Liu et al., 2005
), or of shifting to growth medium supplemented with FBS. Our approach yielded a simpler, more direct model for inducible adipogenesis. Differentiation can be divided into two distinct stages induced by two signaling pathways acting sequentially, but noncommutatively. The initial phase is driven by glucocorticoid signaling and is followed by a second phase driven by cAMP signaling. The significance of the relationship between dex and IBMX in 3T3-L1 cells was recapitulated in C3H10T1/2 cells and in primary MSCs.
The current models of fat ontogenesis do not seem to account for the diversity of adipocyte phenotypes observed in vivo and are simplistic compared with the much better understood hematopoietic system (Schmidt et al., 1990
; Gesta et al., 2007
). In our experiments, we isolated the actions of glucocorticoid signaling during adipogenesis and found that pretreatment with dex primes preadipocytes to respond to IBMX and induces a gene expression profile intermediary between that of preadipocytes and adipocytes. Thus, glucocorticoid signaling appears to define a novel commitment state, the dex-primed preadipocyte. Although the physiological relevance of these findings remains to be determined, it is interesting that patients exposed to excess glucocorticoids present with expansion of a subset of fat depots. Conceivably, certain adipose niches (e.g., visceral fat depot) may be permissive for differentiation of dex-primed preadipocytes, whereas other fat depots may be insensitive to glucocorticoid-induced adipogenesis because they lack the permissive signals that advance dex-primed preadipocytes through the adipogenic program.
In contrast to the outcomes of dex pretreatment of preadipocytes, the increased cAMP signaling resulting from IBMX pretreatment of those cells results in a failure to differentiate upon subsequent dex treatment; these cells are also partially resistant to differentiation induced by the DIM cocktail. Recently, Madsen et al. (2008)
linked increased cAMP signaling in mouse liver and adipose tissues, induced by high-protein diet, to prevention of polyunsaturated fatty acid–induced adipogenesis and obesity. These findings indicate that, in contrast to glucocorticoids, stimulation of cAMP signaling in preadipocytes fails to induce progression toward the adipocyte fate. On the contrary, it seems that IBMX drives preadipocytes toward a state that is off of the adipogenic pathway, but that does not represent a terminal differentiation fate. Accordingly, we found that IBMX-pretreated preadipocytes can differentiate into adipocytes if treated with dex for 48 h followed by IBMX for another 48 h (Supplemental Figure S1). Hence, dex signaling acts dominantly to cAMP signaling to return IBMX-treated preadipocytes to the adipogenesis pathway.
Thus, we suggest that glucocorticoids and cAMP appear to regulate adipogenesis independently by directing preadipocytes toward two cellular states. Dex-primed preadipocytes are on-pathway toward the adipocyte fate; IBMX-treated preadipocytes are off-pathway and differentiate efficiently only if brought back to the adipogenic pathway by dex signaling. These two observations suggest a simple way by which adipogenesis can be spatially and temporally compartmentalized during development and normal adult life and in disease states. Specifically, preadipocytes may integrate order, intensity (e.g., dose of glucocorticoid), and duration of adipogenic signals to adopt distinct cellular states, along an adipogenesis axis, that differ in their sensitivity to further stimulation.
The insulin sensitivity of adipocytes differentiated by treatment with dex followed by IBMX was equivalent to DIM-differentiated adipocytes (A). Accordingly, the two types of adipocytes expressed similar mRNA levels of the glucose transporter GLUT4 (Supplemental Figure S3A). Dex-IBMX adipocytes, however, were less sensitive to induction of lipolysis by the β-adrenergic agonist ISO when compared with DIM-differentiated adipocytes (B). Interestingly, we found that this phenotype correlated with decreased levels of the lipid droplet protein perilipin A on dex-IBMX adipocytes relative to DIM-differentiated adipocytes (Supplemental Figure S3B). Perilipin A is thought to influence adipocyte lipolysis by a mechanism that is dependent on its expression levels and phosphorylation state (Brasaemle, 2007
). When unphosphorylated, perilipin A protects lipid droplets from the activity of cellular lipases and promotes triglyceride storage. Upon ISO stimulation perilipin A phosphorylation is required for full induction of lipolysis and promotes localization of hormone-sensitive lipase to the surface of lipid droplets. Indeed, adipocytes derived from perilipin A null mice display slightly elevated basal lipolysis, but dramatically decreased ISO-stimulated lipolysis (Tansey et al., 2001
). Thus, the lower levels of perilipin A on dex-IBMX adipocytes may underlie the decrease in ISO-stimulated lipolysis, but unchanged basal lipolysis rates when compared with DIM-adipocytes (B and Supplemental Figure S3B).
In addition to being less responsive to the lipolytic effect of ISO, dex-IBMX 3T3-L1 adipocytes stored less triglyceride (C). The triglyceride storage phenotype could reflect the absence of insulin-dependent activation of cyclic AMP response element binding protein (CREB), which in turn stimulates triglyceride accumulation in 3T3-L1 adipocytes (Klemm et al., 2001
; Vankoningsloo et al., 2006
). Additionally, it is consistent with clinical studies showing that excess circulating insulin levels are associated with larger subcutaneous adipocytes. These results indicate that selected aspects of adipocyte metabolic function can be tuned by the mode of differentiation to which preadipocytes are subjected. In an interesting parallel, the human visceral fat depot displays higher rates of catecholamine-induced lipolysis relative to the subcutaneous white fat depots (Arner, 1999
). The similarities between our cell culture results and the diversity of adipocytes in human subjects suggest a clear path for discerning the mechanisms that contribute to the final metabolic phenotype of adipose tissue. Importantly, the differences in expression of perilipin A suggest that its regulation might be part of the mechanism through which adipocyte metabolic diversity is generated.
We applied a candidate approach to infer the mechanism by which the order of exposure to dex and IBMX alters the adipogenesis signal. We found that relative to DIM treatment, IBMX-dex treated cells displayed delayed expression of C/EBPδ and did not substantially increase the protein levels of C/EBPα and PPARγ. Our findings suggest that early expression of C/EBPδ is required for efficient induction of C/EBPα and PPARγ. To explore additional mechanisms underlying the failure of IBMX-dex treatment to induce differentiation, we monitored Pref-1, whose repression by glucocorticoids is necessary for efficient adipogenesis of 3T3-L1 preadipocytes. We found that dex-IBMX treatment indeed represses Pref-1, whereas pretreatment with IBMX prevents stable repression of Pref-1 by dex. These results suggest a mechanism by which preadipocytes sense the order of exposure to dex and IBMX and underscore a role of Pref-1 as a molecular gatekeeper of adipogenesis. In related studies, Feldman et al. (2006)
identified an adipogenesis role for a glucocorticoid receptor target gene, the TGFβ family member myostatin (MSTN). Remarkably, C3H10T1/2 adipocytes differentiated by substitution of dex with MSTN and adipocytes of mice expressing MSTN in adipose tissue are smaller and more insulin sensitive and seem to be immature when compared with wild-type adipocytes. It will be interesting to determine the relationship of those “myostatin adipocytes” to the dex-primed preadipocytes described in our work.
In summary, by temporally uncoupling the signals that comprise the DIM cocktail, we discovered complex interactions of dex and IBMX during adipogenesis in cell culture, which may enrich our understanding of the process and its regulation as played out in intact mammalian organisms. Our results, and those of Feldman et al. (2006)
, imply that an array of intermediate cell types, perhaps reminiscent of those known in hematopoiesis, may punctuate cell fate pathways radiating from mesenchymal stem cells, and in particular proceeding toward mature adipocytes. The distinct metabolic capabilities of dex-IBMX adipocytes compared with DIM adipocytes may reflect a mechanism by which adipogenesis can be fine-tuned spatially, temporally, and functionally to generate the diversity of adipocyte phenotypes described in vivo. In this context, it is intriguing that Pref-1 may, under certain circumstances, act as a sensor of glucocorticoid and cyclic AMP activities, ensuring that preadipocytes advance to differentiation only if a predetermined series of signaling events occurs in the correct order.