It is well known that there is an obesity epidemic in recent years and we commonly talk about this as a worldwide obesity epidemic. While it is true that obesity is on the rise around the world, the epidemic is probably most pronounced in the United States among developed nations. Current statistics show that 34% of US adults are clinically obese (i.e. a body mass index, BMI > 30), about double the worldwide average, and 68% are overweight (BMI > 25).Reference Flegal, Carroll and Ogden¹ The WHO estimates that 700 million adults, about 10% of the world’s population will be obese by 2015.² The main problem with obesity is that it costs a lot of money in added healthcare costs, primarily due to its relationship with metabolic syndrome, diabetes and cardiovascular disease. In 2005, the additional costs ascribed to obesity were estimated at $75 billion; the amount for 2009 was 147 billion. This number is fairly close to the annual cost of implementing health care for all Americans, so obesity is obviously a very significant problem.

If obesity is a significant problem, one might reasonably ask how we get fat? Most doctors adopt the simplistic view that obesity is the result of too many calories in v. too few calories expended – what I call the ‘couch potato syndrome’. More formally, this is called positive energy balance. One classic example of positive energy balance would be sumo wrestlers who are very large men that perform at a high athletic level for a few years but who essentially eat their way into retirement when they get too large to fight competitively any longer.

While it is self-evident that one must consume more calories than are burned to gain weight, the situation is not as simple as the checkbook analogy. Many other factors in addition to positive energy balance have been associated with obesity (reviewed inReference Grun and Blumberg^3, Reference Grun and Blumberg⁴). We know that elevated glucocorticoid levels are linked with obesity and metabolic syndrome.Reference Nyirenda, Lindsay and Kenyon^5, Reference Achard, Boullu-Ciocca and Desbriere⁶ Inadequate sleep, thrifty genes,Reference Hales and Barker⁷ viruses, gut microbes and single nucleotide polymorphisms in a variety of genes can all contribute to obesity in subsets of the population. There are a lot of data regarding prenatal nutrition, the in utero experience and adult disease from the work of Barker and colleagues.Reference Barker and Osmond^8–Reference Barker, Osmond and Golding¹⁰ Maternal smoking is clearly associated with obesity in the exposed offspring.Reference Power and Jefferis¹¹ Overall, many factors in addition to excess calorie consumption contribute to obesity.

Interestingly, there was a paper published a few years ago by Paula Baillie Hamilton where she postulated a role for chemical toxins in obesity.Reference Baillie-Hamilton¹² She noted that the obesity epidemic roughly correlates with the increasing use of industrial chemicals after World War II. Sure enough, there is such a correlation. However, it is important to keep in mind that correlation is not causation and that many factors correlate with obesity, for example, global warming, and the number of dermatologists, climate scientists and SUVs on the road in the United States, among others. It is hard to imagine how any of these correlated factors might be involved in the obesity epidemic.

However, we also know that many chemicals in the environment have effects on the endocrine system, so perhaps there is a kernel of truth to Dr Bailee Hamilton’s argument.Reference Heindel¹³ We know that weight is under hormonal control.Reference Badman and Flier¹⁴ Appetite and satiety are controlled by a set of peptide hormones that control when we feel hungry, when we feel like we have had enough to eat, etc. The thyroid hormone receptor controls the basal metabolic rate, that is, how many calories it takes to maintain bodily functions at rest. The development of fat cells is also under hormonal control.Reference Rangwala and Lazar^15–Reference Lazar¹⁷ The two key nuclear hormone receptors involved in adipogenesis are RXR and peroxisome proliferator activated receptor gamma (PPARγ). PPARγ is considered the master regulator of fat cell development. If you activate this receptor, cells that are capable of becoming fat cells will differentiate into fat cells and existing fat cells will store more fat reviewed in Tontonoz and Spiegelman.Reference Tontonoz and Spiegelman¹⁸

Therefore, one of the questions that we have been studying in my laboratory in recent years is whether disturbances in endocrine signaling pathways are somehow involved in the development of fat cells and in obesity. On the basis of the data described below, we proposed the ‘obesogen hypothesis’.Reference Grun and Blumberg^19, Reference Grun, Watanabe and Zamanian²⁰ That is, that there are molecules called obesogens that inappropriately stimulate the development of fat cells, the storage of fat into existing fat cells, alter metabolic set points or the hormonal control of appetite and satiety leading to increased body fat mass. Are there any existing data that would support such a hypothesis?

We know from the work of Newbold et al.Reference Newbold, Padilla-Banks and Snyder²¹ that exposure to diethylstilbesterol in the perinatal period increases weight at adulthood. Newbold has gone on to show that genistein has a similar effect,Reference Newbold, Padilla-Banks and Jefferson²² Beverly Rubin and Ana SotoReference Rubin, Murray and Damassa²³ have shown that bisphenol-A has a similar effect. We know that there are pharmaceutical obesogens. Thiazolidinediones such as Actos and Avandia that are used to treat type II diabetes increase the number of fat cells at all ages in humans.Reference Larsen, Toubro and Astrup^24, Reference Rubenstrunk, Hanf and Hum²⁵ We know from Rick Stahlhut and Shanna Swann that urinary phthalate levels in men correlate with waist diameter and insulin resistance.Reference Stahlhut, van Wijgaarden and Dye²⁶ Furthermore, there are a variety of cell culture-based studies which show that chemicals such as phthalates,Reference Hurst and Waxman^27, Reference Feige, Gelman and Rossi²⁸ bisphenol-A, alkyl phenols,Reference Heindel¹³ and more recently PFOAReference Hines, White and Stanko²⁹ and organotinsReference Grun, Watanabe and Zamanian^20, Reference Inadera and Shimomura^30–Reference Carfi, Croera and Ferrario³² can all cause the differentiation of fat cells. Taken together, these data indicate that obesogens exist and are worth studying.

We have been working on a compound called tributyltin (TBT), which is the most famous endocrine disrupter that most people have never heard of. I say this because TBT is the single example of an endocrine disrupter for which there is absolutely no controversy about dose and effect. It is found at parts per billion levels and exerts its effects at these levels. The classic effect of TBT is to cause ‘imposex’ the masculinization of female mollusks.Reference Blaber^33, Reference Gibbs and Bryan³⁴ TBT exposed mollusks grow an absolutely gigantic penis which creates a geometric problem for mating; therefore, the animals are sterile. We became interested in TBT when it was shown that TBT could masculinize genetically female Japanese flounder.Reference Shimasaki, Kitano and Oshima³⁵ We asked the question, ‘Which hormone receptor signaling pathway might be activated by TBT and therefore alter the sexual characteristics of both vertebrates and invertebrates?’ We expected to find an effect on one or more steroid signaling pathway. Instead, we found that TBT activated two other nuclear receptors: the 9-cis retinoic acid receptor, RXR and the peroxisome proliferator activated receptor gamma, PPARγ. We went on to show that TBT induced cultured 3T3-L1 cells to differentiate into fat, and TBT caused weight gain and the induction of adipogenic genes in mice.Reference Grun and Blumberg¹⁹ Some of these data are summarized below, followed by our new data on obesogens and stem cells.

Table 1 summarizes the effects of various organotins and other compounds on RXR and PPARγ in receptor activation assay. For RXR, the putative natural ligand 9-cis-retinoic acid and two very good synthetic rexinoids, AGN195203 and LG-100268, have EC₅₀ values in the low nanomolar range. TBT and triphenyltin (TPT) are in the same range. On PPARγ, the thiazolidinedione, troglitazone, has an EC₅₀ of about 1 μM and the organotins are actually much better. Clearly, these compounds are very potent activators for both PPARγ and RXR; do they bind to either receptor? That is important because the organotin structures do not look anything like the known ligands for these receptors. Figure 1 shows the structures of two rexinoids, 9-cRA and LGD100268 and rosiglitazone (ROSI), which is another of the thiazolidinediones, compared with TBT. It is obvious even to the untrained eye that these structures are not particularly similar. Therefore, we tested whether TBT binds to RXR and PPARγ using competitive ligand displacement studies. We found that TBT binds with low nM affinity to both receptors and this has been confirmed in other laboratories.Reference Grun, Watanabe and Zamanian^20, Reference Inadera and Shimomura^30–Reference Carfi, Croera and Ferrario³² It is clear that TBT binds very well to both receptors as an agonistic ligand. We next asked what effects it has in some standard adipogenic models.

Table 1

Activation of nuclear receptor ligand binding domain fusions by candidate ligands

NA, not active; ND, not determined.

The values are EC50 in nM.

Fig. 1

Molecular structures of high-affinity nuclear receptor ligands. The structures of natural (9-cis RA) and synthetic (LGD100268) RXR ligands are given, as is the structure of a high-affinity synthetic ligand for PPARγ (rosiglitazone, ROSI). The structure of TBT is given for comparison.

One very standard adipogenic model utilizes 3T3-L1 cells. These are fibroblasts that can be induced to become adipocytes if treated in just the right way. We found that both TBT and RXR activators could induce 3T3-L1 cells to become adipocytes, as revealed by Oil Red O staining (stains triglycerides) and gene expression.Reference Grun, Watanabe and Zamanian²⁰ This has also been shown by other investigators,Reference Inadera and Shimomura^30–Reference Carfi, Croera and Ferrario³² although there is some controversy about the molecular mechanisms of TBT action.

Therefore, what happens when we expose animals to TBT? We treated pregnant mice with TBT at day 16 of gestation and then examined the effects of this exposure on fat accumulation at birth. We found that animals treated with vehicle control has no discernable triglyceride staining in fat depots, but that TBT treated animals were already storing fat in the liver, testis and mammary adipose depots at birth.Reference Grun, Watanabe and Zamanian²⁰ If we instead allow the prenatally TBT-treated animals to develop, we find that by 10 weeks of age, the TBT-treated animals are smaller, but have stored more fat; therefore, TBT treatment causes increased fat deposition at the expense of overall body mass.Reference Grun, Watanabe and Zamanian²⁰ By 6 months and 9 months, the TBT-treated animals are larger still, up to 15% larger than control animals. Therefore, a single prenatal exposure to TBT has altered the animals such that they are predisposed to weight gain later in life, despite normal diet and access to exercise.

The focus of our recent efforts has been to identify the molecular mechanisms underlying this observed predisposition to weight gain. One can imagine several possibilities. First, the prenatal TBT exposure has altered the hormonal control of appetite and satiety causing the animals to eat more. This is a plausible hypothesis, but we have not seen any evidence to support it. Second, perhaps the TBT treatment has altered the ability of fat cells to store and process lipids. Third, is it possible that TBT treatment has altered the number of preadipocytes or adipocytes? It turns out that this last model is the one for which we find the most support. There is a type of adult stem cell in the body that was previously called a mesenchymal stem cell and is now called a ‘multipotent stromal cell’ or MSC. MSCs can differentiate into multiple lineages in the body, including fat, bone, cartilage and muscle. We know from the literature that the PPARγ agonist ROSI can induce MSCs to differentiate into adipocytes.Reference Carfi, Croera and Ferrario³² MSCs are found primarily in bone marrow and adipose tissue and it is currently controversial whether non-resident MSCs can home to adipose depots.

Our working hypothesis was that prenatal TBT treatment was altering the MSC compartment such that it produces more adipocytes. We first tested the ability of MSCs from either bone marrow or white adipose tissue (WAT) to differentiate into bone, cartilage or adipose tissue in culture. We found that it was possible to isolate relatively pure populations of mouse or human MSCs that expressed appropriate molecular markers and could be induced to differentiate into fat, bone or cartilage in vitro. We next asked the question whether MSCs derived from WAT could be induced to differentiate into adipocytes by TBT and whether TBT exposure altered the potential of MSCs to become bone or cartilage. We found that MSCs were quite responsive to adipogenic induction in vitro and that treatment of MSCs with TBT caused a substantial increase in the number of adipocytes.

We found that both mouse and human adipose derived MSCs (ADSCs) responded strongly to adipogenic induction and that this response was reflected in gene expression.Reference Kirchner, Kieu and Chow³⁶ TBT or ROSI treatment decreased expression of the adipogenesis inhibitor Pref-1 while increasing expression of both early (aP2/Fabp4, PPARγ) and late (leptin) adipocyte markers. Interestingly, TBT or ROSI treatment inhibits the ability of a bone differentiation cocktail to drive ADSCs toward the osteoblastic lineage.Reference Kirchner, Kieu and Chow³⁶

While it appears obvious that ROSI and TBT act through PPARγ to induce adipogenesis, this had never been formally tested. Therefore, we tested the ability of the PPARγ antagonists GW9662 and T0070907 to inhibit TBT or ROSI-induced adipogenesis. As expected, both compounds were able to inhibit the adipogenic effects of ROSI.Reference Kirchner, Kieu and Chow³⁶ In accord with our model that TBT acts through PPARγ, either antagonist was able to block the adipogenic effects of TBT. Addition of either ROSI or TBT to antagonist treated cultures was able to rescue the adipogenesis inducing effects of either compound, further confirming our results.Reference Kirchner, Kieu and Chow³⁶

Summarizing the in vitro effects, TBT induces adipogenesis in MSCs and inhibits the ability of the same cells to be induced into bone cells.Reference Kirchner, Kieu and Chow³⁶ The next question was whether prenatal exposure to TBT had any effect on the differentiation potential of MSCs. We treated pregnant dams at E16.5 with a single oral dose of TBT, ROSI or vehicle and either cross-fostered the pups to CD-1 foster mothers or left them with their dams (Fig. 2). ADSC were harvested at 8 weeks and tested for their ability to become adipocytes, osteocytes or chondrocytes after appropriate induction in vitro. We found that prenatal TBT or ROSI exposure increased the adipogenic potential of the ADSCs and that additional in vitro treatment with TBT or ROSI further induced adipogenesis and the expression of adipogenic genes such as Fabp4 and leptin (Fig. 2).Reference Kirchner, Kieu and Chow³⁶ There was no effect noted on chondrogenesis; however, prenatal TBT or ROSI exposure strongly inhibited calcium deposition and the expression of osteoblast markers (e.g. osteopontin and osteonectin) while simultaneously increasing adipogenesis and the expression of adipogenic markers (Fabp4, leptin; Fig. 2).Reference Kirchner, Kieu and Chow³⁶ Therefore, as with the in vitro experiments, prenatal TBT or ROSI exposure altered the fate of the ADSC compartment, biasing it to favor the adipogenic lineage at the expense of the osteogenic lineage.

Fig. 2

Prenatal TBT exposure predisposes ASDCs to become adipocytes at the expense of osteocytes. The top of the figure shows the experimental design for prenatal exposure. At 8 weeks of age, control or prenatally exposed animals were euthanized and MSCs were harvested from white adipose tissue. These cells were grown in culture and then induced to develop along the adipose or bone differentiation pathways. In each case, further exposure to TBT or ROSI or solvent controls was shown to increase (adipogenesis) or inhibit (bone) differentiation. The degree of adipogenic differentiation was assessed by Oil Red O staining, and the degree of calcium deposition assayed by Alizarin Red staining. Adapted from Kirchner et al.Reference Kirchner, Kieu and Chow³⁶

Of course, the next question is how has the MSC compartment been altered by prenatal TBT or ROSI exposure to favor the production of adipocytes? Our hypothesis was that prenatal obesogen exposure increased the number of preadipocytes; therefore, we analyzed the ADSC population for molecular markers of preadipocytes. Fabp4 is known to be expressed on preadipocytes and macrophages and CD-68 is expressed on macrophages but not preadipocytes. We asked how many cells are Fabp4⁺/CD-68⁻ (i.e. preadipocytes) in the ADSC population from animals treated prenatally with vehicle. We found a 7–15% increase in the number of preadipocytes from obesogen exposed mice compared with controls and concluded that prenatal obesogen exposure increases the number of preadipocytes in the MSC population, an effect that could easily increase the number of fat cells in an adult.Reference Kirchner, Kieu and Chow³⁶ This observation also explains why ASDCs are inhibited from becoming bone – they are already enriched in preadipocytes.

We next asked whether the expression of genes known to be involved in specifying the adipocyte lineage was increased in untreated ADSCs harvested from obesogen treated v. control animals. We showed that Fabp4 and leptin mRNA levels were increased significantly whereas PPARγ mRNA was increased but the increase did not reach statistical significance.Reference Kirchner, Kieu and Chow³⁶ Using methylation sensitive restriction enzyme digestion, we found that CpG islands in the Fabp4 promoter were undermethylated in the prenatally TBT treated animals. This suggests that prenatal exposure has caused an epigenetic alteration in the MSC compartment that increases the fraction of MSCs committed to the adipocyte lineage. Fabp4 is a downstream gene in the hierarchy of adipogenesis; hence, we are currently testing all of the genes known to be involved in adipogenesis to determine which others may be epigenetically altered in MSCs from animals treated prenatally with TBT.