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Endocrine-Mediated Mechanisms of Metabolic Disruption and New Approaches to Examine the Public Health Threat


Abstract and Figures

Obesity and metabolic disorders are of great societal concern and generate substantial human health care costs globally. Interventions have resulted in only minimal impacts on disrupting this worsening health trend, increasing attention on putative environmental contributors. Exposure to numerous environmental contaminants have, over decades, been demonstrated to result in increased metabolic dysfunction and/or weight gain in cell and animal models, and in some cases, even in humans. There are numerous mechanisms through which environmental contaminants may contribute to metabolic dysfunction, though certain mechanisms, such as activation of the peroxisome proliferator activated receptor gamma or the retinoid x receptor, have received considerably more attention than less-studied mechanisms such as antagonism of the thyroid receptor, androgen receptor, or mitochondrial toxicity. As such, research on putative metabolic disruptors is growing rapidly, as is our understanding of molecular mechanisms underlying these effects. Concurrent with these advances, new research has evaluated current models of adipogenesis, and new models have been proposed. Only in the last several years have studies really begun to address complex mixtures of contaminants and how these mixtures may disrupt metabolic health in environmentally relevant exposure scenarios. Several studies have begun to assess environmental mixtures from various environments and study the mechanisms underlying their putative metabolic dysfunction; these studies hold real promise in highlighting crucial mechanisms driving observed organismal effects. In addition, high-throughput toxicity databases (ToxCast, etc.) may provide future benefits in prioritizing chemicals for in vivo testing, particularly once the causative molecular mechanisms promoting dysfunction are better understood and expert critiques are used to hone the databases. In this review, we will review the available literature linking metabolic disruption to endocrine-mediated molecular mechanisms, discuss the novel application of environmental mixtures and implications for in vivo metabolic health, and discuss the putative utility of applying high-throughput toxicity databases to answering complex organismal health outcome questions.
Content may be subject to copyright.
published: 07 February 2019
doi: 10.3389/fendo.2019.00039
Frontiers in Endocrinology | 1February 2019 | Volume 10 | Article 39
Edited by:
Robert Sargis,
University of Illinois at Chicago,
United States
Reviewed by:
Jacqueline M. Stephens,
Louisiana State University,
United States
Mark Andrew Lawson,
University of California, San Diego,
United States
Christopher D. Kassotis
Specialty section:
This article was submitted to
Systems and Translational
a section of the journal
Frontiers in Endocrinology
Received: 28 September 2018
Accepted: 17 January 2019
Published: 07 February 2019
Kassotis CD and Stapleton HM (2019)
Endocrine-Mediated Mechanisms of
Metabolic Disruption and New
Approaches to Examine the Public
Health Threat.
Front. Endocrinol. 10:39.
doi: 10.3389/fendo.2019.00039
Endocrine-Mediated Mechanisms of
Metabolic Disruption and New
Approaches to Examine the Public
Health Threat
Christopher D. Kassotis*and Heather M. Stapleton
Nicholas School of the Environment, Duke University, Durham, NC, United States
Obesity and metabolic disorders are of great societal concern and generate substantial
human health care costs globally. Interventions have resulted in only minimal impacts
on disrupting this worsening health trend, increasing attention on putative environmental
contributors. Exposure to numerous environmental contaminants have, over decades,
been demonstrated to result in increased metabolic dysfunction and/or weight
gain in cell and animal models, and in some cases, even in humans. There are
numerous mechanisms through which environmental contaminants may contribute
to metabolic dysfunction, though certain mechanisms, such as activation of the
peroxisome proliferator activated receptor gamma or the retinoid x receptor, have
received considerably more attention than less-studied mechanisms such as antagonism
of the thyroid receptor, androgen receptor, or mitochondrial toxicity. As such, research
on putative metabolic disruptors is growing rapidly, as is our understanding of
molecular mechanisms underlying these effects. Concurrent with these advances,
new research has evaluated current models of adipogenesis, and new models have
been proposed. Only in the last several years have studies really begun to address
complex mixtures of contaminants and how these mixtures may disrupt metabolic
health in environmentally relevant exposure scenarios. Several studies have begun to
assess environmental mixtures from various environments and study the mechanisms
underlying their putative metabolic dysfunction; these studies hold real promise in
highlighting crucial mechanisms driving observed organismal effects. In addition, high-
throughput toxicity databases (ToxCast, etc.) may provide future benefits in prioritizing
chemicals for in vivo testing, particularly once the causative molecular mechanisms
promoting dysfunction are better understood and expert critiques are used to hone
the databases. In this review, we will review the available literature linking metabolic
disruption to endocrine-mediated molecular mechanisms, discuss the novel application
of environmental mixtures and implications for in vivo metabolic health, and discuss
the putative utility of applying high-throughput toxicity databases to answering complex
organismal health outcome questions.
Keywords: endocrine disrupting chemicals, obesogen, diabetogen, adipogenesis, 3T3-L1, obesity, diabetes
Kassotis and Stapleton Mixtures and Mechanisms of Metabolic Disruption
Endocrine disrupting chemicals (EDCs) have been demonstrated
to directly modulate metabolism in vivo and/or triglyceride
accumulation in vitro through various receptor-mediated
pathways (15), suggesting a potential causative link between
exposure to EDCs and the increasing global prevalence of
metabolic disorders, including obesity (6). Chronic metabolic
health conditions are rapidly increasing in prevalence and
cost to society worldwide: in the US, 39.6 and 9.7% of adults
aged 20 and older are currently classified as obese or have
been diagnosed with diabetes, respectively, with increasing
occurrence in younger age groups as well (710). These
conditions contribute to a rising share of health care costs;
in the US, >$600 million is directed to obesity-related and
diabetes-related illnesses in adults (10,11). These effects are
mirrored in animal populations, with an analysis of >20,000
animals from 24 populations reporting increased weight gain in
numerous species including monkeys, both laboratory and urban
mice, cats, dogs, etc. (12). Notably, attempted interventions
have yielded minimal effects, and analyses have determined that
activity, caloric intake, and genetics are insufficient to explain
the magnitude and speed of this change (13,14). As fat cell
development is driven and modulated by nuclear hormone
receptor signaling (2,1517), EDCs that activate or inhibit
these hormone pathways may be causative agents in promoting
modulation of fat cell development, energy homeostasis, basal
metabolic rate, hormonal control of appetite and satiety, and
brain circuitry controlling food intake and energy expenditure
and ultimately contributing to the development of Metabolic
Syndrome (Figure 1) (18).
Numerous environmental toxicants have been demonstrated
as metabolic disruptors in vivo, supporting EDCs as a causative
factor in these adverse health trends (14). There is a rich literature
demonstrating effects of antibiotics on weight gain in humans
and diverse animal species. Experiments demonstrating their
efficacy in promoting weight gain in agricultural species were
published by the 1950’s, presumably operating through effects
on gut microbiota impacting the processing of carbohydrates in
the diet (19,20). More recent publications have demonstrated
that several weeks of subtherapeutic antibiotics increase fat mass
and weight, particularly when begun during gestation (21,22),
and human epidemiological studies have demonstrated increased
risk of becoming overweight when children were exposed early
in life (23,24). Other notable examples include diethylstilbestrol
(DES), a pharmaceutical provided to pregnant women in the
1940’s through 1970’s in the mistaken assumption it would
reduce rates of abortion, miscarriage, and premature labor (25);
it was later determined to induce a variety of adverse health
effects in both males and females exposed during gestation
(2629). DES has been demonstrated to promote triglyceride
accumulation in vitro, seemingly through an estrogen-receptor
mediated mechanism (30), and both gestational and perinatal
DES exposure increases body weight, body fat, and alters serum
lipid profiles in rodent models throughout life (3133). Increased
risks of obesity in human adults exposed prenatally to DES have
also been reported (34), delineating apparent translational effects.
Our lab has recently demonstrated that common chemicals and
environmental mixtures associated with unconventional oil and
gas (UOG) operations can activate the peroxisome proliferator
activated receptor gamma (PPARγ) and promote triglyceride
accumulation and pre-adipocyte proliferation in vitro (35), and
that gestational exposure to a mixture of UOG chemicals resulted
in increased body weights through weaning in a rodent model
(36,37). UOG development has also been associated with
increased prevalence of low birth weight and small for gestational
age births in the Northeast US (38), and decreased prevalence of
low birth weights and increased risk of higher birth weight babies
in Colorado (39); both low (40,41) and high (42,43) birth weights
are associated with greater risks for obesity later in life.
As costs associated with in vivo screening of putative
metabolism disruptors are prohibitively high, utilizing lower-
order testing, and screening is essential to narrow higher-
order testing to chemicals most likely to be active. Various
pre-adipocyte and mesenchymal stem cell models (both rodent
and human, primarily) have been utilized to assess putative
in vivo metabolic disruptors in vitro; 3T3-L1 mouse pre-
adipocytes have proven reliable as an in vitro screen for
identifying likely obesogenic chemicals in vivo, and other models
such as the OP9 mouse bone marrow-derived stromal pre-
adipocyte cell line (44,45) allow for assessments of varying
molecular pathways important for the process of differentiation.
Additionally, various multipotent mesenchymal cells and cell
lines (46,47) offer the additional ability to assess commitment
to the adipocyte lineage as a distinct process from adipocyte
differentiation (48). However, these assays are lengthy and
their relative abilities to correctly identify chemicals may
depend on both cell line and cell source. As such, there is a
critical need to develop better methods for correctly predicting
metabolic disruptors. Several high-throughput (HTP) screening
programs now exist (Tox21, ToxCast) that report activity across
mechanisms known to modulate metabolic health for thousands
of chemicals. Harnessing these data sets to broadly assess
high-scoring chemicals (across these molecular pathways) for
more targeted in vitro and in vivo testing could provide a
valuable tool for reducing research costs and more broadly
assessing the tens of thousands of commercial chemicals for
potential contributions to adverse health outcomes in humans
and/or animals.
In addition to high-throughput screening, assessments of
mixtures have become more commonplace in recent years.
Tools to evaluate the chemical constituents and biological
activities associated with complex environmental mixtures have
vastly increased the capabilities within this sphere, though
standard approaches to mixtures are still lacking in many
respects, particularly in terms of relevance to human and
animal exposure. One notable mixture that has received
increasing attention is indoor house dust; our laboratory
and others have collected and analytically characterized house
dust from different environments around the world and
routinely report numerous classes of EDCs (known to be
hormonally active), including flame retardants, phthalates,
pesticides, perfluoroalkyl substances (PFAS), and others that
span a wide range of concentrations (4951). Humans, and
perhaps most importantly small children, are chronically
Frontiers in Endocrinology | 2February 2019 | Volume 10 | Article 39
Kassotis and Stapleton Mixtures and Mechanisms of Metabolic Disruption
FIGURE 1 | Representative EDCs Capable of Affecting Adipogenesis. Representative endocrine disrupting chemicals (EDCs) capable of affecting adipogenesis and/or
metabolic health through the specified nuclear receptor pathways listed above. Gross circle size intended to express a general sense of the reported research into
assessing these varying mechanisms; for example, PPARγ, RXRα, and GR have previously received the bulk of the research, whereas others have received less.
Agonists for the receptors are depicted with a (+) following the chemicals, whereas antagonists are denoted with the (). Standard positive and negative control
chemicals for each receptor (for evaluating these pathways) are bolded to distinguish from the other EDC examples. PPAR, peroxisome proliferator activated receptor;
RXR, retinoid X receptor; AR, androgen receptor; ER, estrogen receptor; CAR, constitutive androstane receptor; TR, thyroid receptor; FXR, farnesoid X receptor; LXR,
liver X receptor; GR, glucocorticoid receptor.
exposed to household dust, and thus receive exposure to
EDCs present in the dust. The EPA estimates children ingest
60–100 mg of dust per day from indoor environments (52),
contributing to chronic oral and inhalation exposures to EDCs
(49,53,54), and compounded by other routes of exposure.
Notably, numerous studies have demonstrated clear links
between levels of indoor semi-volatile indoor contaminants
(SVOCs) on hand wipes with levels in house dust (55,56),
with other studies demonstrating clear links with urinary
and serum levels (55,57,58), providing evidence for this
exposure route contributing to an increased body burden of
specific chemicals. As such, environmental matrices such as this
may represent a clear exposure route for humans and could
provide critical information on biological effects of summed
mixture exposures.
While activation of the peroxisome proliferator activated
receptor gamma (PPARγ) is likely the best-described mechanism
through which adipogenesis is initiated/promoted, activation
or inhibition of numerous other receptor systems have
been described to directly or indirectly modulate adipocyte
lineage commitment and/or differentiation of pre-adipocytes
and subsequent accumulation of triglycerides, including
thyroid receptor-beta (TRβ), glucocorticoid receptor (GR),
estrogen receptor (ER), androgen receptor (AR), liver X
receptor (LXR), retinoid X receptor (RXR), and others (59)
(Table 1). Several studies have assessed the expression of
nuclear receptors throughout the differentiation process,
reporting that 30 nuclear receptors were expressed throughout
the differentiation process to varying degrees and at varying
timepoints (15,60). Recent work by Chappell et al. demonstrated
putative GR-mediated effects prior to PPARγactivation after
exposure of 3T3-L1 cells to tetrabrominated bisphenol A
(TBBPA) (61). Notably, EDCs capable of acting through
each of these pathways have been described previously to
modulate metabolic health in vitro, in vivo, or in human
epidemiological studies; though importantly, certain molecular
mechanisms have received far greater research attention than
others (Figure 2).
Peroxisome Proliferator Activated
Receptors (PPARs)
PPARγis often considered the only nuclear receptor whose
activation is necessary and sufficient to initiate adipogenesis
(62,63). Treatment of 3T3-L1 cells as well as other pre-
adipocyte and/or other committed adipocyte lineage cells with
PPARγagonists induces a potent and efficacious increase
in triglyceride accumulation, which has long been realized
(64,65); as such, PPARγagonists such as rosiglitazone
and/or troglitazone are routinely utilized as positive control
ligands for these assays (63). Utilized as therapeutic agents
to treat type 2 diabetes, these thiazolidinediones may act
to improve insulin sensitivity via induction of PPARγin
diverse tissue types, proliferation of smaller adipocytes that
are more insulin-sensitive, or via mediation of the tumor
necrosis factor alpha (TNF-α), leptin, or fatty acid signaling
pathways [reviewed in (66)]. To establish the necessity of
this pathway to adipogenesis, Rosen et al. utilized embryonic
stem cell and chimeric mouse models. They demonstrated
that PPARγ-null cells tended to not generate adipocytes,
suggesting an essential role for this receptor in their formation.
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Kassotis and Stapleton Mixtures and Mechanisms of Metabolic Disruption
TABLE 1 | Major hormone receptor pathways capable of promoting adipogenesis.
Receptor Activity In vitro effects In vivo effects Epidemiological effects
PPARγAgonism Promotes adipocyte differentiation,
also some promotion of
pre-adipocyte proliferation
Increased adipose fat deposition, body
Increased body weights, reverse
hyperglycemia/treat diabetes
PPARβ/δAgonism Promotes adipocyte differentiation Activation improves lipid profiles, depletes
lipid accumulation, increases resistance to
diet-induced obesity
PPARβ/δagonists reduce LDL cholesterol,
triglycerides, insulin, and increase HDL
PPARαAgonism Promotes adipocyte differentiation Activation improves hyperinsulinemia and
hyperglycemia, reduces weight and
PPARαagonists reduce serum
triglycerides and LDL cholesterol, increase
HDL cholesterol
RXRαAgonism Promotes adipocyte lineage
commitment, adipocyte differentiation
Ablated RXR mice are resistant to
diet/chemical-induced obesity
RXR agonists increase plasma
triglycerides, cholesterol, decreased
thyroid hormones
GR Agonism Promotes adipocyte differentiation,
pre-adipocyte proliferation
GR knock-down mice are resistant to
diet-induced obesity, have improved
insulin sensitivity and glucose tolerance,
and increased energy expenditure
Excess glucocorticoids associated with
increased weight, adiposity, and
decreased glucose tolerance/insulin
TR Antagonism Promotes adipocyte differentiation TR null mice exhibit increased
Low thyroid hormone levels promote
weight gain, high levels promote weight
ER Agonism Inhibits adipocyte differentiation,
promotes pre-adipocyte proliferation
ERKO mice exhibit increased adiposity Decreased estrogen in menopause
associated with increased abdominal
AR Antagonism Promotes adipocyte differentiation, no
effect on pre-adipocyte proliferation
AR agonism has anti-adipogenic effects in
Low androgen levels associated with
increased abdominal obesity, reversed
with supplementation
LXR Agonism Promotes adipocyte differentiation,
pre-adipocyte proliferation
LXR knockout mice exhibit less adipose
and are glucose-intolerant; agonist
treatment reduces energy expenditure
LXR agonist treatments increase
triglycerides, cholesterol, and other
negative molecular markers
PXR Agonism Promotes adipocyte differentiation PXR ablation inhibits diet-induced obesity,
insulin resistance, and fatty liver disease;
agonist treatment promotes adiposity in
PXR agonist treatments reported to induce
hyperglycemia and increase diabetes risk
CAR Agonism Promotes adipocyte differentiation CAR agonist treatment enhances insulin
sensitivity, improves glucose and lipid
metabolism, reverses diet-induced obesity
CAR agonist treatment decreases plasma
glucose and improves insulin sensitivity
FXR Agonism Agonists induce adipocyte
differentiation, antagonists reverse
FXR agonist induces weight gain and
glucose intolerance in mice
FXR agonist treatments promote reduced
lipid accumulation and increased glucose
uptake, reduced HDL and increased LDL
cholesterol, improved insulin sensitivity
InsR Agonism Promotes adipocyte differentiation,
triglyceride accumulation
Increased weight gain and glucose
Insulin supplementation promote
increased weight gain, cholesterol, and
blood pressure
IGFR Agonism Promotes adipocyte differentiation,
triglyceride accumulation
Increased weight gain and glucose
Increased weight gain, triglycerides
Descriptive effects for several major hormone receptor pathways that influence the process of adipogenesis and weight maintenance. Summarized evidence is provided for direction
of effects, as well as in vitro, in vivo, and human epidemiological evidence. References and more detailed descriptions can be found within the relevant subsections of the manuscript,
within section Nuclear Receptor Mechanisms Mediating Metabolic Disruption.
*Due to lack of specific, potent, and available ligands, there is minimal reported work in humans. Summarized work describes effects observed in monkey models following treatment
with receptor-specific agonists.
They further demonstrated gene dosage effects in vitro; cells
lacking both copies of PPARγcould not be induced to
differentiate, cells with one copy exhibited an intermediate
degree of differentiation, and wild-type cells exhibited robust
differentiation and efficacious expression of adipocyte-specific
molecular markers (62). Clonal expansion and growth arrest
occurs concurrently with expression of two proteins, PPARγ
and the CCAAT enhancer binding protein alpha (C/EBPα),
and these markers are both important for the differentiation of
pre-adipocytes to adipocytes (63). Further experiments in this
laboratory demonstrated that while C/EBPαis also a primary
marker for the initiation of differentiation, it operates within a
single initiating pathway with PPARγ(67). In cells deficient of
PPARγ, C/EBPαwas not capable of promoting adipogenesis by
itself, suggesting an important but non-essential role in inducing
and maintaining PPARγexpression, as well as an accessory role
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Kassotis and Stapleton Mixtures and Mechanisms of Metabolic Disruption
FIGURE 2 | Mechanisms of EDC Exposure and Potential Human Metabolic Health Effects. Graphical depiction of the potential sources and exposure pathways for
humans to endocrine disrupting chemicals (EDCs), the molecular mechanisms related to metabolic health through which these EDCs may act to drive specific
mechanistic effects, all of which may contribute to potential adverse health risks for humans. Effects reported are representative and are not comprehensive to all
molecular mechanisms and mechanistic effects.
in mediating insulin sensitivity via direct induction of the insulin
receptor (67).
The other PPAR isoforms, αand β/δ, have received
considerably less attention as it relates to adipogenesis,
though gain/loss-of-function experiments suggest putative
roles. Experiments in vitro demonstrated that induction
with a PPARβ/δ-specific ligand induced robust triglyceride
accumulation in wild type cells (68) [and in vivo (69)], while
PPARβ/δ-null cells differentiate and accumulate triglycerides less
efficaciously following PPARγ-mediated induction (68). This
suggests that while PPARβ/δis not necessary for adipogenesis,
the interplay of these isoforms is necessary to induce maximal
differentiation and triglyceride accumulation in adipocytes.
This was supported by other work reporting that PPARβ/δ
activation promotes PPARγexpression, potentially bolstering
adipogenesis, and providing a supportive role (70,71). When
examining the isoforms in isolation, Brun et al. reported
that receptor isoform-specific activation via ligands failed
to induce adipogenesis and triglyceride accumulation for
PPARβ/δ, but did for PPARα, if to a lesser extent and over a
longer time-course than via activation of PPARγ(72). RNA
isolations on day five demonstrated that PPARα-treated cells
had minimal or no induction of various adipocyte markers,
relative to robust induction in the PPARγ-treated cells; however,
both had robust expression by day seven post-induction,
while PPARβ/δexhibited only minimal expression at much
later time points (72). Further experiments demonstrated
that C/EBPαacted cooperatively with PPARγto stimulate
adipogenesis as expected, but not with PPARαor β/δ(72),
suggesting distinct mechanisms. PPARβ/δactivation in mice
has anti-adipogenic effects, improving lipid profiles, reducing
lipid accumulation, and increasing resistance to diet-induced
obesity (73,74). PPARαactivation in mice has similarly been
demonstrated to result in anti-adipogenic effects, including
improved hyperinsulinemia and hyperglycemia, lowered
triglycerides, increased resistance to diet-induced obesity,
and decreased weight and adiposity (7577). Fenofibrates
(PPARαagonists) administered to humans have similarly
been demonstrated to decrease serum triglycerides and LDL
cholesterol and increase HDL cholesterol (78,79). While
absence of good selective PPARβ/δagonists has hindered human
therapeutic examination, limited work with a selective and
potent PPARβ/δagonist in rhesus monkeys reported lowered
LDL cholesterol, triglycerides, insulin, and increased HDL
cholesterol (80).
Retinoid X Receptor (RXRα)
PPARγfunctions as a heterodimer with RXR, suggesting
that this receptor might also have dependent and/or
independent roles in adipogenesis (62). Indeed, more
than a decade ago, it was reported that organotins, potent
activators of both PPARγand RXRα, were also extremely
potent inducers of adipogenesis (81,82). Other studies
have confirmed that receptor-specific activation of RXRα
promotes both adipogenic differentiation and pre-adipocyte
proliferation (60,83,84). Mechanistic experiments have
further determined that of more than 5000 PPARγ:RXR
DNA-binding sites in adipocytes, most are occupied by
non-PPARγ:RXR heterodimers during the early stages of
differentiation and transition to PPARγ:RXR in the later
stages of differentiation (85). Mice with ablated adipocyte-
RXRαare resistant to diet and chemical-induced obesity
and exhibit impaired lipolysis during fasting (86); RXR
agonists have also been demonstrated to sensitize diabetic
and obese mice to insulin (87) and decrease hyperglycemia,
hypertriglyceridemia, hyperinsulinemia, and both weight
gain and food intake in several rodent models (8789).
More recent work from the Blumberg lab elegantly described
RXR activation as an essential signal for commitment of
mesenchymal stem cells to the adipocyte cell lineage, as
well as separately promoting subsequent differentiation (48).
Follow-up investigation determined that RXR activation-
induced adipocyte differentiation created a functionally distinct
adipocyte relative to those induced by PPARγactivation; RXR
activation resulted in decreased glucose uptake, expression of
adiponectin, and did not induce molecular pathways involved
in adipocyte browning, suggesting a dysfunctional white
adipose tissue that could potentially contribute to elevated
obesity and/or diabetes risk (90). Therapeutic treatment
by rexinoids in humans has reported increased plasma
triglycerides, increased plasma cholesterol, and decreased thyroid
hormones (9193).
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Kassotis and Stapleton Mixtures and Mechanisms of Metabolic Disruption
Liver X Receptor (LXR), Constitutive
Androstane Receptor (CAR), Pregnane X
Receptor (PXR), and Farnesoid X Receptor
LXR, CAR, FXR, AND PXR are permissive binding partners
with RXR, forming receptor heterodimers that can be activated
by ligands for either receptor or both (potentially resulting
in a synergistic effect), reviewed in Shulman et al. (94).
LXRαis expressed primarily in the adipose, liver, intestine,
and kidney, while the βisoform is ubiquitously expressed;
LXRs mediate cholesterol transport, stimulating cholesterol
efflux from macrophages, promoting transport in serum and
uptake into liver, increase degradation of cholesterol into bile
acids, inhibit absorption in the intestine, and synthesize fatty
acids and triglycerides (94). Some disparate results have been
reported in vitro: Hummasti et al. reported that LXR agonists
failed to promote triglyceride accumulation and/or adipocyte
differentiation in 3T3-L1 cells and 3T3-F442A cells, though did
regulate adipocyte-specific gene expression (95). However, other
studies have described LXR-mediated promotion of triglyceride
accumulation, adipocyte differentiation, adipocyte-specific gene
expression, and pre-adipocyte proliferation both in vitro and
in vivo (60,96,97), potentially via activation of PPARγ(96,
97). These disparate results could be explained by cells lines
and/or cell sources, as we previously reported different LXR
expression and responsiveness in varying pre-adipocyte sources
(60). Selective knockdown experiments have demonstrated LXRα
as the primary regulator of lipolysis (98), with the βisoform
more involved in cholesterol regulation (99). LXRβ-specific
knockout mice have less adipose, but normal insulin sensitivity
and adipocyte hormones; however, they are glucose-intolerant
and accumulate lipid in pancreatic islets, putatively mediated
by regulation of cholesterol transporters (99). Adipocytes have
been demonstrated to be smaller in LXR deficient mice (97),
and energy expenditure is increased, with reduced triglyceride
accumulation in brown adipose (100); in parallel, energy
expenditure is reduced in LXR agonist-treated wild type mice,
and triglyceride accumulation was increased in brown adipose
(100). In humans, LXR expression is higher in obese individuals,
and receptor isoform polymorphisms have been associated with
increased risks of obesity (101). Therapeutic treatment with LXR
agonists resulted in increased plasma and hepatic triglycerides,
cholesterol, and other negative metabolic markers in humans
as well as primate and rodent models (102), despite some
beneficial effects.
CAR and PXR are two closely-related liver-enriched receptors
that have also been associated with metabolic function, and
were reviewed in detail previously (103,104). While originally
appreciated as regulating xenobiotic metabolizing enzymes, they
have also been demonstrated to help regulate energy homeostasis,
immune function, lipid metabolism, and glucose homeostasis
(103,104). PXR appears to mediate effects through PPARγ, with
PXR activation directly inducing PPARγand other lipogenic
gene expression such as Cd36, though potentially in a species-
specific manner (104). CAR may promote effects on energy
homeostasis through crosstalk with PPARα, or similarly to PXR,
through activation of the free fatty acid uptake transporter
Cd36 and inhibition of sterol regulatory element-binding protein
(SREBP) (105). In animals, PXR ablation inhibits diet-induced
obesity, insulin resistance, and fatty liver disease in various
rodent models, suggesting PXR antagonism as a putative anti-
obesogenic and anti-diabetic pathway (104,106). PXR agonist
treatment in mice promotes hepatic triglyceride accumulation,
and constitutively active PXR mice exhibit enlarged and fatty
liver disease, reviewed in (107). Treatment with CAR agonists,
in contrast, enhances insulin sensitivity, improves glucose and
lipid metabolism, and reverses diet-induced obesity in rodents,
reviewed in (103). In humans, the CAR agonist phenobarbital
has been reported to decrease plasma glucose levels and improve
insulin sensitivity in patients with diabetes (103,108,109), and
though PXR is particularly promiscuous, activation of PXR by
rifampicin, statins, and other pharmaceuticals have been reported
to induce hyperglycemia in patients and increase the risk of
developing diabetes (106). While activation of CAR is seemingly
more therapeutically beneficial relative to PXR, it also carries with
it side effects such as liver hyperplasia and carcinogenesis (103),
among other effects.
Modulation of FXR has also been assessed as it relates
to adipogenesis and a potential therapeutic target in treating
metabolic syndrome, reviewed in (110). Endogenously activated
by bile acids, FXR regulates bile acid synthesis, enterohepatic
circulation, lipid metabolism, and thus indirectly regulates other
bile acid associated receptors, discussed in Prawitt et al. (111).
Researchers have described that FXR is expressed in adipocytes
from adult mice and in differentiated 3T3-L1 cells, but not
in the undifferentiated pre-adipocytes (112). Treatment with
an FXR agonist increased adipocyte differentiation in 3T3-L1
cells, whereas treatment with an FXR antagonist reversed this
(112); FXR agonist treatment also enhanced insulin signaling
and insulin-stimulated glucose uptake (113). Pro-apoptotic and
anti-adipogenic effects of guggelsterone (FXR antagonist) have
also been reported by other researchers (114). Treatment with
an FXR agonist in mice with diet-induced obesity worsened
weight gain and glucose intolerance, seemingly mediated through
reduction of the bile acid pool size and energy expenditure
(115). However, other research in mouse models suggests
beneficial effects for FXR agonist (GW4064) treatment (111,
116). FXR knockout/deficient mice exhibit decreased adipose
tissue, lower leptin concentrations, elevated plasma free fatty
acids, resistance to rosiglitazone-induced obesity, and their
embryonic fibroblasts are also resistant to rosiglitazone-induced
triglyceride accumulation and differentiation due to increased
lipolysis and decreased lipogenesis (113,117,118); despite
these apparent positive metabolic effects, FXR deficient animals
(both mice and rabbits) also exhibit impaired glucose tolerance
and insulin resistance, which are corrected with FXR agonist
supplementation (113,119). FXR expression has also been
demonstrated to be downregulated and/or dysfunctional in
obese humans (110,120), suggesting downregulation may play
a potential role in human obesity. PXR mice exhibit FXR
agonist therapeutic trials in humans have reported reduced liver
lipid accumulation and increased glucose uptake [reviewed in
(110)], reduced HDL cholesterol and increased LDL cholesterol,
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Kassotis and Stapleton Mixtures and Mechanisms of Metabolic Disruption
and improvements in insulin resistance [reviewed in (111)],
suggesting that FXR antagonists and/or selective FXR receptor
modulators might promote more beneficial effects in some tissues
and for specific metabolic endpoints (116).
Thyroid Receptor (TR)
TR also forms a heterodimer with RXR, though in contrast
to other receptors discussed above, it is considered a non-
permissive heterodimer (can be activated only by thyroid
receptor ligands and not RXR ligands), reviewed in Shulman
et al. (94). While less frequently assessed as a contributory
molecular pathway for adipogenesis, one of the defining
characteristics of thyroid hormone action is maintenance of
metabolic health and maintenance of lipid and carbohydrate
metabolism, blood pressure, and body mass [reviewed
in (94,121)]. Hypothyroidism (low thyroxine (T4) and
triiodothyronine (T3), high thyroid stimulating hormone
(TSH)) is characterized by weight gain, while hyperthyroidism
(high T4 and T3, low TSH) is characterized by weight loss
(122,123). As such, thyroid hormones are generally considered
anti-obesogenic, and hypothyroid-associated adiposity can
be reduced with supplementation (124126). TRαprimarily
regulates thermogenesis and TRβprimarily regulates cholesterol
metabolism and lipogenesis, as well as a number of genes
and enzymes necessary for pre-adipocyte proliferation and
adipocyte differentiation, either directly or via PPARγ[reviewed
in (121)]. Studies have demonstrated some disparate findings
regarding the role of TR in adipogenesis itself. For example,
antagonism of TR has been demonstrated to efficaciously
modulate adipocyte differentiation, purportedly via PPARγ,
reviewed in (16); however, we’ve previously demonstrated
that 3T3-L1 treatment with 1–850 (TR antagonist) resulted
in efficacious triglyceride accumulation (60), and TR-null
mice exhibit increased adipogenesis (127). Others, in contrast,
have reported that treatment with triiodothyronine (T3; TR
agonist) promoted adipocyte gene expression and decreased
pre-adipocyte proliferation in Ob L771 mouse pre-adipocytes
(128) or triglyceride accumulation and lipogenic gene expression
in 3T3-L1 pre-adipocytes (60,129). Other work suggested
differing roles at varying levels of treatment; when 3T3-F442A
cells were treated with hyperthyroid T3 levels, the proportion of
adipocytes was increased but expression of lipogenic enzymes
and triglyceride accumulation were decreased, whereas lower
levels stimulated adipose conversion, expression of lipogenic
enzymes, and pre-adipocyte proliferation (130).
Glucocorticoid Receptor (GR)
The GR is intimately connected to lipid metabolism, with a
wealth of in vitro,in vivo, and human epidemiological evidence
supporting its role in adipose formation and maintenance
[reviewed in (131)]. Treatment with dexamethasone induces
a potent and efficacious triglyceride accumulation and pre-
adipocyte proliferation response in various mesenchymal and
pre-adipocyte models, often to greater extents and at lower
concentrations than through direct activation of PPARγ(60,
132), potentially mediated at least in part through activation
of PPARγ(133), though in other cases without meaningful
activation of PPARγ(134); in support, treatment with GR
antagonists inhibits differentiation in various mesenchymal and
pre-adipocyte models (135). Other studies have reported that
glucocorticoids alone were insufficient to promote adipogenesis
either in 3T3-L1 cells (136) or in other models (137), though
stimulated robust differentiation in combination with insulin
(137). As mentioned above, Chappell et al. demonstrated putative
GR-mediated effects prior to PPARγactivation after exposure to
tetrabrominated bisphenol A (TBBPA) (61), which may explain
why isobutylmethylxanthine (IBMX; PPARγligand) treatment
prior to dexamethasone (GR agonist) failed to induce significant
differentiation using the same cell model in another lab,
while dexamethasone treatment before IBMX promoted robust
differentiation (138). The authors posited that glucocorticoid
activation may be necessary for an intermediate commitment
state prior to differentiation via PPARγ(138); however, this
could also be due to differing responsiveness to PPARγand GR
ligands based on 3T3-L1 cell source, which we have reported on
previously (60).
Other research has evaluated the putative role of the
mineralocorticoid receptor (MR), an additional high-affinity
binder of glucocorticoids; treatment of 3T3-F442A and 3T3-L1
cells with the mineralocorticoid agonist aldosterone promoted
adipocyte differentiation, which appeared to be mediated
through PPARγactivation; inhibition and knock-down of
the MR inhibited adipogenesis, whereas knock-down of the
GR did not (139). More recent work, however, demonstrated
that silencing GR, but not MR, inhibited the pro-adipogenic
activity of cortisol, and also decreased leptin and adiponectin,
whereas MR knock-down actually increased leptin (140).
Research in mice investigated knocking out local glucocorticoid
action via 11β-hydroxysteroid dehydrogenase (glucocorticoid
inactivator) overexpression exhibited resistance to diet-induced
obesity/reduced fat accumulation, decreased food intake,
improved insulin sensitivity and glucose tolerance, and increased
energy expenditure (141). Glucocorticoid excess in mice in
contrast resulted in decreased osteogenic gene expression
and mineralization and increased expression of adipogenic
genes (142). Cushing’s syndrome (excess cortisol production)
is associated with increased weight gain, hypertension, type
2 diabetes, and fatty tissue deposits (143,144), suggesting a
pro-adipogenic effect of glucocorticoids in humans as well.
Further, prenatal/antenatal dexamethasone (GR agonist) is often
utilized to promote development of lungs in infants at risk
of being born premature (145,146). Epidemiological studies
have reported that dexamethasone treatment is associated with
reduced birth weight in infants, even after correcting for weeks
of gestation (145,146), and exhibited hypertension and greater
subsequent administration of insulin for hyperglycemia (146).
Estrogens and Androgens
Often considered opposing sex steroids, androgens, and
estrogens have also been described to have opposing effects on
adipogenesis, reviewed in Cooke and Naaz (147). Experiments
comparing differentiation extent in rat pre-adipocytes
determined no effects for either androgens or estrogens in
promoting differentiation in male pre-adipocytes; however,
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estrogens elicited a pro-adipogenic effect (via pre-adipocyte
proliferation) and androgens elicited an anti-androgenic effect
in female cells, potentially mediated by modulation of insulin
growth factor 1 receptor (IGF1R) and PPARγexpression (148).
This promotion of pre-adipocyte proliferation by estrogens
has been successfully replicated in both male and female
omental pre-adipocytes (149), while the inhibitory effect of
estrogens on differentiation/triglyceride accumulation may
be dose-dependent (150). Related work has determined some
of the inhibitory effects of estrogens on adipogenesis appear
to occur through the G-protein-coupled estrogen receptor 1
(GPER) rather than the classical estrogen receptor itself (151),
and that inhibitory effects on adipogenesis are concurrent with
enhancement of osteogenesis (152). Interestingly, estrogen
receptor knock-out (ERKO) mice exhibit increased fat pad
weights, adipocyte size, and adipocyte numbers relative to
wild type control animals, as well as insulin resistance and
impaired glucose tolerance (153). This is mirrored in humans,
as decreased estrogen levels at menopause are associated with
increased abdominal obesity that is ameliorated with estrogen
replacement therapy [reviewed in (154)], an effect also observed
in ovariectomized female mice (155).
Androgens are generally considered anti-obesogenic
[reviewed in (156,157)], and treatment with androgens has
been demonstrated to inhibit adipogenesis in adipose tissue
samples from both sexes (158) and reduce fat mass in humans
[reviewed in (159)]. Dihydrotestosterone inhibits triglyceride
accumulation and adipocyte gene expression in human
mesenchymal stem cells and pre-adipocytes from various depots,
whereas anti-androgen co-treatment attenuated those effects,
and had no apparent impact on pre-adipocyte proliferation in
either model (160). Other research has replicated these findings,
suggesting some of the effects occur through inhibition of the
multipotent stem cell to pre-adipocyte commitment (161,162).
In contrast, anti-androgens have been suggested to act as
obesogens; androgen receptor knock out (ARKO) mice exhibit
increased obesity (163), flutamide has been demonstrated to
modulate lipid profiles in women (164), and hypogonadism
(characterized by testosterone deficiency) is associated with
obesity, hypertension, dyslipidemia, insulin resistance, and
other metabolic effects, which may be corrected with androgen
supplementation (159).
Other Receptors
A variety of other receptors, from the nuclear receptor
family, receptor tyrosine kinase family, and others, have
described roles in adipogenesis and/or lipogenesis. For
example, both the insulin and IGF-1 receptors have widely
accepted roles in growth, tissue-specific hypertrophy, and
weight maintenance (165168). Many others, including the
aryl hydrocarbon, retinoic acid, low density lipoprotein
receptors, among others, have established roles in adipogenesis
but could not be discussed in detail within the scope of
this review. Importantly, while the bulk of study has
assessed activation of PPARγand RXR, numerous other
receptor systems interplay to promote and maintain
adipocytes, and must be taken into account when evaluating
environmental mixtures.
Mitochondria are the major location of fatty acid oxidation,
making them essential in lipid metabolism; as such, dysfunction
can contribute to numerous adverse metabolic health
consequences, including altered lipid accumulation, metabolism,
and insulin resistance (169,170). Mitochondrial function is
intimately connected with metabolic health, as it helps regulate
energy expenditure, production of ATP, and removal of reactive
oxygen species (ROS); ROS reduce oxygen consumption and
inhibit fatty acid oxidation in adipocytes, promoting lipid
accumulation [reviewed in (169)]. ROS production mainly
occurs at complex I and III in mitochondria, and is increased
when excess electrons are provided to the mitochondrial
respiratory chains (when proton gradient is high and ATP
demand is low), as described in Kim et al. (171). Excess
electrons are transferred to oxygen, converted to superoxide,
and subsequently to hydrogen peroxide; this ROS acts to damage
proteins, DNA, and lipids, and activates pathways (via activation
of serine kinases) that phosphorylate insulin receptor substrate
proteins and inhibit insulin signaling, thus promoting insulin
resistance and ultimately resulting in metabolic dysfunction
(171,172). Mitochondrial dysfunction and resultant lipid
accumulation in accessory tissues is also capable of further
impeding insulin signaling and glucose metabolism, promoting
further dysfunction (173); indeed, maternal obesity during
pregnancy in rodents contributes to a transgenerational
mitochondrial dysfunction phenotype (inhibited insulin
signaling for three generations) (174). Notably, chronic
oxidative stress has been well-described in obese individuals,
suggesting a link between ROS production/management and
hyperplasia [reviewed in (175)]. To minimize damage from
these ROS, cells require a balance between ATP synthesis
through oxidative phosphorylation and dissipation of the
proton gradient (169). Mitochondrial dysfunction can also
directly contribute to cardiovascular disease, another hallmark
disease of metabolic syndrome, and myocardial metabolic
function is intimately connected to obesity, diabetes, and altered
insulin signaling [reviewed in (176)]. Research suggests that
decreases in ATP production due to inhibited mitochondrial
respiration, increased oxidative stress, and inhibited calcium
signaling can all contribute to diastolic dysfunction via reduced
velocity of myocardial relaxation velocity and myocardial
compliance (173,176).
Adipocytes are capable of regulating metabolic insults by
altering their number, morphology, as well as the intracellular
mitochondrial distribution (169). Mitochondrial biogenesis is
an essential component of adipogenesis, with mitochondrial
numbers increasing markedly after initiation of pre-adipocyte
differentiation and reaching a maximum toward the end;
this can be noted via treatment with the PPARγagonist
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rosiglitazone, wherein treated cells demonstrate increased
mitochondrial content and function, with increased basal
oxygen consumption, ATP respiration, and proton leak (173,
177,178). ATP levels are naturally reduced with increasing
degree of adipocyte differentiation, putatively due to increased
ATP demands for lipogenesis (179), and reduced levels are
further exacerbated when electron transport chain inhibition
occurs (178). Reduced mitochondrial biogenesis, ATP levels,
and dysfunctional mitochondrial electron transport have been
reported in both humans and animals with metabolic syndrome
(173,179). PPARγco-activator 1α(PGC-1α) is a master regulator
of mitochondrial biogenesis and gene expression and is a potent
co-activator of PPAR isoforms: expression in fat or muscle
cells increases mtDNA content, expression of mitochondrial
genes, and mitochondrial respiration (176). PGC-1α-stimulated
biogenesis in the heart ultimately promotes overt heart failure,
another mechanism through which metabolic dysfunction
can lead to cardiac dysfunction (176). Biogenesis appears
closely linked to adipocyte differentiation, as up-regulation of
mitochondrial biogenesis is well-reported following induction of
adipogenesis and for up to 10 days post-differentiation (177,180),
suggesting that mitochondria are needed to supply the substrates
and factors necessary to support adipogenesis-driven lipogenesis.
Mitochondria also have functionally distinct roles in white vs.
brown adipose tissue. White adipose tissue is composed of
numerous depots of large lipid droplet adipocytes throughout the
body and is essential for maintenance of metabolic health. Brown
adipose tissue, in contrast, is smaller and localized to the neck
and upper-chest in adult humans, and is composed of adipocytes
with large numbers of smaller lipid droplets and more numerous
mitochondria [reviewed in (180)]. In brown adipose tissue, the
heat derived from thermogenesis is produced primarily by the
high mitochondrial content of these cells, via oxidation of fatty
acids and other components (180). Uncoupling proteins (UCPs)
play a key role in this process, serving to uncouple mitochondrial
respiration from ATP generation by inducing a proton leak,
which subsequently allows for energy dissipation as heat (180).
Numerous environmental toxicants have been demonstrated
to promote mitochondrial dysfunction, and these contaminants
may also advance metabolic dysfunction, leading to obesity, and
diabetes. Certain mitochondrial disorders that are characterized
by impaired oxidative phosphorylation are also associated
with disrupted lipid homeostasis: myoclonic epilepsy with
ragged red fibers is associated with triglyceride accumulation
in muscles and multiple symmetrical lipomatosis, a condition
characterized by abnormally small white adipocytes containing
numerous small lipid droplets rather than the classical large
central droplet that displaces the nucleus (170). Mitochondrial
oxidative phosphorylation inhibitors and protein synthesis
inhibitors impair mitochondrial respiration and promote
triglyceride accumulation in 3T3-L1 cells, which retain their
precursor fibroblastic morphology and do not express adipocyte-
specific markers (170,180). Previous research has demonstrated
that treatment of 3T3-L1 cells with rotenone (a complex I
inhibitor), antimycin A, stigmatellin, and myxothiazol (complex
III inhibitors), and oligomycin (ATP synthase inhibitor)
promoted triglyceride accumulation in a dose-dependent
manner (170). Interestingly, these mitochondrial respiration
inhibitors promoted triglyceride accumulation in numerous
small lipid droplets, cells retained their fibroblastic morphology,
and classical adipocyte-specific genes were not expressed in
these cells (170), suggesting a differentiation-independent
mechanism of triglyceride accumulation. Specifically, antimycin
A, which inhibits complex III, induces triglyceride accumulation
in pre-adipocytes via a putative differentiation-independent
mechanism (170); these cells exhibit multi-vesicular lipid
accumulation, reduced expression of standard differentiation
markers (FABP4, C/EBP), and suppression of PPARγand RXR,
supporting other studies suggesting mitochondrial dysfunction
may inhibit adipocyte differentiation (178).
We recently demonstrated a similar phenotype in experiments
with pyraclostrobin, a strobilurin-class fungicide used
on strawberries, spinach, and other produce items, with
production of >2 million pounds per year (181,182).
Pyraclostrobin and other strobilurin fungicides have been
demonstrated to inhibit complex III (183), suggesting a
potential mechanism for metabolic disruption. We previously
reported that pyraclostrobin, azoxystrobin, fluoxastrobin, and
trifloxystrobin all induced both triglyceride accumulation and
pre-adipocyte proliferation in 3T3-L1 cells (184). Previous
research in 3T3-L1 cells and a human adipose-derived stem
cell model suggested this did not occur through activation of
PPARγand that standard differentiation markers were lacking
(47,185), supporting the case for a differentiation-independent
mechanism. Mechanism was further interrogated in our
laboratory through co-exposure experiments in 3T3-L1 cells;
we reported that PPARγantagonists did not protect against
pyraclostrobin-mediated triglyceride accumulation (177).
Instead, pyraclostrobin promoted mitochondrial dysfunction,
including reduced ATP, mitochondrial membrane potential,
basal mitochondrial respiration, ATP-linked respiration, and
spare respiratory capacity (177). In addition, pyraclostrobin-
treated cells exhibited reduced expression of genes regulating
glucose transport, glycolysis, fatty acid oxidation, and lipogenesis
(177). Lastly, co-treatment with a cAMP responsive element
binding protein (CREB) inhibitor reduced pyraclostrobin-
mediated triglyceride accumulation (177). These results all
suggest that toxicants capable of disrupting mitochondrial
function may also have the potential to affect metabolic health,
via modulation of lipogenesis and other metabolic processes.
Similarly, a recent study reported that several samples of oil
sands process-affected water (OSPW), wastewater produced
during the extraction of bitumen from oil sands, exhibited
PPARγagonist activity and promoted triglyceride accumulation
in 3T3-L1 cells (186). Causative ligand characterization
identified several hydroxylated/polyoxygenated carboxylic
acids and hydroxylated sulfates as the major PPARγligands
(186); naphthenic acids, a mixture of carboxylic acids and
natural component of petroleum, are a major component of
OSPW. Interestingly, while these are posited as promoting
adipogenesis via PPARγactivation, a recent publication
demonstrated that naphthenic acids isolated from oil sands
water acted to uncouple oxidative phosphorylation, inhibit
respiration, and increase the production of ROS (187). As noted
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above, these are mechanisms that can promote triglyceride
accumulation in cells, suggesting that this may be an additional
mechanism for the observed adipogenic effects of these waters
in the previous publication (186). Notably, this occurred at
environmentally-relevant concentrations for OSPW, suggesting
that this or a combination of these mechanisms may promote
the environmental sample-induced adipogenicity.
Mitochondrial ROS is produced by pre-adipocytes during
and throughout differentiation, and its presence activates several
early-stage differentiation markers, including C/EBP, PPAR,
and CREB (63,188). Direct impacts on adipogenesis appear
less certain, which has been delineated in greater detail
previously (189); research suggests that ROS may be essential for
adipogenesis, but also may perturb the process. Some research
has demonstrated that ROS promoted mitotic clonal expansion
in 3T3-L1 cells (190), a necessary step prior to induction
of differentiation. Other research has described inhibitory
effects on differentiation, with ROS inhibiting both pre-
adipocyte proliferation and adipocyte differentiation/triglyceride
accumulation (180,191,192). Still other researchers, via co-
treatment experiments utilizing antioxidants, demonstrated
that ROS impacted differentiation but not pre-adipocyte
proliferation: treatment with an antioxidant (reducing ROS)
reduced lipid accumulation in mesenchymal stem cells (193).
However, the varying cell models used for these experiments
may mediate these apparent differences; all studies agree that
ROS appear to modulate early-stage differentiation, though the
mechanisms of this modulation appear to vary based on cell lines,
sources, and experimental details.
Several mitochondrial toxicants have been demonstrated
to promote insulin resistance and/or metabolic syndrome in
epidemiological studies, reviewed in detail previously (172,
194), though this research area has as of yet received limited
attention in the context of metabolic disruption. Several
organochlorine pesticides have been implicated in metabolic
effects via mitochondrial dysfunction. Specifically, atrazine has
been demonstrated to directly inhibit complexes I and III,
reducing oxygen consumption and leading to accumulation of
superoxides; chronic exposure in rats has been demonstrated to
decrease basal metabolic rate, and increase body weight, intra-
abdominal fat, and promote insulin resistance independent of
food intake or activity levels (172,195). Much of the remaining
literature has focused on the role of polychlorinated biphenyls
(PCBs). Several congeners have been demonstrated to promote
mitochondrial dysfunction in vitro (196,197), exposure resulted
in/exacerbated obesity, insulin resistance, and hyperinsulinemia
in mice (198), and higher exposure to PCBs has been linked to
increased risk of obesity, dyslipidemia, and/or insulin resistance
in a number of epidemiological studies (199202).
Numerous in vitro models have been developed and utilized
for the purpose of identifying potential metabolic disrupting
chemicals, reviewed in detail previously (203,204). Generally,
these models can be described as assessing two key parameters
of adipocyte development: commitment to the adipocyte lineage
from multipotent precursor cells (generally through the use
of mesenchymal stem cell (MSC) models) and differentiation
into mature adipocytes (generally through the use of pre-
adipocyte models). MSC models have the additional benefit of
being capable of assessing both endpoints, though are seemingly
less frequently utilized than the available pre-adipocyte models.
In addition, several research groups have begun to report on
the three-dimensional culture of pre-adipocytes, which may
shed additional light on mechanisms in a more physiologically
relevant system. All of these assays are lengthy and their relative
abilities to correctly identify chemicals may depend on both
cell line and cell source. As such, there is a critical need
to develop better methods for correctly predicting metabolic
disruptors. While murine models have historically been used
preferentially, a growing number of species utilized and a
growing movement toward utilization of human models may
help expand our understanding of translational mechanisms and
potential environmental contaminant impacts on human health.
Perhaps the best known pre-adipocyte model is the 3T3-
L1 mouse cell line. First described in the 1970’s, it has proven
reliable as an in vitro screen over several decades for identifying
likely obesogenic chemicals in vivo (205,206). These cells
are already committed to the adipocyte lineage and cannot
develop into other cell types; however, they generally require
activation of particular signaling pathways to promote further
development. Following exposure to adipogenic chemicals, these
cells differentiate into adipocytes, accumulate triglycerides, and
come to resemble a mature human white fat cell (44,46,205,
206). While 3T3-L1 cells have seemingly come to be considered
the de facto model of adipogenesis, some inherent concerns
remain about their utility. As we have described recently (60),
while this line has been well-characterized (207), it is somewhat
unreliable in sourcing. For example, while we know much about
the molecular mechanisms underpinning the development of
mature adipocytes based on this cell line, nuclear receptor
expression related to adipogenesis is markedly different between
different lots and sources of this cell line (60). Moreover, on
investigation into this apparent discord in source, we discovered
that the American Type Culture Collection (ATCC) maintains
five distinct lots of 3T3-L1 cells, which all seemingly have
differing degrees of differentiation success. This issue with cell
line integrity was highlighted in a recent paper (208), suggesting
that these differences can contribute to real discrepancies in the
ability to replicate findings across laboratories. As the current
ATCC cells are meaningfully different in the expression of key
adipogenic pathways from the Zenbio-sourced cells (which are
sourced from the isolating laboratory), it is unclear whether our
understanding of the mechanisms underlying adipogenesis are
from the original cells, the ATCC cells, or where these research
paths diverge. Care needs to be taken to assess reproducibility
across stocks and between laboratories and carefully untangle
where the research underlying this cell line belongs. Other pre-
adipocyte models also exist, including the OP9 mouse bone
marrow-derived stromal pre-adipocyte cell line (44,45), a line
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that allows for considerably faster differentiation, though which
we have demonstrated to exhibit different nuclear receptor
expression and differing degrees of responsiveness to adipogenic
chemicals (60). These varying pre-adipocyte models allow for
assessments of varying molecular pathways important for the
process of differentiation via both source of the cells and species
[discussed further in (204)].
Various multipotent mesenchymal cells and cell lines (46,47)
offer the additional ability to assess commitment to the adipocyte
lineage as a distinct process from adipocyte differentiation (48).
MSC use and applicability in adipogenesis research has been
reviewed in detail previously (209). The variability of these
cell lines are reportedly lower than the pre-adipocyte models,
they are purportedly easier to isolate and culture, and they
have additional utility in that they can be utilized to assess
both differentiation of adipocytes but also commitment to the
adipocyte lineage vs. other cell lineages. For example, many
researchers have utilized these cell lines to evaluate the interplay
between commitment to the osteogenic vs. adipogenic lineages
following exposure to specific environmental contaminants
(210213). Recent work elegantly described a novel protocol for
evaluating both adipogenic lineage commitment and subsequent
differentiation as distinct processes in primary MSCs (48),
which has been described previously for the C3H10T1/2
stem cell model (214,215). These advancements raise the
utility of this model and warrants further investigation into
replicability, reproducibility of this model across laboratories,
and comparisons of translation to human health relative to the
pre-adipocyte models currently utilized.
Lastly, a number of research labs have begun to describe
spheroid cell cultures of adipocyte models (216220), which
may carry some inherent benefits over the standard adherent
monolayer cultures. These studies have suggested that spheroid
culture improves the efficiency, extent, and/or speed of
differentiation (216221), retains the multipotent potential of
these cells (217,222), and transcriptomic analyses have suggested
a potentially more representative model of adipocyte gene
expression relative to known in vivo mechanisms (216). These
models may allow for a more comprehensive understanding of
adipose physiology than was possible via interrogation of the
monolayer cell cultures, and should be evaluated further for
replicability and translation potential relative to the standard
monolayer cultures.
As noted above, the assessments of environmental samples have
proven an interesting new approach to evaluating potential
mixture toxicity. With tens of thousands of chemicals in
use and new chemicals regularly added, there are too many
to characterize individually, and certainly no capabilities to
assess all potential combinations of them (223,224). Body
burden studies have and continue to report human exposure
to hundreds of chemicals on a regular basis (225,226),
demonstrating the problem of realistic mixture exposure studies.
To add to the complexity, research has reported additive
effects on several hormone receptors both in vitro and in vivo
(227231), demonstrating that mixtures can induce effects at
levels below those induced by individual chemicals. From
a toxicological perspective, evaluating whole environmental
samples: wastewater, surface/groundwater, indoor house dust,
air samples, etc. for biological activities has emerged as a
promising tact to assess potential adverse health concerns from
exposure to actual mixtures present in the environment, given
that it can evaluate more realistic environmentally relevant
exposures (Figure 3).
Numerous natural and exogenous contaminants can
contribute to human exposure; as such, measuring the total
receptor bioactivities has proven useful for assessing the total
magnitude of potential effects (232239). While analytical
chemistry techniques and equipment have drastically improved,
allowing for more precise measurements of contaminants at
lower concentrations, recent research has suggested we lack
complete information on all causative bioactive chemicals
present in the environment (240,241). While non-targeted
analytical efforts have improved, we still lack sufficient software
and comprehensive protocols to enable robust and reproducible
non-targeted assessments of contaminants across laboratories.
To address this need of addressing mixture toxicity without
necessarily understanding the full chemical complexity,
bioassays have been utilized to assess biological activities of
actual environmental samples. Reporter gene assays are one
such commonly-utilized tool, assessing total receptor activities
(agonism and antagonism), and valued due to their low cost,
ease of use, reliability, high sensitivity, and ease of adapting
for multiple receptors (227244). These assays provide the
capability to assess the total receptor activity of potentially
numerous low-concentration EDCs (without identifying each
causative chemical) rather than assessing each constituent
chemical individually.
Applying this method to human epidemiological research
has shown great potential; a number of researchers have
rigorously characterized how in vivo mixtures of contaminants
correspond with total hormone receptor bioactivities of human
and animal matrices (serum, tissues, etc.) (245251). Moreover,
some researchers have begun to utilize bioactivities directly
to assess human health outcomes. For example, researchers
have correlated the total placental estrogenic activity with
increased reproductive malformations (252) and impaired
motor development (253), total adipose estrogenic activity with
increased risk for breast cancer (254), and placental estrogenic
activity with increased birth weight in boys (255). Other research
has failed to report significant associations, including a lack
of any association between adipose estrogenic activity and risk
for type-2 diabetes (256), potentially due to a greater role for
other receptors in pathogenesis (257). These studies demonstrate
the potential utility of this method, particularly when targeted
based on a comprehensive understanding of etiology and
molecular mechanisms.
Several studies have begun to apply these techniques to
metabolic endpoints, assessing pertinent receptor bioactivities
(GR, PPARγ, and others) as well as utilizing less high-throughput
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Kassotis and Stapleton Mixtures and Mechanisms of Metabolic Disruption
FIGURE 3 | Utility of Utilizing Environmental Mixtures for Human Health Assessments. Graphical depiction comparing individual chemicals and environmental mixtures
for the assessment of potential human health effects. While environmental mixtures have less use in identifying causative chemicals in all cases (though tools like effect
directed analysis (EDA) and toxicity identification evaluation (TIE) can be used to elucidate this), these mixtures are more relevant in reflecting the suite of chemicals
that people are exposed to on a daily basis than utilizing single chemicals alone, and more often reflect actual environmental exposure concentrations. In this figure,
the blue lines indicate positive relationships and the red lines indicate difficulty for single chemicals or mixtures in assessing the related outcome.
adipogenesis or other assays for predicting in vivo metabolic
disruption potentials. Some of these environmental case studies
are discussed in greater detail below:
Metabolic Disruption Potential of Indoor
House Dust
As noted above, numerous studies have documented the
detection of EDCs from diverse chemical classes in indoor
house dust samples from a variety of sources. A number of
studies have assessed the bioactivities for solvent-extracted house
dust, reporting PPARγ, GR, and ER agonist activities as well
as AR and TR antagonist activities, at concentrations 15
µg dust equivalence per mL (DEQ/mL, mass of extracted dust
per volume of assay medium) (258260). Our laboratory also
assessed the modulation of PPARγby house dust extracts,
reporting that 21 of 24 examined indoor house dust extracts
exhibited significant PPARγbinding at 3 mg DEQ/mL (120
µg dust per assay well) using a relative binding affinity assay
(261) and 15 of 25 extracts activated PPARγat 50% of the
maximal positive control response at concentrations 100 µg
DEQ/mL (4 µg/well) using a commercially-available reporter
assay (262,263). This work demonstrated activation of pathways
known to regulate adipogenesis at very low concentrations, and
subsequently informed our follow-up studies examining higher-
order effects on adipogenesis.
We recently evaluated >40 common SVOCs that are routinely
detected in indoor house dust samples for adipogenic activity
in the 3T3-L1 murine pre-adipocyte cell model. We found
that >two-thirds of these chemicals independently induced
significant triglyceride accumulation and/or pre-adipocyte
proliferation (184). Specifically, pyraclostrobin (strobilurin
fungicide), dibutyl phthalate (DBP), tert-butyl-phenyl diphenyl
phosphate (TBPDP), and the isopropylated triaryl phosphates
(ITPs, mixture of isomers) exhibited near or supra-maximal
triglyceride accumulation relative to the rosiglitazone (positive
control)-induced maximum (184). We further assessed eleven
house dust extracts collected from central North Carolina (NC),
USA households; we found that ten of these 11 extracts exhibited
significant triglyceride accumulation and/or pre-adipocyte
proliferation at <20 µg of dust/well (184). This activity occurred
at orders of magnitude lower concentrations than those the EPA
estimates children to consume each day. As such, this raises
concerns for potential impacts on in vivo metabolic health.
A recent follow-up to this study evaluated the adipogenic
activity of 137 house dust extracts from central NC households
and attempted to determine putative causative chemicals,
molecular mechanisms, and potential impacts on human
metabolic health (264). We reported that 90% of the dust extracts
exhibited significant adipogenic activity, <60% via significant
triglyceride accumulation, and >70% of samples via significant
pre-adipocyte proliferation, with >40% of effects occurring at
<10 µg dust/well (264). Increasing dust-induced triglyceride
accumulation was positively correlated with serum thyroid
stimulating hormone levels in adult residents, and negatively
correlated with serum free triiodothyronine (T3) and thyroxine
(T4) (264). Interestingly, proliferation tended to be positively
correlated with residents’ body mass index (BMI; p<0.10),
potentially suggesting adipogenic chemicals present in the dust
are associated with the weights of residents, but further research
with larger sample sizes are needed to substantiate this. We
further assessed TR antagonism as a potential contributory
causative mechanism in these effects, and found that TRβ
antagonism of these extracts (265) was positively correlated
with triglyceride accumulation (264). Both T3 co-treatment and
siRNA knock-down of TR inhibited the dust-induced triglyceride
accumulation of these extracts, supporting the role of TR
antagonism as a contributory molecular mechanism.
Metabolic Disruption Potential of Oil and
Gas-Associated Wastewaters
Three separate sets of studies have assessed different aspects
of oil and gas operations and metabolic disruption, reporting
in vitro and/or in vivo evidence of metabolic disruption by oil
and gas associated environmental mixtures. The first assessed
three replicate samples of oil sands process-affected water
(OSPW), wastewater produced during the extraction of bitumen
from oil sands (186). They reported that an OSPW sample
activated PPARγat concentrations as low as 0.025x relative
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Kassotis and Stapleton Mixtures and Mechanisms of Metabolic Disruption
water concentration (40-fold dilution relative to pure water). This
sample was further fractionated, with the majority of PPARγ
activity in fractions two and five (five fractions), and fractions
three through five exhibited significant triglyceride accumulation
and induction of adipogenic genes (fatty acid binding protein
and lipoprotein lipase). A pull-down assay and chemical analysis
was further utilized to identify the causative ligands present
in fraction five that were inducing the adipogenic effects; this
analysis revealed hydroxylated/polyoxygenated carboxylic acids
and hydroxylated sulfates as the major PPARγligands inducing
adipogenesis in these samples (186), though the small sample size
requires further substantiation.
Another set of studies assessed the metabolic disruption
potential of crude oil singly or mixed with Corexit oil dispersant
mixture (266,267). To distinguish these mixtures, they utilized
several simpler mixtures in culture media, including: Corexit
9500 +MC252 oil, varying dilutions of MC252 oil, and varying
dilutions of Corexit with corn oil; they found that the Corexit +
oil treatments stimulated PPARγ, while the MC252 oil alone did
not, suggesting a component of Corexit promoting the observed
effects (267). The Corexit +oil mixture was further fractionated
to determine causative ligands, with Tween 80 and dioctyl
sodium sulfosuccinate (DOSS) identified as highly abundant
chemicals in the active fraction (267). DOSS was further
demonstrated to be active in PPAR response element-luciferase
transgenic mice and stimulate triglyceride accumulation and
expression of fatty acid binding protein (Fabp4) in 3T3-
L1 cells (267). Follow-up work assessed the Corexit +oil
mixture and Corexit alone for activation of RXRα, finding
dose-dependent activation, presumably mediated by Corexit
constituents (266). Constituent chemicals were further evaluated,
and DOSS, Span 80, and Tween 80 all demonstrated some degree
of RXRαactivity, with Span 80 also stimulating triglyceride
accumulation and adipocyte gene expression in 3T3-L1 cells.
Interestingly, a combination of DOSS and Span 80 resulted
in putative synergistic effects on adipocyte differentiation,
potentially due to diverging molecular mechanisms (Span 80
exhibited a much more efficacious response for RXRαthan
PPARγ, while DOSS exhibited no RXRαactivity but did activate
PPARγ) (266).
The last set of studies, from our laboratory, evaluated
unconventional oil and gas associated wastewater and chemicals.
Our work on this topic began with receptor activity testing
for 24 common hydraulic fracturing chemicals, reporting that
21 and 7 chemicals antagonized AR and TR in two cell-
based assays, and that mixtures of these chemicals appeared
to act synergistically for TR and additively for AR (36,268).
We further documented AR and TR antagonist activities in
surface, ground, and/or drinking water near UOG operations
in several regions, including CO, WY, WV, and ND [(268),
Kassotis et al., in preparation, (269271)], and evaluated a
mixture of 23 common UOG chemicals via a gestational exposure
experiment in C57 mice, reported putative metabolic effects
(offspring exhibited increased body weights, among other effects)
(36,37). We further interrogated this by evaluating the ability
of this 23-mix, several UOG wastewater samples, and several
UOG wastewater-impacted surface water samples to stimulate
adipogenesis in 3T3-L1 cells and activate PPARγin a reporter
gene assay (35). We demonstrated that UOG wastewater samples
exhibited significant triglyceride accumulation and/or pre-
adipocyte proliferation at relative water concentrations as low as
0.001x, UOG-impacted surface water extracts at concentrations
as low as 0.04x, and the 23-mix at 1 µM; these effects co-occurred
with PPARγactivation for some samples but not others (35),
suggesting differing mechanisms. Related work demonstrated
highly efficacious triglyceride accumulation for various non-ionic
alkylphenol and alcohol polyethoxylates in the absence of PPARγ
activation and potentially mediated by TR antagonism (272).
These compounds are reportedly found at high concentrations
in UOG wastewater (273275) and may be responsible for some
of the observed non-PPARγ-mediated effects observed in the
UOG samples.
The costs and time investments associated with in vivo
examination of putative metabolism disruptors are prohibitively
high; as such, utilizing lower-order testing and screening is
critical to target higher-order testing on chemicals most likely to
be active. Application of numerous in vitro models for assessing
putative “obesogens” or “metabolic disruptors” over the last
several decades has revealed numerous contaminants capable
of affecting metabolic health (18), with recent publications
suggesting that these contaminants are likely common in
indoor and outdoor environments (2,184,272). While
these pre-adipocyte and mesenchymal stem cell models are
useful in determining potential in vivo metabolic disruptors,
they are also time and energy intensive and their relative
abilities to correctly identify chemicals may depend on both
cell line and source. Further, their mechanisms of assessing
adipogenic commitment, adipocyte differentiation, adipocyte
proliferation, and/or lipid accumulation may not capture
the full spectrum of endpoints that compose metabolic
dysfunction more broadly, particularly endpoints related to
“diabetogens”. As such, there is a critical need to develop
better methods for correctly predicting metabolic disruptors,
and while more simplistic models such as activation of PPARγ
are often applied, the vast suite of mechanisms influencing
this process (discussed above) require a more holistic approach
to integrating causative molecular mechanisms. Several high-
throughput (HTP) screening programs now exist (Tox21,
ToxCast) that report activity across numerous molecular
mechanisms for thousands of chemicals, many that are known
to be relevant to metabolic health. Harnessing these data
sets to broadly assess high-scoring chemicals (across relevant
molecular pathways for select endpoints of interest) for more
targeted higher-order testing may provide a valuable tool for
reducing time and research costs and achieving a more broad
assessment of the tens of thousands of commercial chemicals
for potential contribution to adverse health outcomes in humans
and/or animals.
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Kassotis and Stapleton Mixtures and Mechanisms of Metabolic Disruption
This issue of utilizing HTP data in predictive models is
not new and has been applied by a number of researchers
to various in vivo endpoints, with varying degrees of success
(276). Most of these methods have utilized ToxCast Phase
I data, due to the more recent release (October 2015) of
Phase II results, and as a result, some of the inherent issues
reported by these studies have since been addressed. For example,
Schwarzman et al. attempted to build a model to predict
breast carcinogens, though had insufficient data on particular
endpoints critical to altered mammary development (277). Many
of the pathways missing, including prolactin, progesterone, and
estrogen receptor beta effects, among others, are now pathways
with associated assays in the Phase II database. Russell et al.
applied a broad approach to predicting 60 in vivo endpoints, 56
of which were predicted at <55% accuracy (278), though notably
did not aggregate assays to predict in vivo endpoints. Given
that health outcomes are nearly always driven by overlapping
molecular pathways, this is not altogether surprising. Other
researchers utilized assay aggregation and were more successful
in building predictive models that performed with promising
accuracy (>70%). Martin et al. utilized a suite of ToxCast assays
to develop a predictive model for rat reproductive toxicity,
achieving 75% accuracies for training and test sets (279).
Notably, this model incorrectly predicted five of 21 external
validation chemicals as predicted negatives, all of which reduced
early offspring survival with limited accompanying effects on
reproductive performance or reproductive tract development,
suggesting a gap in assays targeting these endpoints. Another
model applied ToxCast data to rat prenatal developmental
toxicity, with >70% accuracy with species-specific models
(280), and found that if they further refined this to more
specific developmental outcomes, they got even better predictive
success (80–90%). Liu et al. utilized both Phase I and Phase
II data to predict hepatotoxicity (hypertrophy, injury, and
proliferative lesions), and reported 53–61% accuracy using only
Phase I data, but >80% when utilizing the expanded Phase II
data (281).
Recently, Auerbach et al. presented predictive models of
putative obesogenic and/or diabetogenic chemicals through
analyzing ToxCast HTP results (282). The researchers,
utilizing experts in a diversity of metabolic health disciplines,
selected known molecular pathways that had been previously
demonstrated to modulate metabolic health, and combined
them into a combined score metric for predicting likely vs.
less-likely metabolic disrupting chemicals. Janesick et al.
recently tested a portion of this method, utilizing a suite of
assays deemed relevant for adipocyte differentiation (16 assays
across 8 molecular mechanisms) to assess 24 chemicals (11
with highest activation scores across the selected assays, 6
with medium activation scores, and 7 presumed negative
controls with low activation scores) for activation of RXRα,
PPARγ, and triglyceride accumulation in 3T3-L1 cells
(47). They reported that 7 of 17 high and medium-scoring
and 2 of 7 low-scoring chemicals were active in 3T3-L1
cells, suggesting poor predictivity (high rates of both false
positives and false negatives). The authors suggested several
potential hypotheses for the poor performance, including:
poor performance of PPARγassays, incorrect selection of
assays for the predictive model, and improper weighting of
endpoints (rather than based on mechanism importance)
and assays within each endpoint (rather than based on assay
We recently undertook an effort to improve the predictive
utility of this model by expanding the pathways and attempting
to incorporate some of the suggestions made by Janesick
et al. (47). Among these, we expanded the outcome by
performing a targeted literature search on all chemicals and
any evidence of effects on metabolic health. This model
performed best when used as a gross metabolic disruption
prediction model, using literature searches to identify any in vitro
or in vivo evidence of adipogenesis or disrupted metabolic
health (weight gain, adipose development, insulin/glucose
signaling, effects on appetite/satiety, etc.). When applied
to a novel set of chemicals for which we had assessed
adipogenic activities in 3T3-L1 cells (60,184), the original
prediction model performed well at predicting gross metabolic
disruption; we observed low rates of both false negatives
(7.9%) and false positives (7.9%), and an apparent accuracy of
84% (283).
We also attempted to bolster this model through inclusion
of additional pathways known to modulate metabolic health,
in hopes of reducing false negatives, though discovered that
expanding the model to incorporate all of these pathways would
produce an inappropriately large and unwieldy model with a
considerably inflated false positive detection rate. Nonetheless,
we determined that additional pathways could be incorporated
into the model if there were a better method for de-selecting
less important or artifactual pathways. Z score corrections were
designed to address this by removing the bioactivities nearest
cytotoxicity as presumed false negatives/non-specific effects. In
our analysis, utilizing the cytotoxicity-derived z score values to
remove putative cytotoxicity-impacted pathways was effective at
reducing false positives, but at the expense of increasing false
negatives. We determined that utilizing Z score corrections (even
with a low threshold) was not an effective option to clarify
important pathways and reduce false positives.
Results from these publications suggest that further
improvements should focus on bolstering molecular pathways
with poor-performing assays or where replicate experiments
and/or assays are not available for a given endpoint within
ToxCast. Ensuring data integrity and robustness is of profound
importance to correct predictions. Efforts such as this have
tremendous putative utility, as screening all chemicals and
mixtures of chemicals for all endpoints is not feasible, and
determining a screen of HTP assays could save tremendous
time and cost and allow for a dramatically narrowed scope
of testing in vivo. Further testing is required to substantiate
this adipogenic prediction model for predicting in vivo
metabolic disruption across a larger chemical space, but these
preliminary results and success with other complex biological
effects demonstrate a clear potential for implementation into
predicting metabolic disruption and potentially helping reduce
and better target in vitro and in vivo chemical assessments in
the future.
Frontiers in Endocrinology | 14 February 2019 | Volume 10 | Article 39
Kassotis and Stapleton Mixtures and Mechanisms of Metabolic Disruption
CK and HS planned and outlined the proposed review. CK wrote
the review, and HS read and bolstered the review via feedback
and guidance.
Project supported by a grant (R01 ES016099) and a
fellowship (F32 ES027320; CK) from the National Institute
of Environmental Health Sciences.
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