Guggulsterone Activates Adipocyte Beiging through Direct Effects on 3T3-L1 Adipocytes and Indirect Effects Mediated through RAW264.7 Macrophages

Background: Plant-derived phytochemicals have been of emerging interest as anti-obesity compounds due to their apparent effects on promoting reduced lipid accumulation in adipocytes. Despite such promising evidence, little is known about the potential mechanisms behind their anti-obesity effects. The aim of this study is to establish potential anti-obesity effects of the phytochemical guggulsterone (GS). Methods: Mature 3T3-L1 adipocytes were treated with GS, derived from the guggul plant native in northern India, to investigate its effects on mitochondrial biogenesis and adipocyte “beiging.” Further, to explore the relationship between macrophages and adipocytes, 3T3-L1s were treated with conditioned media from GS-treated RAW264.7 macrophages. Markers of mitochondrial biogenesis and beiging were measured by western blot. Results: GS treatment in adipocytes resulted in increased mitochondrial density, biogenesis (PGC1α and PPARγ), and increased markers of a beige adipocyte phenotype (UCP1, TBX1, and β-3AR). This upregulation in mitochondrial expression was accompanied by increases oxygen consumption. In GS-treated macrophages, markers of M2 polarization were elevated (e.g., arginase and IL-10), along with increased catecholamine release into the media. Lastly, 3T3-L1 adipocytes treated with conditioned media from macrophages induced a 167.8% increase in UCP1 expression, suggestive of a role of macrophages in eliciting an anti-adipogenic response to GS. Conclusions: Results from this study provide the first mechanistic understanding of the anti-obesity effects of GS and suggests a role for both direct GS-signaling and indirect stimulation of M2 macrophage polarization in this model.


Introduction
Obesity is a complex disease associated with a pathological expansion of white adipose tissue. Adipose tissue has been traditionally classified into the energy-storing white adipose tissue and energy-dissipating, thermogenic brown adipose tissue. In specific environmental conditions, such as elevated beta-adrenergic signaling, white adipose tissue can upregulate mitochondrial biogenesis, uncoupling protein-1 (UCP1) expression, and respiration [1]. This change in the metabolic potential has been termed "beiging" both because of the browning in color of adipocytes in addition to a phenotype that is more metabolically active than white adipose but not to the extent of brown adipose tissue. . 3T3-L1 cells were grown to confluence and induced to differentiate using differentiation media I (DM I) containing DMEM/F12 GlutaMAX with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), 1% PS, 5 µM dexamethasone (Dexa), 1 mg/mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 µM rosiglitazone (Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained in DM I for 3 days followed by another 4-6 days in DM II which contained DMEM/F12 GlutaMAX with 10% FBS, 1% PS, 1 mg/mL insulin, and 1 µM Dexa. Media was changed every other day during the process of differentiation and by day 8, 90-95% of cells were mature adipocytes with lipid droplets. Adipocyte-specific assays are described in the following sections.

Lipid Quantification
Lipid droplets were stained in mature adipocytes treated with varying doses of GS (6-25 µM) or 0.1% DMSO vehicle control during the differentiation period using the AdipoRed™ reagent (Lonza Inc., Walkersville, MD, USA) according to manufacturer's protocol. The microplate was read using the HT Synergy (BioTek, Winooski, VT, USA) microplate reader.

Cell Viability
Mature adipocytes and RAW264.7 macrophages were treated with GS or 0.1% DMSO vehicle control for 24 and 48-h, respectively. Cell viability was measured after treatment using Prestoblue®Cell Viability Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer's protocol. Absorbance of metabolically active cells was measured one hour after incubation using an HT Synergy microplate reader at 570 nm.

Oxygen Consumption
Mature adipocytes were treated with GS (25 µM) and isoproterenol (10 µM), or 0.1% DMSO vehicle for 24 h. The Oxygen Consumption/Glycolysis Dual Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) was used to detect oxygen consumption following manufacturer's protocols. Fluorescence of the MitoXpress®Xtra reagent was measured using the HT Synergy microplate reader.

Immunoblot Analysis
Following treatment with GS for 24 or 48 h, cells were suspended with ice cold RIPA Lysis and Extraction buffer, complete with protease and phosphatase inhibitors (Thermo Fisher Scientific, Grand Island, NY, USA). Whole cell lysate was prepared by centrifuging cells for 10 minutes at 13,300× g. Protein estimation was then determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Grand Island, NY, USA). Proteins were separated on discontinuous SDS 4-20% polyacrylamide gels and transferred onto a polyvinylidene difluoride (PVDF) membrane using a Trans-blot Turbo system (Bio-Rad, Hercules, CA, USA) and then blocked for one hour with Tris buffered saline plus 0.1% Tween 20 (TBS-T) containing 5% bovine serum albumin (BSA). Following blocking, blots were then incubated for one hour in TBS-T plus 5% BSA with primary antibodies. Blots were incubated with secondary antibodies conjugated to IRDye 800 and developed using the Odyssey CLX imaging system (LiCor Biosciences, Lincoln, NE, USA). Signal intensity was determined for each band using the LiCOR imaging system software (Image Studio Ver. 5.2, LiCor Biosciences, Lincoln, NE, USA). For each protein of interest, the density value was normalized to the corresponding density of the loading control to obtain the integrated density values. These values were then normalized to control samples run on the same gel.

Catecholamine Assay
Following 48-h incubation with GS in RAW264.7 cells, the culture supernatant was removed to quantify catecholamine release. Catecholamine levels were determined using ELISA according to manufacturer's instructions (Blue Gene for Life Science, Shanghai, China). The assay plate was read on a HT Synergy microplate reader.

Interleukin-10 (IL-10) ELISA
Following 24-h incubation with GS, the culture supernatant was removed to quantify IL-10 secretion. IL-10 levels were determined using ELISA according to manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). The assay plate was read on a HT Synergy microplate reader at 450 nm.

Data Analysis
All data was normalized to control and expressed as the mean ± S.E.M. Either t-tests or Dunnett's multiple comparisons test was used to determine difference between treatment groups and control where appropriate. Statistical analysis and graphs were made using GraphPad Prism (version 6.07; La Jolla, CA, USA). Statistically significant differences are defined at p < 0.05.

Effect of GS on 3T3-L1 Viability and Apoptosis
Mature 3T3-L1 adipocytes were cultured with GS (6, 12.5, 25 μM) for 24-hours. Following treatment, there was no impact of GS on cell viability ( Figure 2A) or caspase-3 activation ( Figure 2B), indicating that GS does not produce cytotoxic effects in adipocytes. Figure 2. GS treatment does not impact viability of 3T3-L1 adipocytes. GS treatment for 24 h in mature, 3T3-L1 adipocytes has no effect on cell viability (A) or apoptosis as measured by caspase 3 (B). Data presented as mean ± SEM from n = 3-6 replicates per group.

Effect of GS on Mitochondrial Biogenesis and Oxygen Consumption
Mitochondria were quantified following a 24-hour treatment with GS through fluorescence intensity of Mitotracker staining. Mitochondrial density increased by 7.2 ± 2.3% (p < 0.05) at the 25 μM dose ( Figure 3A), which resulted in a significant upregulation of oxygen consumption compared to untreated cells (210 ± 23.9%, p < 0.001; Figure 3B). GS at 25 μM also increased the concentration of PGC1α (105.7 ± 22.2%, p < 0.05; Figure 3C) compared to control, DMSO-treated cells, and a small increase in PPARγ was observed, but did not reach significance (p = 0.07; Figure 3D). GS at the 6 μM

Effect of GS on 3T3-L1 Viability and Apoptosis
Mature 3T3-L1 adipocytes were cultured with GS (6, 12.5, 25 µM) for 24-h. Following treatment, there was no impact of GS on cell viability ( Figure 2A) or caspase-3 activation ( Figure 2B), indicating that GS does not produce cytotoxic effects in adipocytes.

Effect of GS on 3T3-L1 Viability and Apoptosis
Mature 3T3-L1 adipocytes were cultured with GS (6, 12.5, 25 μM) for 24-hours. Following treatment, there was no impact of GS on cell viability ( Figure 2A) or caspase-3 activation ( Figure 2B), indicating that GS does not produce cytotoxic effects in adipocytes.

Effect of GS on Mitochondrial Biogenesis and Oxygen Consumption
Mitochondria were quantified following a 24-hour treatment with GS through fluorescence intensity of Mitotracker staining. Mitochondrial density increased by 7.2 ± 2.3% (p < 0.05) at the 25 μM dose ( Figure 3A), which resulted in a significant upregulation of oxygen consumption compared to untreated cells (210 ± 23.9%, p < 0.001; Figure 3B). GS at 25 μM also increased the concentration of PGC1α (105.7 ± 22.2%, p < 0.05; Figure 3C) compared to control, DMSO-treated cells, and a small increase in PPARγ was observed, but did not reach significance (p = 0.07; Figure 3D). GS at the 6 μM

Effect of GS on Mitochondrial Biogenesis and Oxygen Consumption
Mitochondria were quantified following a 24-h treatment with GS through fluorescence intensity of Mitotracker staining. Mitochondrial density increased by 7.2 ± 2.3% (p < 0.05) at the 25 µM dose ( Figure 3A), which resulted in a significant upregulation of oxygen consumption compared to untreated cells (210 ± 23.9%, p < 0.001; Figure 3B). GS at 25 µM also increased the concentration of PGC1α (105.7 ± 22.2%, p < 0.05; Figure 3C) compared to control, DMSO-treated cells, and a small increase in PPARγ was observed, but did not reach significance (p = 0.07; Figure 3D). GS at the 6 µM concentration failed to increase PGC1α, however the dose increased PPARγ compared to control (22.9 ± 6.1%; p < 0.01).

Discussion
The current study expands on the mechanistic understanding of the anti-obesity effects of GS in adipocytes in vitro. Mature 3T3-L1 adipocytes treated with GS demonstrated increased markers of mitochondrial biogenesis and UCP1 expression, indicative of an induction of a beiging phenotype. As adipose tissue contains macrophages, in addition to adipocytes, this study also explored the effects of GS on RAW264.7 cells. Macrophages treated with GS increased arginase expression, stimulated the secretion of IL-10, an inflammatory cytokine, and upregulated catecholamine release into the media indicative of M2 polarization. Further, conditioned media from GS-treated macrophages were sufficient enough to induce UCP1 expression in adipocytes.
GS has been proposed to be an anti-obesity phytochemical. In vitro treatment of GS results in reduced lipid accumulation, partly by inducing apoptotic pathways [25] and reducing adipogenic signaling in differentiating adipocytes [16,17]. Herein, we demonstrate that a novel anti-obesity property of GS is the induction of beiging. GS shares a structural similarity to many of the steroid hormones and has both agonistic and antagonistic capabilities on a number of steroid receptors including the Takeda G-protein-coupled receptor 5 (TGR5) bile acid receptor, estrogen receptor, and the progesterone receptor. Hence, it is likely that the reason that GS has been suggested to target diseases ranging from cancer to cardiometabolic health is attributable to its ability to interact with a broad spectrum of steroid receptors. Sex hormones have been hypothesized to influence the thermogenic potential of brown adipose tissue, with both estrogen and progesterone having positive effects on UCP1 expression [26]. Unsurprisingly, activation of estrogen receptor alpha in 3T3-L1 adipocytes induces expression of UCP1 and TBX1 through phosphorylation of AMP-activated protein kinase (AMPK) [27], which is a critical sensor of energy balance and an upstream regulator of UCP1 [28]. Further, activation of the TGR5 bile acid receptor can independently induce thermogenesis in brown adipose tissue [21,29]. TGR5 signaling upregulates DIO2 expression, which may increase the UCP1 expression necessary for thermogenesis. Together, this suggests that GS may induce beiging in adipocytes through several potential signaling cascades, which could have been responsible for the upregulation of UCP1 and oxygen consumption observed in the current study. However, this also makes it difficult to understand the specific signaling mechanisms by which GS may be working through in adipocytes.
While we are unable to explain how GS directly induces a beige phenotype in the 3T3-L1 adipocyte, we sought to explore if signals from other peripheral cell types in adipose tissue may influence this phenomenon. Specifically, macrophages can be found in concentrations of upwards of 40% of the cell types in adipose tissue from obese subjects [11] and thus, makes macrophages an important target for anti-obesity compounds. A small subset of studies exist that investigate the effects of GS on macrophages. Perhaps most relevant, a recent study by Che et al. [30] reported that treatment of macrophages with GS in vitro increased markers of M2 polarization in RAW264.7 cells, primary mouse macrophages, and human monocytes. M2 polarization of colonic macrophages was also found in vivo. A shift away from the M1 phenotype in RAW264.7 cells have also been reported in other work [22,31], and together, is supportive of the findings in the current study and the potential for GS to induce beiging through stimulation of resident macrophages.
M2 macrophage polarization occurs during the Th2 response, is activated by IL-4 and glucocorticoids, and release cytokines such as transforming growth factor-β (TGF-β) which can orchestrate the tissue remodeling process. M2 polarization, purportedly as a result of IL-4 stimulation, may also increase norepinephrine production and release [32]. It is hypothesized that cold-induced sympathetic nervous system stimulation induces tyrosine hydroxylase enzyme activity in macrophages, thereby increasing norepinephrine release capable of white adipose tissue beiging. This response has been demonstrated in several rodent models [12], however additional work has recently questioned the relevance of alternatively activated macrophages in adipose tissue [33]. In Fischer et al., macrophage-specific knockout of tyrosine hydroxylase had no impact on body temperature during a cold challenge [33]. Further, in vitro IL-4 stimulation of bone marrow-derived macrophages also failed to induce an adipocyte thermogenic profile in conditioned media experiments, and in vivo IL-4 treatment failed to increase energy expenditure in mice. This is in conflict with the findings in the current study, however, the discrepancies may be related to differences in methodology.
The current study utilized the 3T3-L1 adipocyte line with a differentiation media that contained thyroid hormone, which is important for producing an adipocyte sensitive to UCP1 upregulation [23]. Unlike the aforementioned study by Fischer et al. (2017), which only cultured for 24 h [33], adipocytes in the current study were cultured in the conditioned media for 48 h. The additional time in a primed cell may partially explain our results. Further, it is plausible that continued direct GS signaling from the macrophage-media source may have contributed to the observed UCP1 upregulation. Thus, it is reasonable to hypothesize that it may be necessary to have both systemic and resident macrophage-directed adrenergic signaling to stimulate adipocytes. Lastly, the studies in Fischer et al., failed to see changes in metabolic outputs in mice with deficiencies in either tyrosine hydroxylase or IL-4 [33]. In fact, it may be that alternatively activated macrophages contribute, but are not a primary factor to beiging in vivo. Obesity-related macrophage infiltration in the adipose tissue may further add to this contribution, as what was recently shown in cultured adipocytes treated with conditioned media from primary macrophages obtained from obese donors [34].
The pharmacologic translation of in vitro studies to in vivo remains a challenge in assessing the efficacy of phytochemicals to act as attenuators of disease. In the current study, we used the trans (E)-form of GS for dosing, which we have previously demonstrated has less lipolytic potential than the cis (Z)-form [17]. However, the cis (Z)-form is more cytotoxic [17], which attributed to the selection of the trans (E)-form herein as we wanted to avoid the confounding factor of cell death from influencing the results. It remains plausible that these two isoforms may have differing potentials to beige in vitro, and considering the rapid metabolism of GS in vivo, may also vary widely in a whole-body system. The Cmax for GS is 0.3 µM in rats [35], hence an important limitation of the current study is the supraphysiological doses used. Nonetheless, GS has demonstrated synergistic properties with other phytochemcials such as genistein [18], which would permit for the plausibility of a photochemical blend that contained more physiologic relevant concentrations. Furthermore, hypolipidemic and anti-diabetic effects of GS were demonstrated in humans [36] and rodents [19] suggesting potential beneficial effects of GS under in vivo conditions.

Conclusions
Herein, findings from this work demonstrate that the anti-obesity effects of GS include beiging of adipocytes in vitro. GS has direct activity on 3T3-L1 adipocytes, inducing mitochondrial biogenesis and an upregulation of UCP1 and cellular oxygen consumption, indicative of a beige phenotype. Further, GS appears to have the capability of promoting adipocyte beiging through a macrophage-dependent mechanism. In the current study, treatment with GS in RAW264.7 macrophages promoted M2 polarization and subsequent catecholamine release that was capable of upregulating UCP1 in 3T3-L1 adipocytes. While preliminary results indicate that GS-induced enhancement of beiging may be due to both direct and indirect signaling in cultured adipocytes (summarized in Figure 7). Further studies are required to demonstrate this phenomenon in vivo.

Conflicts of Interest:
The authors declare no competing interests.