*Jun Wu, University of Michigan, USA*

#### *Reviewed by:*

*Zachary Gerhart-Hines, University of Copenhagen, Denmark Sandra Kleiner, Regeneron Pharmaceutical, USA*

#### *\*Correspondence:*

*Sonia M. Najjar, College of Medicine and Life Sciences, University of Toledo, Health Science Campus, 3000 Arlington Avenue, Mail Stop 1009, Toledo, OH 43614, USA<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology*

> *Received: 19 May 2015 Accepted: 13 July 2015 Published: 03 August 2015*

#### *Citation:*

*Lester SG, Russo L, Ghanem SS, Khuder SS, DeAngelis AM, Esakov EL, Bowman TA, Heinrich G, Al-Share QY, McInerney MF, Philbrick WM and Najjar SM (2015) Hepatic CEACAM1 over-expression protects against diet-induced fibrosis and inflammation in white adipose tissue. Front. Endocrinol. 6:116. doi: 10.3389/fendo.2015.00116* CEACAM1 promotes insulin extraction, an event that occurs mainly in liver. Phenocopying global *Ceacam1* null mice (*Cc1–/–*), C57/BL6J mice fed a high-fat (HF) diet exhibited reduced hepatic CEACAM1 levels and impaired insulin clearance, followed by hyperinsulinemia, insulin resistance, and visceral obesity. Conversely, forced liverspecific expression of CEACAM1 protected insulin sensitivity and energy expenditure, and limited gain in total fat mass by HF diet in L-CC1 mice. Because CEACAM1 protein is barely detectable in white adipose tissue (WAT), we herein investigated whether hepatic CEACAM1-dependent insulin clearance pathways regulate adipose tissue biology in response to dietary fat. While HF diet caused a similar body weight gain in L-CC1, this effect was delayed and less intense relative to wild-type (WT) mice. Histological examination revealed less expansion of adipocytes in L-CC1 than WT by HF intake. Immunofluorescence analysis demonstrated a more limited recruitment of crown-like structures, and qRT-PCR analysis showed no significant rise in TNFα mRNA levels in response to HF intake in L-CC1 than WT mice. Unlike WT, HF diet did not activate TGF-β in WAT of L-CC1 mice, as assessed by Western analysis of Smad2/3 phosphorylation. Consistently, HF diet caused relatively less collagen deposition in L-CC1 than WT mice, as shown by Trichrome staining. Coupled with reduced lipid redistribution from liver to visceral fat, lower inflammation and fibrosis could contribute to protected energy expenditure against HF diet in L-CC1 mice. The data underscore the important role of hepatic insulin clearance in the regulation of adipose tissue inflammation and fibrosis.

**Keywords: insulin clearance, liver-adipose tissue axis, insulin resistance, steatosis, CEACAM1, fibrosis, inflammation**

**Abbreviations:** α-Sma, alpha-smooth muscle actin; ApoB48/ApoB100, apolipoprotein B; Col6α3; alpha 3 chain of collagen 6 also known as endotrophin; *Cc1–/–*, global *Ceacam1* null mouse; CEACAM1, carcinoembryonic antigen-related cell adhesion molecule 1; *Ceacam1*, gene encoding CEACAM1 protein; Fasn, fatty acid synthase; FAT/CD36, fatty acid translocase/cluster of differentiation protein 36; FATP, fatty acid transporter protein; HF, high-calorie high-fat diet; Hsl, hormone-sensitive lipase; L-CC1, mice with liver-specific over-expression of wild-type CEACAM1; Lpl, lipoprotein lipase; L-SACC1, liver-specific S503A CEACAM1 mutant mouse; NEFA, non-esterified fatty acids; RD, regular diet; SREBP-1c, sterol regulatory element-binding protein; TNFα, tumor necrosis factor alpha; TGF-β transforming growth factor beta.

### **Introduction**

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes insulin extraction in liver to regulate insulin action. This finding is bolstered by the observation that mice with liver-specific inactivation of CEACAM1 (L-SACC1) or with global null deletion of Ceacam1 gene (*Cc1–/–*) manifest hyperinsulinemia secondary to impaired insulin clearance. This causes insulin resistance in addition to hepatic steatosis (1–3). The latter is mediated by the transcriptional activation of lipogenic genes by sterol regulatory element-binding protein (SREBP-1c) in response to chronically elevated levels of insulin (4). It can also be caused by blunted CEACAM1-mediated downregulation of fatty acid synthase (Fasn) activity under hyperinsulinemic conditions (5).

Both L-SACC1 and *Cc1−/−* mutant mice also display visceral obesity (1–3). Contributing to this metabolic anomaly, is increased redistribution of hepatic triacylglycerol to white adipose tissue (WAT), in particular that mice are propagated on the C57/BL6J genetic background, which favors redistribution to adipose tissue (6).

We have recently shown that high-fat (HF) intake causes insulin resistance in part by reducing CEACAM1 mRNA and protein levels in liver (7). Conversely, transgenically protecting hepatic CEACAM1 in L-CC1 mice prevented diet-induced insulin resistance and limited hepatic steatosis in response to HF diet (7). Additionally, it blunted the negative effect of HF diet on energy expenditure and spontaneous locomotor activity. This protective effect of hepatic CEACAM1 gain-of-function could be mediated, at least in part, by inducing FGF21, which in turn, enhances fatty acid oxidation in liver (8, 9) and increases brown adipogenesis (10, 11).

 Because CEACAM1 is barely detectable at the protein level in adipose tissue (12), we hypothesized that extra-adipocytic factors brought about by overexpressing CEACAM1 in liver are involved. To test this hypothesis, we investigated whether altered CEACAM1 dependent insulin clearance pathways regulate adipose tissue biology in response to HF diet.

### **Materials and Methods**

#### **Mice Generation**

As recently described (7), L-CC1 transgenic mice with liverspecific over-expression of FLAG-tagged WT rat CEACAM1 were generated using human apolipoprotein A1 (APOA1) promoter/enhancer element (13), and propagated on the C57/BL6J background (Jackson Laboratories), as described (7). The "minigene" construct was obtained by subcloning the proximal APOA1 promoter 5*′* of a *Ceacam1* rat minigene-containing intron 1 into pCMV-3Tag-3A plasmid at *Not1* site.

Starting at 2 months of age, male mice were fed *ad libitum* either a standard diet (12:66:22% calories from fat:carbohydrate:protein) or a HF diet (45:35:20% calories from fat:carbohydrate:protein) (Catalog #D12451, Research Diets). Mice were kept in a 12-h-dark/light cycle. This study was carried out in accordance with the recommendations of the Institutional Animal Care and Utilization Committee (IACUC), which approved all procedures.

#### **Metabolic Parameters**

Mice were fasted overnight and their retro-orbital venous blood drawn at 11:00 hours the following day. Plasma was stored at *−*80°C until levels of insulin (Linco Research), FFA (NEFA C, Wako), and triacylglycerol (Pointe Scientific Triglyceride, Canton) were assessed. Hepatic triacylglycerol content was measured as previously described (1, 3).

#### **Body Composition**

Whole body composition was assessed by nuclear magnetic resonance (NMR; Bruker Optics).

#### **Immunofluorescence of Visceral White Adipose Tissue**

Visceral WAT was formalin-fixed, cut into 2–3 mm sections, and transferred to Dulbecco's phosphate buffered saline (PBS, Sigma Aldrich) for 48 h at 4°C. Adipose tissue was permeabilized in 1% Triton X-100 (Fisher) in PBS for 10 min before being stained with rat anti-mouse F4/80 (Invitrogen, Carlsbad) to mark macrophages and detected with donkey anti-rat IgG conjugated to Alexa Fluor488 (Invitrogen). Tissues were incubated with primary antibodies overnight at room temperature (RT) in the dark and washed 3*×* with staining buffer before being subjected to secondary stain for 2 h and wash (3*×*) in PBS. Stained samples were then counterstained for 15 min at RT with 5 μM BODIPY 558/568 (Molecular Probes, Inc.) to visualize lipid. Adipose tissue samples were placed directly on a coverslip with buffer and visualized.

#### **Laser-Scanning Confocal Microscopy**

Samples were imaged using a Leica TCS SP5 laser-scanning microscope (Leica Microsystems) equipped with conventional solid state and a Ti-sapphire tunable multi-photon laser (Coherent). Images were acquired in the 3D XYZ plane in 2.5 μm steps with a 20*×* objective (NA 0.70) using the sequential scan mode to eliminate any spectral overlap in the individual fluorophores. Specifically, AlexaFluor488 (Invitrogen) was excited at 488 nm with collection at 500–558 nm. The BODIPY 558/568 dye (Molecular Probes, Inc.) was excited at 561 nm and collected at 567–609 nm. Selected images are a 2D representation of the 3D laser-scanning confocal microscopy (LSCM) image *z*-stack, as labeled.

#### **Gomori's Trichrome Staining**

Adipose tissue (*n* = 5 per mouse group) was fixed in 10% formalin and replaced by 70% ethanol before undergoing blocking in paraffin. Sections were deparaffinized at 60°C and hydrated in deionized water. Antigens were unmasked in Bouin's Fluid at 56°C for 45 min. Upon rinsing in deionized water, nuclei were stained with Working Weigert's Iron Hematoxylin at RT for 10 min. Trichrome stain was performed using the Thermo Scientific Richard-Allan Scientific Chromaview-advanced Testing (Cat. No. 87020). In brief, rinsed slides were trichrome stained at RT for 15 min and dehydrated sequentially in 1% acetic acid solution for 1 min, 95% ethanol for 30 s, and 100% ethanol for 1 min (twice). Sections were then cleared in three changes of clearing reagent for 1 min each and mounted.

#### **Western Analysis**

Tissues were lysed and proteins analyzed by SDS-PAGE followed by immunoprobing with polyclonal antibodies against Fasn (α-Fasn) (Assay Designs), fatty acid translocase/cluster of differentiation protein 36 (FAT/CD36) (Santa Cruz Biotechnology), apolipoprotein B (Chemicon International), and total and phospho-Smad 2 and Smad 3 (Cell Signaling). For normalization, monoclonal antibodies against α-actin (Santa Cruz) were used. Blots were incubated with horseradish peroxidase-conjugated anti-goat IgG (Santa Cruz Biotechnology), anti-mouse or antirabbit IgG (Amersham) antibodies, and proteins detected by enhanced chemiluminescence (ECL; Amersham) prior to quantification by densitometry (Image J software).

#### **Semi-Quantitative Real-Time RT-PCR**

Total hepatic RNA was isolated with PerfectPure RNA Tissue Kit (5*′* ), and total adipose tissue RNA was isolated with RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer's protocol. cDNA was synthesized by ImProm-II™ Reverse Transcriptase (Promega), using 1 μg of total RNA and oligo dT primers (**Table 1**). cDNA was evaluated with real-time quantitative PCR (Step One Plus, Applied Biosystems). The relative amounts of mRNA were calculated by comparison to the corresponding standards and normalized relative to 18S.

#### **Statistical Analysis**

Data were analyzed with SPSS software by two-way analysis of variance (ANOVA) or two-tailed Student's *t*-test with GraphPad Prism 4 software. *P <* 0.05 was statistically significant.

## **Results**

#### **Lipid Metabolism**

As expected, HF diet caused hyperinsulinemia and fed hyperglycemia (**Table 2**), markers of insulin resistance, in WT mice. In support of the positive effect of hyperinsulinemia on lipogenesis (4), qRT-PCR (**Table 3**) and Western blot analysis showed increased mRNA and protein levels of Fasn, respectively, in the liver of HF-fed WT mice relative to mice fed a regular diet (RD) (**Figure 1A**). HF diet also induced hepatic mRNA (**Table 3**) and protein levels of CD36 fatty acid translocase (**Figure 1A**), in addition to inducing the mRNA level of fatty acid transport protein-1 (Fatp-1) (**Table 3**). Together with increased lipogenesis, elevated lipid transport could contribute to increased hepatic triacylglycerol content in HF-fed WT mice (**Table 2**).

Increased production of hepatic triacylglycerol drives output, as supported by elevated plasma apolipoprotein B (ApoB48/ApoB100) protein levels in WT mice (**Figure 1B**). With fasting plasma triacylglycerol levels being intact (**Table 2**), it is likely that triacylglycerol was redistributed to the WAT, as expected from mice on the C57/BL6J genetic background that favors substrates partitioning to the adipose tissue (6). Supporting this notion, mRNA levels of Fatp-4 and lipoprotein lipase (Lpl) **TABLE 1 | Real-time PCR primer sequences from mouse genes**.

that are critical in lipid transport to the adipocyte were elevated in HF-fed relative to RD-fed WT mice (**Table 4**). This appears to drive adipogenesis, as suggested by higher Srebp-1c mRNA levels (**Table 4**), and ultimately, increased visceral obesity (**Table 2**). Elevated mRNA levels of hormone sensitive lipase (Hsl) (**Table 4**) and the rise in fasting plasma NEFA levels (**Table 2**) are consistent with increased lipolysis in HF-fed WT mice.

In L-CC1 mice, however, hepatic triacylglycerol content was neither modified by HF intake (**Table 2**) nor were mRNA levels of Fasn, Fatp-1, and Cd36 (**Table 3**). Consistent with normal hepatic lipogenesis, plasma ApoB48/ApoB100 protein levels in L-CC1 mice were not significantly altered by HF diet (**Figure 1B**). Moreover, HF did not modulate the mRNA level of genes involved in lipid metabolism in the adipocyte (Lpl, Fatp-4, and Srebp-1c) (**Table 4**). Consistent with normal fasting plasma NEFA (**Table 2**), HF did not alter Hsl mRNA levels in the WAT of L-CC1, as it did to WT mice (**Table 4**).

#### **Visceral Obesity**

High-fat feeding time dependently increased body weight gain in WT mice (**Figure 2A**). In contrast, it took *≥*6 weeks before HF induced a statistically significant body weight gain in L-CC1 (**Figure 2A**). NMR analysis (**Figure 2B**) revealed persistently lower fat mass in L-CC1 relative toWT mice under normal feeding conditions until ~5 months of age, at which point this difference became statistically insignificant (**Figure 2B**, starting at about 11 weeks of experimental feeding).

While HF feeding caused an increase in total fat mass relative to RD feeding in both mouse groups, this effect was delayed (at 5 vs. 3 weeks in L-CC1 vs. WT) (**Figure 2B**). Furthermore, fat accumulation in L-CC1 mice did not become significantly higher than RD-fed WT until after 7 weeks of HF feeding but remained lower than HF-fed WT even after 4 months of HF, as shown by NMR (**Figure 2B**), and by 1H-magnetic resonance spectroscopy (7).

#### **TABLE 2 | Effect of high-fat diet for 4 months on plasma and tissue biochemistry**.

*Mice (n > 8/feeding group/genotype) were fed an HF diet for 4 months starting at 2 months of age. Fat depots were excised and weighed. Intra-abdominal visceral adipose tissue fat refers to combined weight of mesenteric and gonadal fat. The relative mass of fat depots was expressed as percentage of body weight (% BW).*

*Values are expressed as mean ± SEM.*

*\*P < 0.05 HF vs. RD per genotype.*

*†P < 0.05 L-CC1 vs. WT per feeding group.*

#### **TABLE 3 | Effect of high-fat diet on mRNA levels of lipid metabolism genes in liver**.

*Tissue mRNA levels were analyzed in duplicate per mouse (n ≥ 6 mice/feeding group/genotype).*

*Values were normalized to 18S and expressed as mean ± SEM.*

*\*P < 0.05 HF vs. RD per genotype.*

*†P < 0.05 L-CC1 vs. WT per feeding group.*

#### **FIGURE 1 | Western analysis of proteins involved in hepatic lipid**

**homeostasis**. **(A)** Liver lysates from wild-type mice fed a regular (RD) or a high-fat diet (HF) for 4 months were analyzed by immunoblotting with α-Fasn and α-CD36 antibodies, followed by reprobing with α-actin for normalization. A representative gel of three different experiments performed on two mice per feeding group is included. **(B)** Plasma from both WT and L-CC1 mice was diluted and analyzed by 4–10% gradient SDS-PAGE and immunoblotting with an antibody against apolipoprotein B (ApoB), which recognizes both ApoB48 and ApoB100. A representative gel of two independent experiments performed on two different pairs of mice per feeding group per genotype is included.

*Mice (n > 5 per feeding group per genotype) were fed an HF diet for 4 months starting at 2 months of age.*

*Tissue mRNA levels were analyzed in duplicate per each mouse and normalized to 18S. Values are expressed as mean ± SEM.*

*\*P < 0.05 HF vs. RD per genotype.*

*†P < 0.05 L-CC1 vs. WT per feeding group.*

#### **Increased Macrophage Recruitment to White Adipose Tissue in Response to High-Fat Diet**

Histological analysis of H&E stained sections from WAT showed a significant expansion of adipocytes in both groups of mice in response to 4 months of HF intake, but to a lower extent in L-CC1 mice (**Figure 3**).

Moreover, immunofluorescence analysis of F4/80, a macrophage marker, showed multiple crown-like structures (CLS) containing macrophages in WAT from HF-fed WT mice (**Figures 4A,A'**, green). As previously shown (14), moving through the individual *z*-stack sections shows numerous small pieces of lipid inside the macrophages in CLS (**Figures 4A,A'**), indicating adipocyte degradation in HF-fed WT mice. In contrast, HF-fed L-CC1 mice exhibited minimal CLS formation with little evidence of lipids inside the macrophages in these structures. Consistently, HF diet-induced F4/80 mRNA levels by ~2.5-fold in WT as compared to ~1.5-fold in WAT of L-CC1 mice, and mRNA levels of TNFα adipokine in WT mice (by ~2-fold), but

high-fat (HF) diet and their body weight (grams) was weighed for 0–24 weeks. Values expressed as mean *±* SEM. \**P <* 0.05 HF vs. RD and †*P <* 0.05 L-CC1 vs. WT per feeding group. **(B)** Whole body fat mass was determined using NMR and expressed as percentage of total body weight (*n >* 10 mice/feeding group/genotype). Values expressed as mean *±* SEM. \**P <* 0.05 HF vs. RD, †*P <* 0.05 L-CC1 vs. WT per feeding group and §*P <* 0.05 HF-fed L-CC1 vs. RD-fed WT mice.

not in L-CC1 mice (**Table 4**). Together, this demonstrates that protecting hepatic CEACAM1 levels against HF diet limited the development of inflammation in the WAT of L-CC1 mice.

#### **Induction of Fibrosis and TGF-**β **Activation in White Adipose Tissue by High-Fat Diet**

In agreement with reports showing TNFα inhibition of the expression of Smad 7 (15), a negative regulator of TGFβ–Smad2/3 signaling pathway (16, 17), HF markedly repressed

(by ~5-fold) the mRNA level of Smad 7 in WT, but not L-CC1 mice (**Table 4**). Subsequently, HF induced Smad 2 and Smad 3 activation in the WAT of WT, but not L-CC1 mice, as assessed by Western analysis of Smad 2 and Smad 3 phosphorylation (**Figure 5A**).

In adipose tissue derived from WT, but not L-CC1 mice, HF induced significantly the transcript levels of two main players in fibrosis (**Table 4**): α-smooth muscle actin (α-Sma) (by *≥*10-fold) and endotrophin/Col6α3 (by ~2-fold), a protein that promotes metabolic derangement and fibrosis in adipose tissue (18). This translated into a higher induction of collagen deposition in the WAT of WT than L-CC1 mice by HF feeding, as shown by trichrome staining (**Figure 5B**).

#### **Discussion**

Phenocopying L-SACC1 mice with liver-specific inactivation of CEACAM1 and global *Cc1−/−* null mice (1–3), HF diet represses hepatic CEACAM1 levels to impair insulin clearance and cause hyperinsulinemia, which in turn, activates *de novo* lipogenic

pathways and elevates lipid production in liver (7). The current studies show that this drives lipid output (manifested by a rise in plasma ApoB100/ApoB48) and redistribution to WAT to provoke visceral obesity. This is consistent with a positive correlation between liver steatosis, hyperinsulinemia, and high plasma ApoB levels in humans and rodents (19–23). For instance, mice with conditional null mutation of the insulin receptor in liver exhibit impairment of insulin clearance and hepatic insulin resistance with elevated lipogenesis, and a rise in plasma ApoB100/ApoB48 levels in parallel to low plasma triacylglycerol levels and obesity (24).

In support of the important role that reducing hepatic CEA-CAM1 level plays in the pathogenesis of diet-induced metabolic derangement, protecting hepatic CEACAM1 levels by means of transgenic induction preserves insulin clearance and prevents insulin resistance and hepatic steatosis in response to HF feeding (7). The current studies show that it also causes a delay in the progression of fat accumulation and limits the expansion of adipocytes in response to HF diet. Together, this supports an important role for altered CEACAM1-dependent insulin clearance pathways in the pathogenesis of diet-induced hepatic steatosis and visceral obesity. These findings lend a mechanistic underpinning for the marked reduction of hepatic CEACAM1 observed in insulin-resistant obese subjects (25).

Immunofluorescence analysis showed less recruitment of CLScontaining macrophages to the WAT of L-CC1 and less adipocyte degradation inside these macrophages compared to WT mice. In addition to normal mRNA levels of TNFα, an adipokine that blunts insulin action (26), this could explain the protected insulin sensitivity along the liver-WAT axis (7).

In addition to inflammation (26), visceral obesity and insulin resistance are associated with increased fibrosis and activation of TGF-β signaling pathways inWAT in rodents (18, 27) and humans (28). Consistently, HF feeding induced more collagen deposition and activation of the TGF-β–Smad 2/3 pathway (16, 17) in WT than L-CC1 mice. This could prevent adipose tissue remodeling and contribute to the more metabolic derangement in HF-fed WT mice (29).

In summary, the current studies demonstrate that gain-offunction of CEACAM1 in liver restricts visceral obesity caused by HF diet, and that this is mediated, at least in part, by reducing lipid output from liver and limiting inflammation and fibrosis in WAT. Together with increased FGF21 production (7), this could mediate in part, preserved energy expenditure in L-CC1 mice (7, 28, 30). Because CEACAM1 protein is not produced to a significant extent in WAT (12), preserving adipose tissue biology could be attributed to extra-adipocytic factors caused by inducing CEACAM1 expression in liver. Although the underlying mechanisms are not fully delineated, the current studies provide a proof-of-principle of the importance of hepatic CEACAM1-dependent insulin clearance in the regulation of visceral obesity, and adipose tissue inflammation and fibrosis in response to HF diet.

### **Author Contributions**

SL researched data, designed experiments, and wrote a first draft of the manuscript, LR, SG, SK, EE, TB, GH, and QA researched data. AD designed and generated the targeting vector for the generation of the L-CC1 mouse line, screened and propagated the mouse line, and reviewed the manuscript. WP generated the L-CC1 mouse. MM participated in scientific discussions, in detecting CLS, and in the revision of the manuscript. SN was responsible for study design, conceptualization, data analysis and results interpretation, reviewing, and revising the manuscript. SN had full access to all the data of the study and takes responsibility for the integrity and accuracy of data analysis and the decision to submit and publish the manuscript.

#### **Acknowledgments**

This work was supported by grants from the NIH [R01 DK054254, R01 DK083850, and R01 HL112248 (to SN) and R15 DK103196 (to MM)]. The work was also supported in part by the Wolfe Innovation Fund (University of Toledo Foundation). SG is supported by the Middle East Diabetes Research Center.

#### **References**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Lester, Russo, Ghanem, Khuder, DeAngelis, Esakov, Bowman, Heinrich, Al-Share, McInerney, Philbrick and Najjar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Unlock the Thermogenic Potential of Adipose Tissue: Pharmacological Modulation and implications for Treatment of Diabetes and Obesity

*Xiao-Rong Peng1 \*, Peter Gennemark2 , Gavin O'Mahony3 and Stefano Bartesaghi1*

*1Cardiovascular and Metabolic Diseases IMED Biotech Unit, Diabetes Bioscience Department, AstraZeneca R&D, Mölndal, Sweden, 2Cardiovascular and Metabolic Diseases IMED Biotech Unit, Drug Metabolism and Pharmacokinetics Department, AstraZeneca R&D, Mölndal, Sweden, 3Cardiovascular and Metabolic Diseases IMED Biotech Unit, Medicinal Chemistry Department, AstraZeneca R&D, Mölndal, Sweden*

*Edited by:*

*Jun Wu, University of Michigan, USA*

#### *Reviewed by:*

*Bin Xu, Virginia Tech, USA Qin Yang, University of California Irvine, USA*

*\*Correspondence: Xiao-Rong Peng<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology*

*Received: 26 September 2015 Accepted: 28 October 2015 Published: 19 November 2015*

#### *Citation:*

*Peng X-R, Gennemark P, O'Mahony G and Bartesaghi S (2015) Unlock the Thermogenic Potential of Adipose Tissue: Pharmacological Modulation and Implications for Treatment of Diabetes and Obesity. Front. Endocrinol. 6:174. doi: 10.3389/fendo.2015.00174*

Brown adipose tissue (BAT) is considered an interesting target organ for the treatment of metabolic disease due to its high metabolic capacity. Non-shivering thermogenesis, once activated, can lead to enhanced partitioning and oxidation of fuels in adipose tissues, and reduce the burden of glucose and lipids on other metabolic organs such as liver, pancreas, and skeletal muscle. Sustained long-term activation of BAT may also lead to meaningful bodyweight loss. In this review, we discuss three different drug classes [the thiazolidinedione (TZD) class of PPARγ agonists, β3-adrenergic receptor agonists, and fibroblast growth factor 21 (FGF21) analogs] that have been proposed to regulate BAT and beige recruitment or activation, or both, and which have been tested in both rodent and human. The learnings from these classes suggest that restoration of functional BAT and beige mass as well as improved activation might be required to fully realize the metabolic potential of these tissues. Whether this can be achieved without the undesired cardiovascular side effects exhibited by the TZD PPARγ agonists and β3-adrenergic receptor agonists remains to be resolved.

Keywords: brown adipose tissue, thermogenesis, uncoupling protein 1, drug discovery, PPAR**γ** agonists, thiazolidinediones, **β**3-adrenergic receptor agonists, FGF21 analogs

## INTRODUCTION

According to the International Diabetes Federation (IDF), 8.3% of adults worldwide – 370 million people – have type-2 diabetes (T2D), and the number of people with the disease is set to rise beyond 592 million in under 25 years (1). Although there are many drugs available for diabetes, none of them safely and durably prevent or reverse disease progress and its associated comorbidities. Poor diet, sedentary lifestyle, and obesity are considered major risk factors for diabetes. Inappropriate fuel handling by adipose tissue, liver, and skeletal muscle, combined with ectopic lipid deposition in key metabolic organs (such as liver, pancreas, muscle, and heart) have been hypothesized to play a significant role in the development of insulin resistance. Insulin resistance increases the overall burden on β-cells, which over time leads to β-cell failure and development of T2D.

Adipose tissue can be grossly divided into two major depots, white adipose tissue (WAT) and brown adipose tissue (BAT). WAT stores excess energy as triglycerides (TGs), which can be mobilized by lipolysis to generate free fatty acids (FFAs) for use by other tissues. BAT, on the other hand, is the main site of non-shivering thermogenesis (NST), which requires a brown adipocyte-specific protein called uncoupling protein 1 (UCP1).

Non-shivering thermogenesis by BAT is an interesting target for the treatment of metabolic disease due to the high metabolic capacity of BAT. BAT is highly vascularized and richly innervated by sympathetic nerves, and its activation is predominantly regulated by the sympathetic nerve system via β-adrenergic receptors (β-ARs). Enhancing energy expenditure (EE) through activation of NST by β3-adrenergic receptor (β3-AR) agonists has been investigated as an alternative to inhibition of food intake for bodyweight loss. This has, however, been unsuccessful in human clinical trials. This lack of effect on EE was partly attributed to negligible BAT function in adult humans compared to the situation in rodents.

The rediscovery of BAT in the adult human in 2007, and the subsequent demonstration of functional involvement of human BAT in NST have revitalized this area (2–5). In addition, the presence of brown-like adipocytes in WAT [referred to as beige or brown-in-white (brite) adipocytes] further increased the interest in brown adipocyte biology, as WAT mass is relatively large and any increase in cellular energetics in this tissue may have a significant impact on whole-body metabolism and EE. The beige nomenclature will be used for this review. Utilization of FFAs during NST could lead to depletion of brown and or beige adipocytes' lipid stores, which may result in redistribution of fuels [including glucose and non-esterified fatty acids (NEFA)] toward brown and beige adipocytes. In turn, this could lead to a reduced fuel over-supply to other metabolic organs (heart, skeletal muscle, and liver) and, thus, improved insulin sensitivity (**Figure 1**).

also expressed in committed skeletal muscle precursors (19, 20). The developmental origin of the beige adipocyte remains to be elucidated. The whole-body NST will have contributions from

It is now beyond doubt that BAT is present in adult humans and plays a role in NST (3). BAT activity can be detected by 18F-fluorodeoxyglucose (18F-FDG) uptake using PET–CT (2, 4, 5). Retrospective analysis of populations that have undergone PET–CT examination indicates that the prevalence of BAT varies between 1 and 5%. For example, one study on 4011 asymptomatic individuals (<5% obese subjects) showed that BAT prevalence is 5% in female and 1.3% in male (21). The BAT-positive subjects had lower body mass index (BMI), less visceral and subcutaneous fat areas, lower fasting glucose and TG levels, and increased HDL cholesterol concentrations compared to the BAT-negative subjects. Similar results were reported for 56 healthy volunteers (22), and for cancer patients (2, 23). The inverse correlation between BAT activity and BMI was further confirmed in non-diabetic subjects over a wide range of body compositions (BMI ranging

) (4, 24–27). A recent retrospective analysis of 18F-FDG uptake data (analysis of the neck regions of two relatively large cohorts of individuals) reports that the average body weight of BAT-positive individuals is approximately 5 kg lower compared

Using a combination of MRI and molecular analysis, Enerbäck's group clearly demonstrated that iBAT in human infants consists of classical brown adipocytes (15). However, the molecular signature of brown adipocytes isolated from the neck regions of adult humans resembles that of the rodent beige phenotype rather than classical brown adipocytes (15, 28–31). What causes the BAT phenotype transformation between infant and adult humans is not understood. It is conceptually important to unravel the function, regulation, and differentiation of beige and classical brown adipocytes in order to be able to pharmacologically enhance thermogenesis in humans. For example, if the adipocytes with various white and beige appearances in adult human neck region are, in fact, simply dormant brown adipocytes, they may be readily re-activated by cold or sympathomimetics. In addition, it is important to understand the translational aspects of BAT biology, i.e., whether the same or different pharmacological agent(s) will show desirable effects in preclinical animal species and man?

BAT AND METABOLIC SIGNIFICANCE

The contribution of BAT to whole-body metabolism in rodents has recently been examined using tools such as radioactive tracers and PET–CT imaging. Bartelt et al. showed that BAT is the major site of triglyceride-rich lipoprotein (TRL) clearance during acute cold exposure (32). Cold exposure also dramatically increased the glucose disposal to BAT tissue. The remarkable capacity of BAT to take up substrates is illustrated by the ratio of BAT mass to the total glucose and TG uptake by BAT compared to that of other major organs in mice under cold challenge. Labbe et al. extended this observation through PET–CT analysis of the rate of substrate flux and oxidation in the iBAT of both warm- and cold-adapted rats (33). The rate of glucose uptake into iBAT was relatively low at 27°C but increased 10-fold upon acute cold exposure and increased 46-fold

both classical brown and beige adipocytes.

from 22 to 48 kg/m2

to that of BAT-negative subjects (21, 23).

Transcriptional and hormonal regulation of the "browning" program of adipose stem cells and characterization of the molecular signature have been reviewed extensively elsewhere (6–12). This paper focuses on the metabolic potential of BAT and beige adipocytes, how these systems can be manipulated by pharmacological means, and how to assess if a brown adipocyte phenotype has been achieved by pharmacological intervention. Finally, we discuss the challenges of drug discovery in this area by reviewing three classes of clinically investigated pharmacological agents that regulate various aspects of BAT and beige adipocyte function: thiazolidinedione (TZD) PPARγ agonists, β3-AR agonists, and fibroblast growth factor 21 (FGF21) analogs.

#### BROWN ADIPOSE TISSUE IN ADULT HUMANS

Typically, BAT is located in the interscapular (iBAT), cervical, auxiliary, perirenal and paraaortic areas of animals or human infants (13–15). In adult humans, BAT depots have a diffuse anatomic distribution, with mixtures of white and beige adipocytes, seeming to coexist in close proximity. Beige adipocytes have also been reported to arise in what are normally considered WAT depots (such as inguinal adipose in rodents) in response to various stimuli such as cold, TZDs, and β-AR agonists (9). In humans, beige adipocytes were found in the WAT of pheochromocytoma patients due to the presence of catecholamine-secreting tumors (16, 17) and in the subcutaneous adipose of severely burned patients where heat loss is increased and who experience prolonged adrenergic stress (18). Both lineage-tracing studies and transcriptional profiling of classical brown and beige adipocytes indicate that these two cell types seem to originate developmentally from distinct cell lineages. Classical brown adipocytes in interscapular BAT arise from precursors that are *myf5*+, a gene known to be

partitioning.

also expressed in committed skeletal muscle precursors (19, 20). The developmental origin of the beige adipocyte remains to be elucidated. The whole-body NST will have contributions from both classical brown and beige adipocytes.

It is now beyond doubt that BAT is present in adult humans and plays a role in NST (3). BAT activity can be detected by 18F-fluorodeoxyglucose (18F-FDG) uptake using PET–CT (2, 4, 5). Retrospective analysis of populations that have undergone PET–CT examination indicates that the prevalence of BAT varies between 1 and 5%. For example, one study on 4011 asymptomatic individuals (<5% obese subjects) showed that BAT prevalence is 5% in female and 1.3% in male (21). The BAT-positive subjects had lower body mass index (BMI), less visceral and subcutaneous fat areas, lower fasting glucose and TG levels, and increased HDL cholesterol concentrations compared to the BAT-negative subjects. Similar results were reported for 56 healthy volunteers (22), and for cancer patients (2, 23). The inverse correlation between BAT activity and BMI was further confirmed in non-diabetic subjects over a wide range of body compositions (BMI ranging from 22 to 48 kg/m2 ) (4, 24–27). A recent retrospective analysis of 18F-FDG uptake data (analysis of the neck regions of two relatively large cohorts of individuals) reports that the average body weight of BAT-positive individuals is approximately 5 kg lower compared to that of BAT-negative subjects (21, 23).

Using a combination of MRI and molecular analysis, Enerbäck's group clearly demonstrated that iBAT in human infants consists of classical brown adipocytes (15). However, the molecular signature of brown adipocytes isolated from the neck regions of adult humans resembles that of the rodent beige phenotype rather than classical brown adipocytes (15, 28–31). What causes the BAT phenotype transformation between infant and adult humans is not understood. It is conceptually important to unravel the function, regulation, and differentiation of beige and classical brown adipocytes in order to be able to pharmacologically enhance thermogenesis in humans. For example, if the adipocytes with various white and beige appearances in adult human neck region are, in fact, simply dormant brown adipocytes, they may be readily re-activated by cold or sympathomimetics. In addition, it is important to understand the translational aspects of BAT biology, i.e., whether the same or different pharmacological agent(s) will show desirable effects in preclinical animal species and man?

## BAT AND METABOLIC SIGNIFICANCE

The contribution of BAT to whole-body metabolism in rodents has recently been examined using tools such as radioactive tracers and PET–CT imaging. Bartelt et al. showed that BAT is the major site of triglyceride-rich lipoprotein (TRL) clearance during acute cold exposure (32). Cold exposure also dramatically increased the glucose disposal to BAT tissue. The remarkable capacity of BAT to take up substrates is illustrated by the ratio of BAT mass to the total glucose and TG uptake by BAT compared to that of other major organs in mice under cold challenge. Labbe et al. extended this observation through PET–CT analysis of the rate of substrate flux and oxidation in the iBAT of both warm- and cold-adapted rats (33). The rate of glucose uptake into iBAT was relatively low at 27°C but increased 10-fold upon acute cold exposure and increased 46-fold after cold acclimation at 10°C. Similarly, NEFA levels rose 6-fold upon acute cold exposure, and ~100-fold after cold acclimation. The metabolic activity of the iBAT reached levels similar to that of heart and liver after 6 h of cold exposure. In spite of these results, it should be kept in mind that although BAT glucose uptake per unit volume of tissue is important, the bulk of glucose turnover during cold exposure is mediated by skeletal muscle metabolic activation even when shivering is minimized (7).

It is more challenging to determine the specific contribution of beige adipocytes to whole-body metabolism. Bartelt et al. showed that acute cold exposure also increases TRL uptake in inguinal WAT (iWAT), but to a smaller extent compared to iBAT (32, 34). Seale's group reported an aP2-PRDM16 transgenic mouse that exhibited a highly favorable metabolic phenotype, in which iBAT remained unchanged but with extensive browning in iWAT. This suggests that beige adipocytes may contribute to the overall metabolic phenotype observed in this mouse (35).

On the whole-body level, Reitman's group dissected the relative contributions of cold-induced, diet-induced, and physical activity-associated EE in mice in relation to the basal metabolic rate (BMR) at various temperatures (36). This work clearly illustrated that at 22°C, the temperature at which most reported metabolic studies have been conducted, mice expend a relatively large amount of energy to generate heat (120% of BMR). Adult humans, on the other hand, live in or near their thermoneutral zone, with a relatively small contribution from adaptive thermogenesis to EE (5% of BMR) (11). Interestingly, in mice housed at thermoneutrality (30–32°C), the relative contributions of BMR, diet, physical activity, and adaptive thermogenesis in mice to overall EE are reported to be ~60, 12, 25, and 0%, respectively (36). These figures are very similar to the relative contributions seen in humans with low activity levels, and might represent experimental conditions more suitable for translational research in this field. The EE increase in relation to external temperature is conceptually depicted in **Figure 2** (not scaled to real data).

Numerous studies that address the role of human BAT activation and its quantitative impact on whole-body metabolism have been published. Based on the heat production capacity of mouse BAT, Rothwell and Stock proposed in the 1980s that 40–50 g of BAT, if maximally activated, could account for 20% of daily EE in human (37). More recently PET–CT measurements estimated the average active BAT volume in healthy humans to be 137 cm3 , corresponding to a conservative estimate of around 50 g BAT mass (11). Virtanen et al. estimated the EE of human BAT to be 55 W/ kg (5) based on the rate of glucose uptake during cold exposure, as measured by dynamic PET–CT. These conservative estimates suggest that the EE of fully activated BAT could amount to 2–5% of BMR. Recent cold exposure experiments confirmed that the cold-induced NST-associated increase in EE accounts for 0–15% of BMR (11). Using a human body-composition model (38), we further extrapolated that a 4% increase of BMR could lead to a 3% bodyweight reduction per year, assuming that the effects are sustained. A key assumption in such an extrapolation is that functional tolerance can be avoided (39). In support of this assumption, some pieces of evidence suggest that increased thermogenesis is not always fully compensated for and negated by an increase in food intake, for reasons that are not fully understood (40).

FIGURE 1 | White adipose tissue stores excess energy as triglycerides that can be mobilized by lipolysis to generate FFA for use by other tissues. BAT is the main site of NST, which is carried out by UCP1. Beige adipocytes have uncoupling capabilities similar to brown adipocytes, but are found in what is normally considered WAT. Appropriate partitioning and oxidation of fatty acids into BAT, WAT, beige adipocytes, and other metabolic organs can reduce ectopic fat deposition in metabolic organs, resulting in improved insulin sensitivity. Green arrows indicate appropriate partitioning and red arrows indicate inappropriate

partitioning.

Cypess et al. recently reported a 13% increase in BMR as a result of BAT activation upon administration of an acute dose of the β3-AR agonist mirabegron (41). Again, assuming that this BMR increase could be sustained upon chronic treatment, modeling suggests that this could potentially lead to an 8% bodyweight loss per year in BAT-positive healthy men. Besides potential functional tolerance, it remains uncertain if chronic β-AR-mediated stimulation of BAT is possible without encountering the side effects often associated with β-AR agonists.

Bodyweight reduction has been shown to have positive effects not only on preventing the progression of pre-diabetes to diabetes, but also leads to a reduction of hemoglobin A1c (HbA1c) (42). More recently, Hanssen et al. showed that 10-day cold acclimation in obese T2D patients could increase BAT activity, which was in turn associated with a reduction of BAT TG content and increased EE (43). One very encouraging observation is the 43% improvement of glucose infusion rate during a clamp study. Importantly, the improvement in insulin sensitivity of both adipose and skeletal muscle appeared before any bodyweight change could be seen (43). As indicated by Hanssen et al., the cold-induced improvement in insulin sensitivity exceeds that which was observed after long-term exercise training. To place this in a pharmacological context, this improvement in insulin sensitivity is similar in extent to that seen after 2-week treatment with dapagliflozin (Farxiga/Forxiga), which affords an 18% improvement in tissue glucose disposal (44).

### FUNCTIONAL BROWN/BEIGE ADIPOCYTES

The remarkable metabolic capacity of classical brown adipocytes in the activated state has been well characterized using rodent brown adipocytes. Functional characterization of beige adipocytes from the inguinal adipose tissue of mice or differentiated beige adipocytes from human adipose stem cells suggests that these cells are functionally similar to classical brown adipocytes (45, 46). The developmental origin of brown and beige adipocytes is currently an area of intense research and has been extensively reviewed elsewhere (47, 48).

Uncoupling of the mitochondrial transmembrane proton gradient in brown adipocytes in order to generate heat in response to cold is a complicated but well-orchestrated event. The key metabolic and signaling pathways in the brown or beige adipocyte are summarized in **Figure 3**. To be able to carry out this function, brown adipocytes need to be equipped with a complex machinery that is able to (i) signal through norepinephrine (NE) or other catecholamines via β-ARs, (ii) generate intracellular FFAs through hydrolysis of TGs from lipid droplets (lipolysis), and (iii) uncouple the ATP-generating process via UCP1 activation (13, 49). Chronically, cold challenge leads to a recruitment process in which cell proliferation, mitochondrial biogenesis, and angiogenesis are enhanced. Given the broad range of cellular processes that are involved in these events, it is not surprising that large numbers of genes/factors have been described to regulate the differentiation and function of brown and beige adipocytes (9, 34).

The β3-AR has emerged as a leading molecular target for the activation of brown or beige adipocytes (18, 41). Rodent and human adipose tissue display different β-AR expression profiles. (50–53). In mouse, the β3-AR is highly expressed in both BAT and WAT with several-fold higher abundance than the β1-AR, whereas in the adult human, the β3-AR is only expressed in BAT. In human WAT, β1-AR has been reported to be 50-fold more abundant than β3-AR (52). The β3-AR subtypes also differ in their potency toward various ligands, G-protein coupling, and desensitization. The binding affinity of NE to β3-AR was reported to be in the low micromolar range, while the potency for cAMP accumulation is in the low nanomolar range when measured in intact cells, suggesting that the coupling efficacy to adenylyl cyclase of β3-AR was higher than that of β1-AR (52, 54). In addition, β1-AR desensitizes more rapidly than β3-AR upon exposure to agonists. This led to the hypothesis that β1-AR may mediate the NE response to low levels of sympathetic stimulation. On the other hand, the activation of β3-AR may require higher levels of sympathetic stimulation, but once activated this receptor is likely to deliver a more sustained effect (52). It is important to note that circulating NE levels are generally in the low nanomolar concentration range, and it is likely that NE concentrations at the synaptic clefts will be much higher during cold response, providing sufficiently high local concentrations in BAT to enable local activation of β3-AR in spite of the low plasma concentration. Recently, several studies have shown that systemic administration of adrenergic activators, such as isoproterenol (ISO) and ephedrine, fail to elicit BAT activation in man (55–57). To properly interpret these clinical data, it is crucial to understand if the plasma concentrations of the adrenergic agonists used reached a sufficiently high concentration to enable activation of lipolysis and thermogenesis in human BAT.

Norepinephrine-mediated β-AR activation results in an increased intracellular cAMP concentration, which in turn stimulates lipolysis in brown adipocytes via activation of the protein kinase A pathway. Lipolysis is a stepwise process with different enzymes acting at each step: TGs are hydrolyzed by desnutrin/adipose triglyceride lipase (ATGL) to form diacylglycerol (DAG). DAG is then hydrolyzed by hormone-sensitive lipase (HSL) to monoacylglycerol and, subsequently glycerol, with a fatty acid released at each stage. Intracellular FFAs are the direct activators of UCP1 (58). In humans, the BAT radiodensity (which is indicative of intracellular TG stores) is inversely correlated with NST, strongly suggesting that depletion of intracellular TG occurs during cold exposure. In rats, inhibition of lipolysis by nicotinic acid-mediated GPR109a agonism significantly reduced the oxidative capacity of iBAT in response to cold, again suggesting a key role for lipolysis in UCP1-mediated thermogenesis (33). Additionally, adipose tissue-specific knockout of ATGL led to the formation of "whitened" brown adipocytes and resulted in impaired lipolysis and defective thermogenesis in BAT (59–62).

The brown adipocytes' cold-depleted energy stores are replenished by *de novo* lipogenesis and glycogen synthesis, which requires cellular uptake of circulating glucose and FFAs (derived either from TRLs or from lipolysis in WAT). In brown adipocytes, glucose uptake is mostly mediated by glucose transporter 1 (GLUT1) and glucose transporter 4 (GLUT4) and subsequently stored as glycogen or converted to lactate through anaerobic glycolysis. As shown in **Figure 3**, FFAs are transported into the cell by cluster of differentiation 36 (CD36) and TGs are subsequently synthesized through re-esterification by a series of enzymes, including glycerol-3-phosphate acyltransferase (GPAT) and diacylglycerol O-acyltransferases (DGATs) (49). The process of recruitment of new brown or beige adipocytes and the maintenance of mature adipocyte function during acute and chronic cold acclimation are subject to complex transcriptional and hormonal regulation, for example, by PPARγ, bone morphogenic proteins (BMPs), the thyroid axis, and FGF21. These regulatory mechanisms have been extensively reviewed elsewhere (8, 47, 48, 63).

The functional activity of BAT is reduced by chronic warm acclimation, old age, obesity, and diabetes. Rodent models of genetic deficiency of leptin (*ob/ob*, *db/db*, and obese *fa/fa* rats) are cold sensitive, and their brown adipocytes have a white adipocyte appearance with reduced expression of UCP1 protein (64, 65). Old age and lack of cold challenge also reduce the thermogenic activity of the brown adipocytes (66). Brown adipocytes isolated from guinea pigs housed at 30°C appear unilocular, although these cells appear to retain their brown adipocyte identity and are able to respond to NE with a robust increase of thermogenesis, which is not the case for adipocytes isolated from WAT (67). In humans, BAT activity is inversely correlated with age, fat mass, and BMI. Insulin-stimulated glucose uptake is also compromised in the BAT of diabetic individuals (68). Recent reports that both chronic cold acclimation and weight loss can enhance BAT activity in humans are encouraging (27, 69, 70), indicating that reduced BAT function may be restored. In this respect, adipocytes or adipose precursor cells are highly plastic and able to adapt to the functional needs, as has been shown in rodent using lineage tracing experiments (71).

### THIAZOLIDINEDIONES

The TZDs [represented by rosiglitazone (Avandia) and pioglitazone (Actos)] are a chemical class of PPARγ agonists used as insulin sensitizers for the treatment of T2D (72). The primary site of action of the TZDs is adipose tissue, where they improve several functional aspects, including the uptake and storage of plasma NEFA. They also increase FFA mobilization under fasting conditions and enhance postprandial suppression of FFA mobilization by insulin (73).

Thiazolidinediones have been shown to increase UCP1 expression and BAT mass in rodents. Rosiglitazone has been shown *in vitro* by several laboratories to induce UCP1 in rodent brown adipocytes and differentiated adipose stem cells (74–77). In addition, chronic treatment of human subcutaneous adipose stem cells with rosiglitazone upregulated several components of the mitochondrial electron transport chain, which is consistent with what has been observed in human (78). The UCP1 protein in human adipocytes differentiated *in vitro* in the presence of rosiglitazone is only functional when cells have been allowed to differentiate for a longer time than is typically reported in mouse studies (45). Although TZDs have the ability to recruit the "browning program" in both mouse and human adipocytes, the thermogenic capacity of UCP1-expressing cells cannot be unleashed without subsequent activation (e.g., by β-AR agonists). An increase in the oxygen consumption rate (OCR) of human adipocytes differentiated *in vitro* in the presence of rosiglitazone could only be observed when the cells were stimulated with isoproterenol or in the presence of exogenously provided FFAs. The increase of OCR in these beige cells is completely UCP1 dependent, as UCP1 knock-down abolishes the effect (45).

Various *in vivo* rodent models of insulin resistance have been used to show that the TZDs increase UCP1 mRNA in iBAT and overall iBAT weight. However, this is not associated with a subsequent increase in thermogenesis or whole-body EE. In addition, TZD treatment leads to the brown adipocytes becoming lipid filled (79–81). This led to the hypothesis that obese and diabetic animals or humans could first be primed by a PPARγ

agonist to expand the BAT capacity, followed by its activation via a β-AR agonist. This concept was tested pre-clinically in *ob/ob* mice by pre-treating with a PPARγ agonist (COOH, a non-TZD PPARγ agonist) followed by treatment with the β3-AR agonist CL-316,243. Synergistic effects on EE and bodyweight reduction were indeed observed in this single study (82). However, a later study using rosiglitazone followed by acute cold exposure failed to reproduce these effects (83). A possible explanation for the lack of increased EE in spite of increased TG storage, UCP1 expression and BAT mass induced upon PPARγ activation by rosiglitazone (84) may be the downregulation of β3-AR and iodothyronine deiodinase type II (DIO2) expression caused by TZD treatment (85, 86). In addition, as COOH belongs to a different structural class of PPARγ agonists to the TZDs, it is not unlikely that different outcomes could be observed between the two compounds. In humans, 12 weeks of pioglitazone treatment has been shown to generate a small increase in UCP1 mRNA levels in subcutaneous adipose tissue (87). However, combined pioglitazone and ephedrine treatment for 12 weeks in obese human subjects failed to deliver significant bodyweight reduction (88).

When comparing the observed outcomes of studies employing PPARγ agonists, it must be kept in mind that even structurally closely related compounds may exhibit very different PPARγdependent pharmacodynamic profiles. This is, in part, due to ligand-dependent modulation of the PPARγ protein that leads to recruitment of different coactivator and corepressor proteins, in turn resulting in a unique transcriptional profile for each compound (89). Since the identification of PPARγ as the molecular target of the TZDs (90), significant effort has been invested by the pharmaceutical industry in PPARγ agonist drug discovery (91–96) albeit largely without commercial success (97). The availability and high throughput of PPARγ LBD-based ligand binding assays and chimeric PPARγ–GAL4 reporter gene/transactivation assays enabled the generation of many highly potent, structurally diverse, selective PPARγ full and partial agonists. Despite this, since the launch of pioglitazone and rosiglitazone, no new selective PPARγ agonists have survived clinical testing and remain on the market, mainly due to preclinical and clinical safety issues (98–101). The central role of PPARγ in adipose biology has, however, not diminished due to these failures and PPARγ remains an attractive but challenging drug target.

The lack of translation between *in vitro* receptor binding and the functional (both *in vitro* and *in vivo*) effects of the compounds and inter-species differences in PPARγ biology are considered major obstacles in this field. This is due to a number of factors, including the combinatorial nature of the activation of the PPARγ:RXR heterodimer (102, 103), subtle differences in coactivator/corepressor recruitment between superficially related compounds (104–107), ligand effects on the extent of posttranslational modifications (108, 109), and non-transcriptional effects of the ligands (72). This suggests that a reductionist approach to PPARγ agonist discovery based on the use of isolated protein domains and chimeric reporter gene assays is unlikely to provide compounds with the desired functional or clinical outcome.

Since the "traditional" approach of optimizing receptor binding and agonist potency has not borne fruit, a radical change in the preclinical approach to PPARγ drug discovery is needed in order for the functional potential (for example, browning of WAT) of small-molecule PPARγ activation to be realized. For example, the application of phenotypic screening in relevant cell systems [i.e. primary human cells (110)] is one approach to frontload the functional assessment of compounds, with traditional *in vitro* assessment of PPARγ activity included in a secondary wave of assays. In addition, the recent widespread availability of omics techniques (such as *RNA*omics and proteomics) makes the preclinical identification of PPARγ agonists with a desirable functional profile a realistic prospect. A combination of such approaches avoids focus on a single receptor-dependent pathway or mechanism and allows pleiotropy to be accounted for. However, whether the perceived target-related risk associated with PPARγ agonism is considered acceptable in proportion to the potential commercial viability of a safe PPARγ agonist remains to be seen.

## BETA-3 ADRENERGIC RECEPTOR

Several sympathomimetic β3-AR agonists that selectively stimulate rodent brown and white adipocyte lipolysis were discovered from the mid-1980s onward (e.g., BRL-37344, CL-316,243, and CGP-12177A) (50, 51, 111). Early optimization of these compounds was mostly performed in rodent tissue or cell models, as the human β3-AR was not cloned until 1989 (112). The compounds showed potent anti-obesity and anti-diabetic effects in rodent models of obesity and diabetes, but none of these compounds advanced beyond the clinical phase II due to lack of efficacy. Specifically, compounds optimized using rodent β3-AR did not effectively translate to human. Several β3-AR agonists were synthesized and evaluated after the cloning of human β3-AR cDNA (113). A summary of studies investigating the effect of β3-AR agonism on EE in man is given in **Table 1**. Note that many binding and adenylyl cyclase activity assays were performed using isolated membrane preparations, returning β3-AR binding affinities in the micromolar range for the compounds tested. Lower potencies (in the nanomolar range) have been reported for the same compounds when measured in whole-cell assays (cAMP, lipolysis, or respiration) (52), suggesting a G-protein coupling efficiency for the β3-AR that is only captured in a whole-cell context.

Most early human trials of β3-AR agonists used bodyweight reduction as a clinical endpoint, to be achieved through increased EE. Data on BAT activation and metabolic parameters are scarce, with the exception being for the CL-316,243 study (124). In this study, treatment of lean healthy men for 4 weeks did not increase EE, but resulted in a 45% increase of insulin-mediated glucose disposal and a reduced 24-h respiratory quotient (24-h RQ), indicating enhanced fat oxidation. Intriguingly, CL-316,243 significantly increased fasting FFA levels in parallel with improved insulin action. These data are in line with recently published improved glucose infusion rates in T2D patients after 10 days of mild cold exposure (43). In both cases, improved insulin action preceded any significant weight loss. This improved action of insulin may be due to partitioning of FFAs toward BAT, which in turn reduces the fatty acid burden on other metabolic tissues (see **Figure 1**). Indeed, improved glucose uptake was seen in the skeletal muscle of T2D patients after cold exposure (43).

TABLE 1 | *In vitro* properties of **β**3-adrenergic receptor agonists that have been tested in humans, and their effect on energy expenditure in man.

#### PHYSICAL AND PHARMACOKINETIC PROPERTIES

BAT: Unlock the Potential

Similarly to CL-316,243 treatment, chronic dosing of the β3- AR agonists ZD7114 and ZD2079 did not significantly increase EE, although the latter compound showed a tendency toward a non-statistically significant increase of 2% (115).

Data have been reported for both acute and chronic dosing of β3-AR agonist L-796568 in overweight to obese men. Acutely, van Baak et al. (125) reported a significant increase (8%) in EE after 4 h for the highest dose used. However, no average increase was observed over the period of observation, 0–4 h. Chronic dosing of L-796568 for 4 weeks did not result in any significant change in EE (121). The authors discuss possible reasons for this lack of effect, e.g., declining plasma exposure levels over time, insufficient agonist-induced BAT proliferative capability of β3- AR-responsive tissues in humans during chronic stimulation, potential downregulation of β3-AR receptors, or other functional feedback. We believe that the small effect on EE observed in the acute study is another reason.

A chronic β3-AR agonism study (using TAK-677) in obese humans with no observed effect on EE or diabetic parameters was reported by Redman et al. (122). Whether BAT activation truly occurred after administration of compound was not carefully assessed in this study, which makes data interpretation challenging.

In contrast to the above-mentioned β3-AR agonism studies, Cypess et al. recently reported a ~13% increase in resting metabolic rate (RMR) upon acute treatment with high doses of β3-AR agonist mirabegron (41). In this study, healthy young male subjects with detectable BAT were selected in order to provide proof of concept (PoC) for β3-AR agonist-mediated BAT activation. Importantly, it was found that BAT metabolic activity was a significant predictor of the changes in RMR. This study also reports a weak correlation between cold- and drug-induced detectable BAT activities. Generally, such data from the same individuals are important to help put the large quantity of cold-induced BAT activation data into a drug-discovery context. It remains to be investigated if the reported energy-expenditure effect is sustained upon chronic dosing and if the efficacy of mirabegron will persist in females and other patient subpopulations, such as those with different ages and BMIs.

The failure of β3-AR agonists to show clinical effects on weight loss decreased interest in the mechanism as a means of treating the metabolic syndrome. However, the recent rediscovery of BAT in adult humans as well as the demonstration of functional activation of BAT by a β3-AR agonist may lead to a resurgent interest in β3-AR agonists for the treatment of metabolic disorders. The observation that improved insulin action preceded any significant weight loss upon β3-AR agonist treatment is particularly encouraging.

Clearly, important questions remain unanswered with respect to the role of the β3-AR and the clinical profile of β3-AR agonists. In order to make significant progress in β3-AR drug discovery, the lack of translation from rodent to human (i.e., receptor expression and functional differences) and from *in vitro* to *in vivo* for the human setting needs to be resolved. The clinically assessed β3-AR agonists exhibit structural and physicochemical property diversity; however, clinical plasma exposures relative to EC50 are similar for the chronic and acute studies (**Table 1**), and activation

TABL

E 1 |

Continued

*cClaimed to be a full agonist, but only 80% TE in in vitro assay.*

of BAT would therefore be expected in the chronic studies. The lack of effect on EE observed in the chronic studies may either be due to not measuring a mechanism-relevant clinical endpoint or that the patients recruited lacked sufficient BAT to lead to an effect on overall EE. The activation of pre-existing BAT by β3-AR agonists may require a personalized healthcare approach, unless combined with a compound or mechanism to expand the BAT depot.

Finally, cardiovascular side effects have been associated with β3-AR agonist treatment, usually attributed to insufficient selectivity toward the β1- and β2-ARs. However, the *in vitro* functional activity of many of the clinical compounds assessed does not explain the increase in heart rate observed – several of the compounds are functional β1-AR antagonists (β1-AR blockers), and as such could be expected to have the opposite effect to that clinically observed. The complex nature of AR biology needs to be further investigated if this mechanism is to be reconsidered as a means of activating BAT.

## FIBROBLAST GROWTH FACTOR 21

Fibroblast growth factor 21 (FGF21) is a member of the endocrine FGF 19/21/23 family. FGF21 protein is expressed in liver, pancreas, and adipose tissue and is regulated by fasting, ketogenic diet, low protein diet, PPARγ, and PPARα activation as well as glucagon action (126). It acts through binding to a cell-surface receptor complex composed of conventional FGF receptors (FGFR1c/2c/3c) and the co-factor β-Klotho, leading to activation of FGF receptor substrate 2α and ERK1/2 phosphorylation. While FGF receptors are ubiquitously expressed, β-Klotho expression is restricted to a few tissues, including BAT, WAT, and liver (127, 128), which are the main sites of FGF21 action. When administered pharmacologically, FGF21 enhances EE and insulin sensitivity, reduces bodyweight, glucose, and lipids, and thus has the potential to be used for treatment of the metabolic syndrome via multiple mechanisms (129–131).

Functional enhancement of existing BAT and recruitment of beige adipocytes have been hypothesized to be the mechanism behind the EE increase, bodyweight loss, and improved glucose and lipid homeostasis induced by FGF21. FGF21 expression is increased in BAT upon cold challenge (132, 133). Adiposederived FGF21 acts in an autocrine/paracrine manner to increase expression of UCP1 and other thermogenic genes, such as *PGC1*α, *PRDM16*, *BMP8b*, and *DIO2* in iBAT and iWAT (134, 135). In neonates, FGF21 expression in liver is increased by suckling, which occurs via activation of PPARα, leading to induced BAT thermogenesis (136).

Attempts have been made to directly assess the contribution of BAT and beige adipocytes to FGF21-mediated effects. Surgical resection of iBAT in two different mouse models showed that FGF21-mediated EE increase and bodyweight reduction were retained (137, 138). However, one of the studies showed that several beige adipocyte markers, including *PPARγ*, *PRDM16*, *PGC1α*, and *CIDEA* tended toward upregulation in subcutaneous adipose tissue, and perigonadal adipose weight also was reduced (138). The authors propose that residual BAT and beige adipocytes may have compensated for the loss of iBAT. Recently, the role of UCP1 in mediating the pharmacological effects of FGF21 was assessed by treating UCP1 knockout (UCP1 KO) mice with either recombinant FGF21 or FGF21 fused with fragment crystallizable region (Fc-FGF21) (139, 140). Surprisingly, several FGF21-mediated effects were largely retained in the absence of UCP1. Again, compensatory mechanisms seem to be activated in UCP1 KO mice treated with recombinant FGF21. In FGF21-treated UCP1 KO mice, reduced food intake offset the decrease in EE and resulted in a similar bodyweight reduction to that observed in FGF21 treated wild-type mice. In addition, genes regulating fatty acid metabolism were upregulated in liver and epididymal adipose tissue, suggesting that FGF21 recruits UCP1-independent pathways in these tissues to compensate for the lack of UCP1. An intriguing observation was a two- to threefold increase in FGF21 secretion in iBAT when UCP1 KO mice were challenged by cold, suggesting that FGF21 may be one of the factors that recruit alternative thermogenic mechanisms when iBAT/UCP1 fails to generate heat. It should be noted that many previous publications have showed remodeling of the WAT in UCP1 KO mice, and that alternative thermogenic mechanisms have been discussed (141, 142).

In spite of the attractive metabolic effects of FGF21, development of FGF21-based therapeutics has encountered several technical challenges, including short *in vivo* half-life and poor biophysical properties of FGF21 protein (e.g., it is prone to aggregation). To date, three different analogs of FGF21 have advanced to concept testing in human: (130, 143, 144) (i) LY2405319 (an aggregation-resistant FGF21 analog), (ii) PF-05231023 (FGF21 linked to a Fab fragment of a scaffold antibody), and (iii) ARX-618 (PEGylated FGF21). For both LY2405319 and PF-05231023, the effects on TG (close to 50% reduction in 28 days), LDL cholesterol, and HDL cholesterol were substantial and bodyweight reduction was significant. However, the failure of FGF21 analogs to achieve clinically meaningful glucose-lowering effects was unexpected, and the mechanism behind this remains to be understood. Recently, Genentech reported a bispecific monoclonal antibody agonist that binds to both FGFR1c and β-Klotho, with sustained effects on NST, EE, and bodyweight over a 35-day period after a single administration to diet-induced obese mice (145). The effects on glucose and lipids were similar to those observed after administration of recombinant FGF21, and mostly attributed to peripheral FGF21 action as CNS exposure of the antibody was reported to be minimal. Together, these data further support the importance of BAT function in FGF21 action. As for the β3-AR agonist story, it may be important for future clinical concept testing to assess BAT recruitment/activation in order to understand if sufficiently high FGF21 levels have been achieved in patients. In addition, it is critical to constantly monitor potential safety concerns of this treatment principle in all studies, including bone density, growth hormone resistance, and female fertility.

## PERSPECTIVES

The understanding of BAT physiology has increased rapidly in recent years. Data generated in both rodents and humans in cold/warm acclimation studies suggest that both BAT and beige adipocytes demonstrate recruitability and plasticity, which opens the door to pharmacological intervention. As obese and T2D patients are likely to have reduced BAT function, in order to realize the full potential of NST, it may be necessary to first restore the function of "whitened" brown or beige adipocytes before activation (**Figure 2**). In this respect, future studies using rodent models require consideration of external temperature in order to more closely mimic human physiology. Cold acclimation for a short duration in obese and diabetic humans indicates that it is possible to enhance BAT activity and achieve an improved metabolic profile before significant bodyweight reduction occurs. The improved insulin action in the absence of bodyweight change seen in the 4-week treatment of healthy individuals by CL-316,243 (124) suggests that restoration of BAT function may at least lead to improved metabolic control in patients with T2D. Acute treatment of BAT-positive human subjects with the β3-AR agonist mirabegron resulted in a significant increase in EE. If this effect is sustained upon chronic dosing and without eliciting cardiovascular side effects, this mechanism may finally lead to significant weight loss.

PPARγ remains an interesting target for the recruitment of BAT or beige adipocytes, although discovery of a compound that does not impair the adrenergic sensitivity or thyroid function of the adipocyte is extremely challenging. Phenotypic screening,

#### REFERENCES

for example a screen based on human brown adipocyte function, may however facilitate the discovery of such a compound.

Finally, if an FGF21 analog with appropriate half-life and potency could be developed, it would enable PoC testing in humans in order to understand if the potential beneficial metabolic effects of this hormone can be realized.

#### AUTHOR CONTRIBUTIONS

XP: conception, structure and content of the text, including the figures. PG: mathematical modeling of the literature data and critical examination and interpretation of pharmacokinetic and pharmacodynamic data on the β3-AR agonists in clinical studies. Major contribution to the Table 1. GO'M critical review and interpretation of PPARγ and β3-AR agonists structure, physiochemical properties and assays used for generating the data of all compounds included in Table 1. SB: critical review of bioenergetic of cell biology, general contribution of bioscience-related text, and critical reading and editing of review.

### ACKNOWLEDGMENTS

The authors would like to thank Nick Oakes (AstraZeneca) for his contribution to **Figure 1**.

tissue in healthy adult humans: effects of cold exposure and adiposity. *Diabetes* (2009) **58**:1526–31. doi:10.2337/db09-0530

electronic medical record data. *Diabetes Res Clin Pract* (2014) **103**:402–11. doi:10.1016/j.diabres.2013.12.038

of brown-fat mitochondrial uncoupling protein in adult dogs. *Proc Natl Acad Sci U S A* (1991) **88**:10774–7. doi:10.1073/pnas.88.23.10774

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Peng, Gennemark, O'Mahony and Bartesaghi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The therapeutic potential of brown adipocytes in humans

*Craig Porter1,2\*, Maria Chondronikola1,3 and Labros S. Sidossis1,2,4,5\**

*1Metabolism Unit, Shriners Hospitals for Children-Galveston, Galveston, TX, USA, 2Department of Surgery, University of Texas Medical Branch, Galveston, TX, USA, 3Department of Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, TX, USA, 4Department of Nutrition and Dietetics, Harokopio University of Athens, Athens, Greece, 5Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USA*

Obesity and its metabolic consequences represent a significant clinical problem. From a

#### *Edited by:*

*Jun Wu, University of Michigan, USA*

#### *Reviewed by:*

*Elahu Gosney Sustarsic, University of Copenhagen, Denmark Sona Kang, Beth Israel Deaconess Medical Center, USA*

#### *\*Correspondence:*

 *Craig Porter and Labros S. Sidossis, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77550, USA<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology*

*Received: 30 June 2015 Accepted: 18 September 2015 Published: 13 October 2015*

#### *Citation:*

*Porter C, Chondronikola M and Sidossis LS (2015) The therapeutic potential of brown adipocytes in humans. Front. Endocrinol. 6:156. doi: 10.3389/fendo.2015.00156*

thermodynamic standpoint, obesity results from a discord in energy intake and expenditure. To date, lifestyle interventions based on reducing energy intake and/or increasing energy expenditure have proved ineffective in the prevention and/or treatment of obesity, owing to poor long-term adherence to such interventions. Thus, an effective strategy to prevent or correct obesity is currently lacking. As the combustion engines of our cells, mitochondria play a critical role in energy expenditure. At a whole-body level, approximately 80% of mitochondrial membrane potential generated by fuel oxidation is used to produce ATP, and the remaining 20% is lost through heat-producing uncoupling reactions. The coupling of mitochondrial respiration to ATP production represents an important component in whole-body energy expenditure. Brown adipose tissue (BAT) is densely populated with mitochondria containing the inner mitochondrial proton carrier uncoupling protein 1 (UCP1). UCP1 uncouples oxidative phosphorylation, meaning that mitochondrial membrane potential is dissipated as heat. The recent rediscovery of BAT depots in adult humans has rekindled scientific interest in the manipulation of mitochondrial uncoupling reactions as a means to increase metabolic rate, thereby counteracting obesity and its associated metabolic phenotype. In this article, we discuss the evidence for the role BAT plays in metabolic rate and glucose and lipid metabolism in humans and the potential for UCP1 recruitment in the white adipose tissue of humans. While the future holds much promise for a therapeutic role of UCP1 expressing adipocytes in human energy metabolism, particularly in the context of obesity, tissue-specific strategies that activate or recruit UCP1 in human adipocytes represent an obligatory translational step for this early promise to be realized.

Keywords: adipose tissue, mitochondria, uncoupling protein 1, thermogenesis, obesity

#### Introduction

Obesity and its associated metabolic complications (hyperlipidemia, insulin resistance, and glucose intolerance) have become significantly more prevalent in the United States and around the world in recent years (1, 2). One third of Americans are either obese (1) or exhibit symptoms of pre-diabetes (3). Worldwide, excessive adiposity is responsible for more than three million Porter et al. Adipose tissue thermogenesis

deaths and is a significant cause of disability (4). Increasing adiposity is associated with derangements in glucose and lipid metabolism and in the development of insulin resistance (5, 6). Therapeutic strategies aimed at preventing obesity or treating its metabolic complications typically target dietary intake and/or physical activity, but to date do not seem to exhibit a great deal of long-term efficacy. Mitochondrial proton leaks, i.e., respiration uncoupled from ATP production, accounts for around 20% of total mitochondrial respiration (7). The physiological role ascribed to this uncoupling of oxidative phosphorylation is heat production (thermogenesis). While thermogenesis represents a significant portion (~20%) of whole-body energy expenditure (7, 8), its therapeutic role in energy metabolism has only recently begun to receive attention.

Brown adipose tissue (BAT) is the major tissue responsible for non-shivering thermogenesis in mammals (9). The recent (re)discovery of BAT in human adults (10–12) has triggered scientific interest in the role of BAT in human energy metabolism. Unique features of BAT include an abundance of mitochondria that contain the transmembrane carrier protein thermogenin, most commonly referred to as uncoupling protein 1 (UCP1) (13). Upon activation by long-chain fatty acids, UCP1 acts as an inner mitochondrial proton carrier (14), allowing protons to reenter the mitochondrial matrix independently of ATP synthase, thus uncoupling mitochondrial respiration from ATP production (9). This process alters mitochondrial energy transduction, where mitochondrial membrane potential is lost as heat (**Figure 1**). The purpose of this review is to summarize the current evidence regarding the role of the human adipose tissue as a therapeutic target against obesity and its related metabolic complications.

### The Role of Brown Adipose Tissue in Energy Balance and Obesity

Although the role of BAT in energy metabolism and obesity has been studied in detail in rodents (15–17), our understanding of the role BAT plays in human energy metabolism remains in its infancy. For obvious reasons, it is more difficult to manipulate and study BAT in humans. Retrospective reviews of medical records and positron emission tomography–computed tomography (PET/CT) scans, originally performed for diagnostic purposes, demonstrate an association between BAT and body mass index (BMI) (10, 18, 19) and with non-alcoholic fatty liver disease (20). The retrospective nature of these studies along with the fact that the PET/CT scans were performed under non-standardized conditions limit the generalizability of these data. However, these findings are bolstered by prospective studies which report a correlation between BAT volume and activity with BMI (11, 21), total fat mass (11, 21), and both abdominal subcutaneous and visceral adiposity (21). Moreover, weight loss via bariatric surgery has been linked to increased BAT activity (22), further suggesting that BAT may contribute to energy balance in human beings.

Acute (non-shivering) cold exposure studies have been recently performed in an attempt to estimate the contribution of BAT to cold-induced energy expenditure. However, the reported results have been highly variable. These studies reported that acute cold exposure (2–4 h at 16–19°C) induces BAT activation, resulting in a 13–27% increase in resting energy expenditure (REE) (11, 23, 24). In contrast, Ouellet et al. reported an 80% increase in REE with acute cold exposure (for 2 h at ~18°C) (25), a result over threefold higher than those of other studies, possibly attributable to muscle shivering. Muzik et al. used oxygen 15 (15O)-PET/CT in an attempt to more directly quantify the contribution of BAT to energy expenditure (26, 27). In contrast to previous studies, these investigators found that BAT minimally contributed to the reported increase in energy expenditure (15–25 kcal/day). Although these two studies shed doubt on the role of BAT in energy expenditure in humans, the rather short cold stimulation protocol (30 min at 18°C) may not have been adequate to fully activate BAT. Indeed, BAT recruitment via daily mild cold exposure (17°C for 6 weeks) was associated with increased thermogenesis and decreased body fat, supporting a relationship between BAT activity and adiposity (28). Similarly, Lee et al. performed a crossover cold acclimation study in five healthy participants. These researchers showed that BAT recruitment was reversibly associated with increased post-prandial energy expenditure (diet-induced thermogenesis) after cold exposure, further supporting the role of BAT in energy expenditure (29).

When interpreting the results of these studies, one important point to consider is that chronic weight gain can result from even a small discordance in daily energy balance (e.g., an energy surplus of as little as 25 kcal/day in humans can result in a weight gain of 1 kg/year). Thus, even sporadic BAT activation for limited periods during the course of the day could conceivably have a significant cumulative impact on energy balance and adiposity over several months or years.

## Can Human Brown Adipose Tissue Alter Glucose Homeostasis and Insulin Sensitivity?

Brown adipose tissue has been touted as therapeutic tissue which may protect against hyperglycemia in insulin-resistant individuals. In murine models of extreme cold exposure, BAT is responsible for the majority of whole-body glucose disposal (30), while transplantation of BAT into the abdominal visceral adipose tissue improves glucose tolerance and insulin sensitivity (31). In humans, the involvement of BAT in systemic glucose metabolism is evident from 18F-FDG uptake images from PET/ CT scans (**Figure 2**) performed for diagnostic purposes (32). Moreover, evidence from retrospective medical record review studies indicates an inverse relationship between BAT activity, diabetes, and glycemia (10, 19, 33, 34). Further, prospective studies have shown that acute cold exposure increases glucose uptake in BAT, where BAT glucose uptake rate per unit of tissue was higher than that of muscle (25, 35, 36), underscoring the oxidative potential of BAT. Specifically, Orava et al. reported that cold exposure resulted in a 12-fold increase in glucose disposal in BAT only, but not in other tissues (35). Similar results have been reported by Ouellet et al. (25). These data suggest that upon

electron transport chain (ETC). Pyruvate also participates in mitochondrial anaplerosis and the production of reducing equivalent by being decarboxylated to acetyl-CoA by pyruvate dehydrogenase (PDH) or being carboxylated by pyruvate carboxylase (PC), forming oxaloacetate (OAA).

FIGURE 2 | Coronal (left) and transverse (right) 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG) positron emission tomography (PET)-computed tomography (CT) images from a volunteer during cold exposure. The intense orange color in the supraclavicular area corresponds to brown adipose tissue. Image from Ref. (24). Reprinted with permission.

activation, BAT clears plasma glucose. However, Ouellet et al. demonstrated that BAT minimally contributed to whole-body plasma glucose utilization, which might be due to the short duration of cold exposure (3 h at ~18°C) and the presence of mild shivering during cold exposure (25).

The opposing results of the aforementioned studies question the physiological significance of human BAT in systemic glucose metabolism. In an attempt to address this, we studied glucose metabolism in individuals with BAT (BAT+) or without BAT (BAT−) under cold exposure and thermoneutral conditions by infusing isotopically labeled glucose in the fasted state and during a hyperinsulinemic-euglycemic clamp. We found that cold exposure increased whole-body glucose disposal in the fasted and insulin-stimulated state only in BAT+ individuals (24), suggesting that BAT can indeed play a significant role in systemic glucose metabolism in humans. Theoretically, if BAT remained chronically active, it could dispose ~23 g of glucose in 24 h [equating to approximately 8.4 kg of glucose (33,600 kcal) in a year]. Similarly, Lee et al. recently reported that increased BAT activity via cold acclimation improved post-prandial glycemia (29). Collectively, these studies support the notion that upon activation, BAT can indeed play a significant role in glucose homeostasis. Therefore, BAT may represent a novel therapeutic target in the management of hyperglycemia.

### Does Human Brown Adipose Tissue Play a Role in Systemic Lipid Metabolism?

Free fatty acids (FFAs) constitute the primary substrate for BAT (37). Moreover, FFAs bind to and activate UCP1, allowing proton transfer from the intermembrane space to the mitochondrial matrix resulting in heat production (9). Upon activation, BAT initially oxidizes intracellular lipid stores to produce heat (9). In contrast, prolonged BAT stimulation leads to increased uptake of FFA derived from circulating FFA and lipolysis of circulating lipoproteins, presumably when local lipid stores become depleted (9). BAT activation has been recently reported to increase the systemic clearance of TG-rich lipoproteins in rodents, lending credence to the notion that BAT may protect against hyperlipidemia (30, 38). Moreover, rodent BAT has been shown to release factors that increase vascular permeability in BAT, allowing TG-rich lipoproteins to enter the interstitial space (30).

The role of BAT in human lipid metabolism remains largely unexplored. Results from some (11, 35), but not all (23), cold exposure studies report a reduction in the respiratory gas exchange ratio during cold exposure, indicating increased fat oxidation in humans during BAT activation. Ouellet et al. (25) studied the role of BAT in lipid metabolism in more detail. Using PET/CT and a fatty acid tracer, 18F-fluorothiaheptadecanoic acid (18FTHA), they showed that 3 h of cold exposure increased BAT FFA uptake but that this accounted for <1% of the total FFA turnover (25). The conclusion of this study was that thermogenic BAT relies predominantly on the limited intracellular substrates (i.e., triglycerides stored inside BAT) to fuel mitochondrial thermogenesis (25), which questions the physiological significance of human BAT in whole-body lipid metabolism. However, the relatively short duration of the study might have been insufficient to deplete the intracellular lipid stores underestimating the contribution of BAT in the systemic lipid metabolism.

We recently studied BAT+ and BAT− individuals under mild cold exposure and thermoneutral conditions while infusing stable isotopes of glycerol and palmitate in order to directly trace lipid turnover in the fasted state and during a hyperinsulinemiceuglycemic clamp. We found that during cold exposure BAT+ individuals had greater whole-body lipolysis and FFA oxidation compared with BAT− participants, indicating that increased lipid mobilization from WAT provides FFA to sustain thermogenesis in BAT (39). Moreover, BAT+ individuals were protected from the cold-induced adipose tissue insulin resistance noted in the BAT− group (40). Our data suggest that BAT may indeed play an important role in the regulation of systemic lipid metabolism. The discord between our preliminary findings and those of others (25) may be related to the duration of cold exposure. For example, we studied individuals after ~5 h of cold exposure, while Ouellet et al. performed a 3 h cold exposure study. Whether there is a time-dependent depletion of BAT lipid stores, at which point BAT switches to using systemic FFAs as a fuel source, remains unknown.

## Can Human WAT Really *Brown*?

More than 30 years ago, Young et al. demonstrated the appearance of brown adipocytes in the parametrial fat pad of mice following acclimation to severe cold exposure (41). This was confirmed when Cousin et al. demonstrated that severe cold exposure or treatment with a β3 adrenoreceptor agonist induced brown adipocytes within WAT (42). More recently, Shabalina et al. demonstrated that acclimation to cold (~5°) results in the browning of inguinal WAT. Importantly, these researchers showed the presence of UCP1 protein while demonstrating its function (via GDP-sensitive respiration) in inguinal WAT of cold-acclimated mice (43), thus demonstrating that these mitochondria had functional UCP1. Interestingly, per milligram of tissue, inguinal WAT from cold-acclimated mice developed a thermogenic capacity ~50% of that of intrascapular BAT (43). This striking plasticity of murine subcutaneous WAT to develop a thermogenic phenotype has potential implications for humans, given that most individuals have only 100 g or less of BAT, but many kilograms of subcutaneous WAT. If WAT in humans were able to exhibit the same plasticity as in mice, this would have huge implications for energy expenditure and substrate (glucose, lipids, and potentially amino acid) metabolism.

Unlike the inguinal subcutaneous WAT depot of mice, there is no evidence to suggest that human WAT has a comparable ability to alter its thermogenic capacity in response to an environmental stress. Indeed, chronic (10 days), albeit mild cold exposure, which activates BAT, does not alter the phenotype of WAT in humans (44). This suggests that a more profound and/ or chronic adrenergic stress is likely needed to induce browning in humans. Indeed, this is perhaps reasonable considering that classic rodent models of browning typically acclimate rodents to extremely cold temperatures (~5°C) for several days, if not weeks (41–43). Considering that a thermoneutral temperature for a mouse is somewhere close to 30°C, it would be reasonable to theorize that chronic exposure to a temperature 25°C below thermoneutrality would be required to induce browning of WAT in humans.

Since cold exposure protocols used in rodents cannot be replicated in humans, researchers have looked to pathologies associated with chronic adrenergic stress. Over 30 years ago, it was reported that patients with pheochromocytoma, a tumor of the adrenal medulla which results in a chronic elevation in catecholamine secretion, had BAT surrounding their kidneys (45). More recently, Frontini et al. (46) reported the existence of multilocular adipocytes that stained positive for UCP1 within the omental adipose tissue of pheochromocytoma patients. This evidence suggests that human WAT may contain brown adipocytes, but unfortunately only histological and genomic measurements were made on these WAT samples. Whether these omental adipocytes, which showed morphological similarities to that of brown adipocyte and immune-reactivity to UCP1, had functionally thermogenic mitochondria was not addressed.

Burn trauma represents a unique injury model as it is accompanied by prolonged adrenergic stress and a hypermetabolic state. To determine if subcutaneous WAT can acquire a brown fat phenotype, we collected biopsy samples of WAT from severely burned individuals. We found that when prospectively following patients after burn injury, UCP1 mRNA and UCP1 protein were induced in subcutaneous WAT at ~2 weeks post-injury (47). Moreover, this was accompanied by a similar time-dependent increase in mitochondria respiratory capacity and whole-body metabolic rate (47). Thus, it appears that human subcutaneous WAT has the ability to brown. In the context of burn trauma, this makes physiological sense, since following the destruction of their skin barrier burn victims have difficulty in conserving heat and maintaining core temperature.

To the best of our knowledge, our recent data provide the first evidence of browning of WAT in humans (47). However, we should note that while we did indeed see induction of UCP1, morphological adaptations, and increased mitochondrial respiratory in permeabilized WAT samples in a time-dependent manner after burn, the increase in thermogenic capacity (~threefold) was rather modest when compared with those seen in rodent WAT after chronic cold exposure. For example, Shabalina et al. (43) showed that the mitochondrial respiratory capacity of mitochondria isolated from the BAT of mice kept at 30°C was ~10-fold greater than that of mitochondria isolated from inguinal WAT. However, when acclimated to 5°C, the respiratory capacity of BAT mitochondria was only ~1.7-fold greater than that of inguinal WAT mitochondria. Indeed, BAT mitochondrial respiratory capacity was only marginally greater in cold-acclimated mice compared with thermoneutral mice. In contrast, WAT mitochondrial respiratory capacity was ~500% greater in inguinal WAT of cold-acclimated mice compared with thermoneutral animals. This remarkable plasticity, where murine inguinal WAT can adopt a thermogenic capacity akin to that of BAT, does not appear to be conserved in other mammals such as humans. Indeed, while we saw a threefold increase in subcutaneous WAT respiratory capacity after burn injury, respiration per milligram of tissue was still ~20-fold lower than what we have measured in human intrascapular BAT (47).

### The Therapeutic Potential of UCP1-Positive Mitochondria: Should we Activate BAT or Brown WAT?

Much emphasis has been placed on manipulating UCP1 in order to increase thermogenesis and thus energy expenditure, which over time would reduce adiposity and consequently correct metabolic abnormalities associated with obesity. Broadly speaking, manipulation of UCP1 can be classified into two distinct ways: (i) acute activation of existing UCP1 and (ii) induction of UCP1. Induction of UCP1 can take several forms, i.e., the expansion of existing BAT depots, producing more UCP1 per gram of BAT, or inducing UCP1 within WAT (browning). However, the significance of either activating UCP1 within classical BAT depots or browning WAT in humans in terms of energy expenditure remains unclear. Using available data on organ-specific metabolic rates, and our own data on (a) the metabolic rate of human BAT following acute cold exposure and (b) human WAT that has undergone browning, we present theoretical calculations to estimate the potential impact of UCP1 on energy expenditure in humans (**Tables 1** and **2**).

Since UCP1 is inactive in the presence of inhibitory purine nucleotides such as ATP (50), BAT makes a negligible contribution to whole-body REE given that ~100 g of BAT represents ~0.1% of total body mass of a 70 kg individual. Similarly, in an individual with ~20% total fat mass, WAT accounts for a small (~4%) portion of whole-body REE (48) (see **Table 1**). Following acute (5 h) acclimation to mild cold (5 h at ~18°), our data (24) suggest that on average there is a 7.5% increase in REE. This acute intervention transforms BAT from being a quiescent tissue to one that makes a comparable contribution to whole-body REE as the heart or the kidneys (**Table 1**). In a 70 kg individual, this activation of BAT increases REE by 127 kcal (**Table 1**), which if maintained for 30 days would combust 0.5 kg of adipose tissue.

Burn trauma results in a significant increase in REE (51), which persists for months if not years post injury (52). While many ATP-consuming processes increase after burn, increased ATP turnover only explains 50–60% of burn-induced hypermetabolism (53). This means that thermogenesis represents a significant portion of hypermetabolism in burn victims, which makes sense since these patients have a compromised skin barrier and inability to thermoregulate. In order to estimate the

TABLE 1 | Impact of acute non-shivering cold exposure and severe burns on whole-body energy expenditure.

*Values for each organ/tissue are presented as kilocalories/day with the percent contribution to whole-body energy expenditure in brackets.*

*a Values for thermoneutral conditions are taken from Rolfe and Brown (7).*

*bValues for white adipose tissue (WAT) are taken from Gallagher et al. (48). c Values for cold exposure (5 h at ~18°) were derived from Chondronikola et al. (24),*

*assuming a 7.5% increase in resting energy expenditure following acute cold exposure, which was attributable to brown adipose tissue (BAT) activation.*

*dValues for burn victims are taken from Wilmore and Aulick (49) for a patient with fullthickness burns encompassing* ≥*50% of their total body surface area, which results in an 80% increase in resting energy expenditure.*

*e As chronic cold exposure results in a 50% increase in BAT volume [van der Lans et al. (44)], BAT values derived from Chondronikola et al. (24) were multiplied by a factor of 1.5.*

*f WAT values for healthy individuals derived from Gallagher et al. (48) were multiplied by a factor of 3 to account for the increase in leak respiratory capacity of WAT seen in burn victims (47).*

*Values for acute cold exposure and severe burn injury reported in Table 1 are presented as the change (*Δ*) from healthy values and also as a percentage (%) of the increase in whole-body energy expenditure.*

potential contribution of BAT used burn injury as a model of severe adrenergic stress which is accompanied by hypermetabolism. We made calculations of whole-body and tissue-specific energy expenditure based on a patient with a burn covering 50% of their total body surface area, which resulted in an 80% increase in REE (49).

While the impact of burn trauma on BAT is largely unknown, we theorized that like chronic intermittent cold exposure, prolonged adrenergic stress following severe burn trauma would expand BAT depots. Assuming an ~50% increase in BAT volume as seen with 10 days of mild cold exposure (44), BAT would account for 191 kcal of total REE (**Table 1**). Here, while BAT makes a greater contribution to REE in absolute terms, since burn injury results in a large increase in REE, the relative contribution of BAT to whole-body REE is slightly lower (6.3%) when compared to cold exposure (7%) (**Table 1**). Perhaps more strikingly, when considering WATs contribution to REE in burn victims, the threefold increase in thermogenic capacity we found in subcutaneous WAT of burn victims would increase WATs contribution to whole-body REE to ~7% (**Table 1**). Thus, WAT and BAT may make similar absolute and relative contributions to whole-body REE in burn survivors. Again though, we should point out that these calculations are based on an individual with around 100 g of BAT and 15 kg of WAT, underscoring the profound difference in thermogenic potential per gram of tissue between BAT and WAT.

While the chronic adrenergic stress accompanying severe burn trauma provides a unique model where human WAT undergoes browning, it also offers significant insight into the physiological impact of achieving UCP1 activation and recruitment. In **Table 2**, we present the values reported in **Table 1** as the change from normal and also as a percentage in which each tissue contributes to the increase in whole-body REE. While the 127 kcal increase in REE following acute cold exposure is attributable to BAT, several organs contribute to the massive (1230 kcal) increase in REE following burn severe trauma. For example, while burn trauma may result in large absolute increases in BAT and WAT metabolic rates, their relative contributions to total REE are less impressive. Indeed, skeletal muscle (28%), the liver (24%), and the heart (16%) contribute most to burn-induced hypermetabolism, followed by BAT (14%) and WAT (10%) (**Table 2**).

We make the point above to underscore the fact that while the chronic stress response to burns may augment BAT and WAT thermogenesis, the systemic milieu which brings these adaptations in adipose tissue energy expenditure likely impacts many other organs. While our data on burn victims (47) provide novel evidence that human WAT can indeed undergo browning under very severe and extended adrenergic stress, this extreme pathophysiological state has an impact on nearly every organ system in the body. Thus, with all the available evidence to date, we suggest that the therapeutic potential of UCP1-positive mitochondria in combating obesity and its metabolic complications will likely be realized in humans by the acute activation of existing BAT and/ or the expansion of BAT depots. If WAT browning holds any therapeutic potential in the context of obesity, the development of safe tissue-specific agents to induce browning is needed before this potential can be realized.

#### Summary

Adipocytes with an abundance of UCP1-positive mitochondria are unique in that gram for gram they have a respiratory capacity akin to that of muscle or liver, yet only minimal ability to produce ATP. The physiological role ascribed to this adaptation in mitochondrial energy transduction is heat production, facilitating thermoregulation in mammals. In the context of the global obesity epidemic, there is now much interest in the environmental and/or pharmacological exploitation of this thermoregulatory mechanism, since increased metabolic rate will combat obesity and the deleterious metabolic phenotype it incurs. In humans, a growing body of evidence suggests that acute activation of BAT can indeed have a meaningful impact on REE and intermediary glucose and lipid metabolism. However, whether brown adipocytes can be recruited in WAT and whether this alters REE or macronutrient metabolism remains unclear. Few human models mimic the extremity of confining rodents to near-freezing temperatures for several weeks, and in our study of severely burned patients, while prolonged stress does indeed brown WAT, several other organs are also significantly affected. Thus, to avoid the fate of the mitochondrial uncoupler dinitrophenol, future strategies aimed at augmenting UCP1 content and function in humans must strive to be tissue specific.

### Acknowledgments

This work was supported by grants from the National Institutes of Health (P50-GM60338, R01-GM05668, T32 GM008256, H133P110012, and a Clinical and Translational Science Award UL1TR000071) as well as grants from the Shriners Hospitals for Children (85310 and 84090), the John Sealy Memorial Endowment Fund for Biomedical Research (66992), ADA (1-14-TS-35), and the Sealy Center on Aging at UTMB. CP was supported, in part, by a National Institute of Disability and Rehabilitation Research Training Grant (H133P110012). MC is supported by the Onassis Foundation.

### References

diabetic status determine the prevalence, mass, and glucose-uptake activity of 18F-FDG-detected BAT in humans. *J Clin Endocrinol Metab* (2011) **96**(1):192–9. doi:10.1210/jc.2010-0989

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Porter, Chondronikola and Sidossis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Biodegradable Polymeric Microsphere-Based Drug Delivery for inductive Browning of Fat

*Chunhui Jiang1,2† , Liangju Kuang1,2† , Madeline P. Merkel2,3 , Feng Yue4 , Mario Alberto Cano-Vega1,2 , Naagarajan Narayanan1,2 , Shihuan Kuang4,5\* and Meng Deng1,2,6,7\**

*1Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN, USA, 2Bindley Bioscience Center, Purdue University, West Lafayette, IN, USA, 3College of Pharmacy, Purdue University, West Lafayette, IN, USA, 4Department of Animal Sciences, Purdue University, West Lafayette, IN, USA, 5Center for Cancer Research, Purdue University, West Lafayette, IN, USA, 6School of Materials Engineering, Purdue University, West Lafayette, IN, USA, 7Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA*

#### *Edited by:*

*Jun Wu, University of Michigan, USA*

#### *Reviewed by:*

*Sean Bong Lee, Tulane University, USA Wei Zhang, Johns Hopkins University School of Medicine, USA*

#### *\*Correspondence:*

*Shihuan Kuang<EMAIL_ADDRESS>Meng Deng<EMAIL_ADDRESS>
*† Chunhui Jiang and Liangju Kuang have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology*

> *Received: 19 September 2015 Accepted: 23 October 2015 Published: 09 November 2015*

#### *Citation:*

*Jiang C, Kuang L, Merkel MP, Yue F, Cano-Vega MA, Narayanan N, Kuang S and Deng M (2015) Biodegradable Polymeric Microsphere-Based Drug Delivery for Inductive Browning of Fat. Front. Endocrinol. 6:169. doi: 10.3389/fendo.2015.00169*

Brown and beige adipocytes are potent therapeutic agents to increase energy expenditure and reduce risks of obesity and its affiliated metabolic symptoms. One strategy to increase beige adipocyte content is through inhibition of the evolutionarily conserved Notch signaling pathway. However, systemic delivery of Notch inhibitors is associated with off-target effects and multiple dosages of application further faces technical and translational challenges. Here, we report the development of a biodegradable polymeric microsphere-based drug delivery system for sustained, local release of a Notch inhibitor, DBZ. The microsphere-based delivery system was fabricated and optimized using an emulsion/solvent evaporation technique to encapsulate DBZ into poly(lactide-*co*-glycolide) (PLGA), a commonly used biodegradable polymer for controlled drug release. Release studies revealed the ability of PLGA microspheres to release DBZ in a sustained manner. Co-culture of white adipocytes with and without DBZ-loaded PLGA microspheres demonstrated that the released DBZ retained its bioactivity, and effectively inhibited Notch and promoted browning of white adipocytes. Injection of these DBZloaded PLGA microspheres into mouse inguinal white adipose tissue depots resulted in browning *in vivo*. Our results provide the encouraging proof-of-principle evidence for the application of biodegradable polymers as a controlled release platform for delivery of browning factors, and pave the way for development of new translational therapeutic strategies for treatment of obesity.

Keywords: Notch inhibition, browning, PLGA, microspheres, drug delivery, obesity

## INTRODUCTION

The obesity epidemic has posed a major concern for public health in modern society due to its association with a spectrum of metabolic diseases, including type 2 diabetes (T2D), heart diseases, hyperglycemia, and multiple cancers (1, 2). Obesity is morphologically characterized by the excessive lipid storage in the white adipose tissue (WAT) and featured as a complex disorder of energy imbalance wherein intake outpaces expenditure. Considerable efforts have been devoted to explore the pathological mechanisms as well as the cellular and molecular therapeutic targets to combat obesity.

Currently, there are few medications available for obesity treatment (3). Mostly, those therapies are dedicated to decreasing energy intake by either suppressing appetite through stimulating the central nervous system or reducing nutrient digestion and absorption within the gastrointestinal tract (4). However, those medications only produce modest effects and are usually accompanied with unpleasant, potentially harmful side effects (5). Thus, alternative approaches to overcoming obesity are under extensive exploration. One such approach is through boosting energy expenditure. Recently, this strategy through stimulating thermogenesis has been emerging as an appealing alternative.

Brown adipose tissue (BAT) is the primary site of thermogenesis through uncoupling mitochondrial respiration and ATP synthesis via uncoupling protein 1 (Ucp1). While producing heat, BAT consumes not only free fatty acid but also large amounts of glucose (6, 7), thus providing benefits to metabolic health. Classical BAT is abundant in neonates but diminishes with age leading to limited quantities in adult humans. This compromises the therapeutic value of BAT in clinical intervention of obesity.

The recent discovery of inducible BAT (iBAT), also called beige adipose tissue, in WAT depots reignites the promise of obesity treatment through stimulating energy expenditure (8, 9). Beige adipocytes also possess robust thermogenic capacity, which has been confirmed recently by the transplantation of an engineered beige adipose tissue (10). More importantly, iBAT can be stimulated to burn energy in response to multiple pharmacological and genetic manipulations. Various factors have been identified to induce the transformation from white to beige adipose tissue (termed "browning") over recent years (11, 12).

We have recently reported that inhibition of the Notch signaling pathway leads to browning of WAT, and thus ameliorates obesity (13). Notch signaling is well known to play fundamental roles in organ development and cell fate determination, but its regulatory roles in energy homeostasis has previously been underappreciated. We showed that genetic deletion of Notch1 receptor or the nuclear mediator of Notch signaling (Rbpj) in mice improved their glucose tolerance and insulin sensitivity through inducing browning of WAT. Importantly, Notch inhibition through intraperitoneal injection of dibenzazepine (DBZ), a γ-secretase inhibitor, similarly improved insulin sensitivity and glucose homeostasis. However, it remains elusive whether this systemic administration of DBZ causes any uncontrolled drug distribution and potential off-target side effects as Notch has been recognized to play multifaceted roles in various types of cells (14). Therefore, it is imperative to develop an effective drug delivery system to locally and continuously exert effects of Notch inhibition specifically on WAT.

Polymer-based drug delivery systems provide a robust technology platform to enable sustained, spatio-temporally controlled drug release (15). Biodegradable polymers are of utmost interest, because they can be broken down and excreted or resorbed by the body without the necessity for removal interventions or surgical procedures (16, 17). Biodegradable polymer microspheres with predetermined degradation and drug release profiles have been a primary research focus for development of effective drug delivery systems for a variety of applications, including cancer, cardiovascular diseases, and vaccine development (18, 19). Among a variety of polymers that have been employed for drug delivery, poly(lactide-*co*-glycolide) (PLGA), copolymers of lactic acid and glycolic acid, have been particularly prominent due to biocompatibility, biodegradability, and convenient processability (20). PLGA microspheres constitute a versatile class of drug delivery vehicles from which the drug release kinetics can be controlled by modulating the microsphere size, molecular weight, and composition of PLGA (21, 22).

In this study, we aimed to develop a PLGA microsphere-based DBZ delivery system to promote browning of WAT for the treatment of obesity (**Figure 1**). PLGA with a 50:50 lactide to glycolide ratio was utilized since this specific polymer has a relatively fast degradation rate among the PLGA family, which is advantageous to induce browning. We formulated the DBZ-loaded PLGA microspheres using an emulsion/solvent evaporation technique and characterized the morphology and release profiles of those microspheres. Moreover, we demonstrated that the DBZ-loaded PLGA microspheres inhibited Notch, and consequently promoted browning both *in vitro* and *in vivo*. To the best of our knowledge, this is the first time that an effective bioengineered system was developed to deliver therapeutic agents to convert white adipocytes to beige adipocytes. This study not only contributes to our understanding of the underlying mechanism of Notch inhibition in the browning process but also paves the way for development of novel therapeutic strategies to counteract obesity and its associated metabolic syndrome in humans.

### MATERIALS AND METHODS

#### Materials

PLGA (lactide:glycolide 50:50; Mw = 75,000) was obtained from lakeshore Biomaterials. Poly(vinyl alcohol) (PVA; 87–90% hydrolyzed, average Mw = 30,000–70,000), fluorescein isothiocyanatelabeled dextran (FITC-dextran) (Mw = 70,000), and DBZ were obtained from Sigma. Methylene chloride (DCM), dimethyl sulfoxide (DMSO), and acetonitrile were purchased from Fisher Scientific.

#### Animals

All the mice used in this study were wild-type lean mice kept under normal maintenance in a clean facility at Purdue University. All procedures regarding animal maintenance and experimental use were conducted following the regulation presented by Purdue University's Animal Care and Use Committee.

## FITC-Dextran-Loaded Microsphere Formulation and Characterization

Fluorescein isothiocyanate-labeled dextran-loaded PLGA microsphere was prepared by a water-in-oil-in-water (WOW) double emulsion technique (23). Briefly, 0.4 g of PLGA was dissolved in 4 mL DCM. Into this organic phase (O), 330 μL of aqueous solution (W1) containing ~3.7 mg of FITC-dextran was emulsified using a vortex mixer operating at 1,000 rpm for 3 min to form the W1/O emulsion. This primary emulsion was injected into 400 mL of

an aqueous phase containing 1% (w/v) PVA (W2). The resulting W1/O/W2 emulsion was stirred at 400 rpm for overnight with an overhead magnetic stirrer to allow solvent evaporation and microspheres hardening. The microspheres were then isolated by centrifugation, washed three times with distilled water, and were dried in a vacuum oven for 24 h. Final products were stored in a desiccator. FITC-dextran-loaded PLGA microspheres were imaged by fluorescence microscopy (EVOS FL).

## DBZ-Loaded Microsphere Formulation and Characterization

PLGA microspheres loaded with DBZ were prepared by an oil-inwater (O/W) emulsion/solvent evaporation technique (24). The oil phase consisted of 540 mg of PLGA and 10.8 mg of DBZ dissolved in 2.7 mL DCM. This oil phase was injected into 400 mL of an aqueous phase containing 1% (w/v) PVA, which was stirred at 600 rpm to achieve an O/W emulsion system. The resulting emulsion was stirred overnight with an overhead magnetic stirrer to allow complete evaporation of the solvent and solidification of the droplets into microspheres. The microspheres were then isolated by centrifugation, washed three times with distilled water, and dried in a vacuum oven for 24 h. Final products were stored in a desiccator. The microsphere surface structure was investigated by scanning electron microscopy (SEM) (Nova Nano SEM). The microsphere diameter was measured using Image-J software, and about 130 microspheres were randomly selected for analysis.

The content of DBZ in microspheres was analyzed by a precipitation method. Five milligrams of DBZ-loaded PLGA microspheres were completely dissolved in chloroform. Once dissolved, the chloroform solution was added dropwise into 4 mL of methanol in a centrifuge tube to dissolve DBZ and precipitate PLGA. DBZ in the supernatant was collected and concentrated under nitrogen stream. Then, DBZ was dried in the vacuum oven for overnight and dissolved in 10 mL of 2:1 acetonitrile to Millipore water. The product was filtered for high-performance liquid chromatography (HPLC) analysis (Thermo HPLC). The samples were analyzed using a mobile phase of acetonitrile to 0.1% phosphoric acid (50:50) at a flow rate of 1 mL/min on a pentafluorophenylpropyl column and UV detection at 232 nm. Each sample was measured in duplicates. Actual drug loading and drug encapsulation efficiency were calculated using the following equations:

$$\text{Theoretical DBZ loading } \%= \frac{\text{DBZ (Total)}}{\text{DBZ (Total)} + \text{PLGA}} \times 100\% \tag{1.1}$$

$$\text{Actual DBZ loading } \%= \frac{\text{DBZ (Experimental)}}{\text{DBZ (Total)} + \text{PLGA}} \times 100\% \tag{1.2}$$

$$\text{Encapsulation efficiency } \%= \frac{\text{Actual DBZ loading}}{\text{Theoretical DBZ loading}} \times 100\%$$

$$\bf (1.3)$$

## *In vitro* FITC-Dextran Release from Microspheres

Fluorescein isothiocyanate-labeled dextran released from microspheres was measured by suspending ~20 mg microspheres in 5 mL PBS buffer at pH 7.4. The samples were placed in a shaker maintained at 37°C and shaken at 200 rpm. At predetermined time intervals, the samples were removed from the shaker and centrifuged at 1,000 × *g* for 2 min. Two milliliters of medium was aliquoted for analysis and fresh medium of equal volume was added thereafter. The precipitated microsphere pellets were resuspended in the medium and placed back in the shaker. *In vitro* release of FITC-dextran was studied in triplicate. The FITC-dextran concentration in the aqueous phase was determined fluorometrically (excitation: 485 nm, emission: 520 nm, Synergy H1 microplate reader) using a standard calibration curve.

## *In vitro* DBZ Release from Microspheres

The DBZ-loaded PLGA microspheres were characterized for drug release for 6 days in PBS at pH 7.4 and 37°C. DBZ release was measured using HPLC. In brief, 20 mg samples were placed in individual centrifuge tubes and filled with 2.5 mL of PBS. The samples were then placed in a shaker maintained at 37°C and shaken at 200 rpm. At specific time points, 1 mL medium was removed, saved for analysis, and replaced with same amount of fresh PBS. Time points were chosen such that perfect sink conditions were maintained. Each supernatant sample was extracted with 1 mL chloroform. The organic layer was then separated and allowed to evaporate. The dried DBZ was then reconstituted with 1 mL of a 40% solution of acetonitrile in water to provide a suitable solution for HPLC analysis (Thermo HPLC).

## Isolation of Primary Preadipocytes

Primary preadipocytes were collected from limb subcutaneous WAT depots and minced into 2–5 mm2 pieces. Then, these pieces were subject to 1.5 mg/mL collagenase digestion with agitation at 37°C for 1.5–2 h. The digestion was terminated with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). After that, the floating mature adipocytes were removed and the cell suspension was filtered through 100 μm mesh, followed by brief centrifugation at 450 × *g* for 5 min. The pellet was resuspended and seeded onto tissue culture plates.

## Co-Culture of Primary Preadipocytes with Microspheres

Primary preadipocytes were cultured in growth medium containing DMEM, 20% FBS, and 1% penicillin/streptomycin at 37°C with 5% CO2. The medium was changed every other day. Upon confluence, cells were subject to induction medium containing DMEM, 10% FBS, 2.85 μM insulin, 0.3 μM dexamethasone, and 0.63 mM 3-isobutyl-methylxanthine for 4 days, followed by differentiation medium containing DMEM, 200 nM insulin, and 10 nM triiodothyronine for four more days. Meanwhile, 5 mg polymer microspheres were added in permeable transwell inserts suspended in each well of 24-well plates while cells were induced for adipogenic differentiation. Other parallel treatment groups included cells in DBZ-containing medium (10 μM) and cells in DMSO vehicle control. To monitor adipogenic differentiation, the lipid droplets and nuclei of adipocytes during culture were counterstained with BODIPY and DAPI, respectively.

## Quantitative Polymerase Chain Reaction

Total RNA was extracted from cell culture through Trizol. Random hexamer primers were utilized for the reverse transcription to synthesize cDNA. The quantitative polymerase chain reaction (qPCR) was performed with a Roche Light Cycler 96 machine (Roche). The 18S rRNA was applied as an internal control for normalization. For qPCR result analysis, the 2−ΔΔct method was used to calculate the fold change.

## Protein Extraction and Western Blots Analysis

Total protein was extracted from cells or tissue samples using RIPA buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% Non-idet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). Protein concentrations were measured by using Pierce BCA protein assay reagent (Pierce Biotechnology, Rockford, IL, USA). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Billerica, MA, USA). Membranes were blocked in 5% milk for 1 h and incubated with primary antibodies at 4°C overnight. The PGC1-α (sc-13067) and GAPDH (sc-32233) antibodies were purchased from Santa Cruz Biotechnology, and both were diluted 1:1,000. The horseradish peroxidase (HRP) conjugated secondary antibody (anti-rabbit IgG, 7074S, Cell Signaling) was diluted 1:5,000. Signals were detected with a ChemiDoc™ Touch Imaging System (Bio-Rad). The lanes were analyzed for densitometry quantification with Bio-Rad Image Lab V5.2.1.

### *In vivo* Injection of PLGA Microspheres

Mice were first anesthetized by a ketamine–xylazine cocktail and then either FITC-dextran-loaded PLGA microspheres or DBZ-loaded PLGA microspheres (20 mg/30 g body weight) were injected into the inguinal WAT depot in one side of the body in a 1 mL solution of 0.5% Methocel E4M (wt/vol; Dow Chemical) and 0.1% Tween-80 (wt/vol; Sigma) in water. Similarly, PLGA microspheres were injected into the contralateral inguinal WAT depot. Adipose tissues were harvested after 24 h and 14 days post-injection for the study with FITC-dextran-loaded PLGA microspheres and DBZ-loaded PLGA microspheres, respectively. Oil Red O staining was performed to label the adipocytes for the study with FITC-dextran-loaded PLGA microspheres whereas hematoxylin and eosin (H&E) and immunohistochemistry staining was conducted for the study with DBZ-loaded PLGA microspheres.

## H&E and Immunohistochemistry Staining

Adipose tissues were fixed in 10% formalin for 24 h at room temperature. Then, the tissues were embedded into paraffin and cut into 4 μm thick slices, deparaffinized, and rehydrated using xylene, ethanol, and water by standard methods. Immunohistochemistry was performed on a Dako Autostainer (Dako). Slides were incubated with 3% hydrogen peroxide and 2.5% normal horse serum (S-2012, Vector), followed by incubation with rabbit polyclonal anti-Ucp1 primary antibody diluted 1:200 in 2.5% normal horse

serum (S-2012, Vector) for 60 min. Signals were detected with an anti-rabbit IgG Polymer Detection Kit (MP-7401, Vector). Labeling was visualized with 3,3′-diaminobenzidine (DAB) as the chromogen (SK-4105, Vector). Slides were counterstained with Harris hematoxylin (EK Industries), and whole-slide digital images were collected with an Aperio Scan Scope slide scanner (Aperio).

## Statistical Analysis

Quantitative data were reported as mean ± SEM *P*-values were calculated by a two-tailed Student's *t*-test. *P*< 0.05 was considered to be statistically significant.

## RESULTS

## Formulation and Characterization of PLGA Microspheres

Using PLGA 50:50, drug-loaded PLGA microspheres were prepared and characterized for drug distribution and morphological characteristics. We first encapsulated FITC-dextran, a commonly used fluorescent probe, into PLGA microspheres to assess the distribution of FITC-dextran in microspheres by fluorescent microscopy. It was observed that green fluorescence was uniformly distributed throughout the microspheres (**Figure 2A**). Next, we formulated DBZ-loaded PLGA microspheres by optimizing the process parameters, such as PVA concentration and stirring speed, to obtain a narrow microsphere size range of 50–150 μm (**Figure 2B**). Higher magnification SEM micrograph revealed the smooth surface morphology of the microsphere (**Figure 2C**). The size distribution of the DBZ-loaded PLGA microspheres resembled a Gaussian distribution with more than 50% in the range of 90–110 μm (**Figure 2D**). The actual DBZ loading percentage was characterized to be ~1.4% (w/w), equivalent to ~68% DBZ encapsulation efficiency.

## PLGA Microsphere System Enables FITC-Dextran and DBZ Release

To examine the cargo drug release capabilities from the PLGA microspheres, we first placed the FITC-dextran-loaded PLGA microspheres in the PBS at 37°C and release was quantified fluorometrically. The FITC-dextran-loaded PLGA microspheres showed a rapid release profile, which resulted in about 50% of total FITC-dextran released after 24 h (**Figure 3A**). This is presumably due to the hydrophilic nature of the FITC-dextran. We further characterized the release profile of DBZ from DBZloaded PLGA microspheres (**Figure 3B**). In general, the release