Phenylbutyrate Ameliorates High-Fat Diet-Induced Obesity via Brown Adipose Tissue Activation

Abstract

Obesity, which is characterized by an excessive accumulation of body fat, is one of the critical factors causing metabolic syndrome. Many studies have been performed to identify appropriate agents to control obesity, but toxicity remains a problem. Herein, we identified that phenylbutyrate (PBA), which has been used to treat urea cycle disorder with very low toxicity for a long time, efficiently inhibited high fat-induced body weight gain in a diet-induced obesity mouse model (DIO model). PBA treatment decreased body fat mass and increased lean composition. Moreover, PBA increased brown adipose tissue (BAT) activity by increasing glucose uptake, thereby improving glucose tolerance and insulin tolerance. Interestingly, PBA could induce the expression of liver type phosphofructokinase (PFKL), a key enzyme in the glycolytic pathway, and knocking down PFKL dramatically repressed the expression level of Ucp1 as well as those of Prdm16, Cidea, Pgc1α, and Pparγ, which are marker genes for BAT activation. These results strongly suggested that PBA could increase energy expenditure by increasing BAT activity via the induction of PFKL. Taken together, PBA could be used as a therapeutic agent for people with obesity to prevent the development of metabolic syndrome.

INTRODUCTION

The imbalance between energy expenditure and food intake is a major factor in obesity, which is characterized by increased body fat accumulation. Adipose tissue is a functional organ that regulates energy homeostasis by alternating between energy storage in the form of triglycerides and energy supply in the form of free fatty acids, depending on the energy status of the body. Continued energy intake stimulates fat cells to proliferate to store excess energy and makes the cells enlarged, leading to inflammation in fat tissue.¹⁾

Recent studies on overcoming obesity have focused on activating signaling pathways of fat consumption, in which generated energy is dissipated as heat. Uncoupling protein 1 (UCP1) is a well-known key molecule that dissipates energy as heat by uncoupling protons in the inner membrane of mitochondria. UCP1 is most abundant in brown adipose tissue (BAT), which is an organ specialized to consume excess energy as heat. In addition, recent studies have identified that UCP1 expression is inducible in white adipose tissue (WAT) upon cold exposure and beta-adrenergic receptor agonist treatment, converting the basic function of WAT from fat storage tissue to fat consumption tissue called beige fat. In either case, UCP1 is indispensable for the consumption of excess energy. The discovery of functional BAT in human adults has resulted in the explosion of BAT studies, and BAT has been identified as a promising target to treat patients with obesity-related disease. UCP1-ablated mice showed an obesity phenotype even when fed a control diet and did not induce diet-induced thermogenesis.²⁾ In contrast, UCP1 transgenic mice driven by aP2 promoter showed resistance to diet-induced obesity.^3–5)

Phenylbutyrate (PBA) has been used to treat urea cycle disorder, which is a genetic disorder caused by a deficiency of one of the enzymes in the urea cycle. Its application has been expanded to various diseases, such as malignancies,^6–8) cystic fibrosis⁹⁾ and sickle cell anemia.¹⁰⁾ As a histone deacetylase inhibitor, PBA has recently been shown to ameliorate endoplasmic reticulum (ER) stress-mediated metabolic disease.¹¹⁾ PBA reduced body weight gain and adipogenesis in diet-induced obesity mouse model.¹²⁾ PBA administration also improved obesity-induced chronic inflammation in adipose tissue¹³⁾ protected against liver ischemia reperfusion injury by inhibition of ER stress¹⁴⁾ and liver steatosis by inhibition of de novo lipogenesis.¹⁵⁾ Additionally, PBA has been tested in patients with spinal muscular atrophy and shown beneficial effects,¹⁶⁾ indicating that PBA has various beneficial effects on metabolic organs.

In this study, we demonstrate that PBA ameliorates high-fat diet-induced obesity by increasing the activities of brown adipose tissue. Therefore, PBA could be a therapeutic agent for patients with obesity-related disease by targeting brown adipose tissue.

MATERIALS AND METHODS

Animals

Male C57BL/6J mice (8 weeks old) were fed with Low-fat diet (LFD-10% fat, Research Diets D12450B) or High-fat diet (HFD-60% fat, Research Diets D12492) for 8 weeks. After that, each group of mice was subjected to gavage every 12 h with vehicle (distilled water) or sodium PBA (500 mg/kg,^11,17) Santa Cruz Biotechnology, U.S.A., sc-200652A) for additional 8 weeks while being fed LFD and HFD for monitoring body weight gain. Body weight and food intake were monitored on a weekly basis. The mice were acclimatized to a 12 h light–dark cycle at 22–24°C. All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory animals (NIH Publication No. 8023, revised 1978).

In Vivo Positron Emission Tomography/Computed Tomography (PET/CT) Analysis and in Vitro Glucose Measurement Using 18F-Fluorodeoxyglucose (18F-FDG)

Mice were administered intravenously with 7.4 MBq of 18F-FDG, and then a 20-min scan was performed with a Triumph II PET/CT system (LabPET8, Gamma Medica-Ideas Inc., Waukesha, WI, U.S.A.) at 30–60 min post injection. A CT scan was done with an X-ray detector right after the acquisition of PET imaging. PET images were reconstructed by three dimensional (3D)-ordered subset expectation maximization (OSEM) iterative image reconstruction, and reconstructions for CT images were performed using filtered back-projections. All mice were anesthetized using 1–2% isoflurane gas during imaging. Functional PET images were coregistered with anatomical CT images using 3D image visualization and analysis software VIVID (Gamma Medica-ideas, Northridge, CA, U.S.A.). The uptake (%ID/cc) for the volumes of interest (VOIs) from each image were determined by manually drawing the brown adipose tissue of interest in coregistered CT images using PMOD 3.5 software (PMOD Technologies, Zurich, Switzerland).

For in vitro glucose measurement using 18F-FDG, immortalized brown adipocyte (IBA) cells were seeded in a 24-well plate and differentiated. For measurement of FDG uptake, the differentiated IBAs were incubated with Dulbecco’s modified Eagle’s medium (DMEM) 5.5 mM glucose containing 0.5% fetal bovine serum (FBS) medium for 12 h. The medium was changed to Krebs–Ringer N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) (KRH) buffer (50 mM HEPES, 137 mM NaCl, 4.7 mM KCl, 1.85 mM CaCl₂, 1.3 mM MgSO₄, and 0.1% bovine serum albumin (BSA)) for 2 h, and the chemicals were administered for 30 min.

Fat Composition Analysis

After sodium PBA administration for 10 weeks, fat mass and lean body mass were measured and analyzed by nuclear magnetic resonance using a Bruker Mouse Minispec (Bruker-LF50 Model) following the manufacturer’s instructions.

Glucose Tolerance Tests and Insulin Tolerance Test

For the glucose tolerance test (GTT), the mice that were administered sodium PBA for 8 weeks were fasted for 16 h, then received D-glucose (1.5 g/kg) via an intraperitoneal (i.p.) injection. Additional sodium PBA was treated 2 h before measurement. For the insulin tolerance test (ITT), the mice that were administered sodium PBA for 9 weeks were fasted for 6 h, then received insulin (0.75 U/kg) via an i.p. injection. Additional sodium PBA was treated 2 h before measurement. Blood glucose levels were measured by an ACCU-CHECK Active blood sugar test meter (Roche, Switzerland) at the indicated time points (0, 15, 30, 45, 60, 90 and 120 min).

Total mRNA Extraction and Quantitative RT-PCR

Total RNA from cells was extracted using QIAzol reagent (Qiagen, Valencia, CA, U.S.A.) and cDNA was synthesized using a RevertAid™ First Strand cDNA Synthesiskit (Fermentas, Vilnius, Lithuania) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using SYBR Green (SYBR Green Master Mix, Applied Biosystems, Warrington, U.K.) with a StepOne™ Real-Time PCR system (Applied Biosystems). The primers used were as follows: UCP1, forward: 5′-GGT CGT GAA GGT CAG AAT GCA-3′, reverse: 5′-GCA TTG TAG GTC CCC GTG TAG-3′; PGC1a, forward: 5′-TGC GGG ATG ATG GAG ACA-3′, reverse: 5′-GCG AAA GCG TCA CAG GTG TA-3′; PRDM16, forward: 5′-AAC CAG GCA TCC ACT CGA AT-3′, reverse: 5′-TGG ACA CGG TGG GTT GCT-3′; Cidea, forward: 5′-TGA CCC CCC TCA TAC ATC CA-3′, reverse: 5′-GGC TAC TTC GGT CAT GGT TTG-3′; peroxisome proliferator-activated receptor γ (PPARγ), forward: 5′-CAC AAG AGC TGA CCC AAT GGT-3′, reverse: 5′-GAT CGC ACT TTG GTA TTC TTG GA-3′; phosphofructokinase (PFKL), forward: 5′-ATC GGC CTG CGG AAG AAG-3′, reverse: 5′-CGG TGC TCA AAA TCA GTC TCT TT-3′; 36B4, forward: 5′-ACC TCC TTC TTC CAG GCT TT-3′, reverse: 5′-CTC CAG TCT TTA TCA GCT GC-3′.

Metabolic Phenocages

To measure the metabolic parameters, which included VO₂, VCO₂, physical activity, food consumption and water consumption, the mice were fed LFD or HFD for 21 weeks with vehicle (distilled water) or PBA sodium PBA (500 mg/kg, twice/d by oral gavage) for last 13 weeks. After then the mice were housed individually in an indirect calorimetric cage (TSE PhenoMaster) as previously reported.¹⁸⁾ Each mouse was monitored during 48 h in the fed state and all results were analyzed for 24 h on average. The energy expenditure was calculated using VO₂ and VCO₂.

Primary Culture from BAT

The interscapular brown adipose tissues of 3- to 4-week-old mice were minced in a collagenase buffer and digested at 37°C at 160 rpm for 25 min. The digested fat tissue was filtrated through a 100-µm cell strainer and centrifuged at 1500 rpm for 5 min. The supernatant was removed, and pellet containing the stromal vascular fraction cells was filtrated through a 40-µm cell strainer. After centrifugation, the pellet was plated in high glucose-DMEM supplemented with 10% bovine serum. For brown adipocyte differentiation, confluent precursor cells were incubated in medium supplemented with 5 µg/mL insulin, 0.5 µM dexamethasone, 0.25 mM IBMX, 2 nM T3, 0.065 mM indomethacin, 1 µM rosiglitazone and 10% fetal bovine serum. After 2 d, the medium was replaced with medium supplemented with 1 µg/mL insulin, 2 nM T3 and 10% FBS and then changed every 2 d with medium containing 10% FBS and 2 nM T3.

IBA Culture

The interscapular immortalized brown adipocytes were established using SV40T retrovirus as previously described.^19,20) IBA differentiation was performed as previously reported.²¹⁾ When cells were reached more than 90% confluence, the medium was replaced to DMEM with 10% fetal bovine serum and cells were treated with insulin (0.5 µg/mL), T3 (1 nM), indomethacin (0.125 mM), dexamethasone (2 µg/mL) and IBMX (0.5 µM) for 2 d to differentiate cells to mature brown adipocyte. The medium was changed to DMEM with 10% fetal bovine serum and cells were treated with insulin (0.5 µg/mL) and T3 (1 nM) in every 2 d until 6 d.

Western Blot Analysis

Total cell lysates were prepared using lysis buffer containing 20 mM Tris (pH 7.4), 5 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), 10 mM Na₄P₂O₇, 100 mM NaF, 2 mM Na₃VO₄, 1% NP-40, 0.1 mM PMSF, and proteinase and phosphatase inhibitors. Protein concentration was measured by BCA protein assay reagent (Thermo Fisher Scientific; 23225). The PFKL primary antibodies were purchased from Abcam (Cambridge, U.K.). Detection of each protein was performed using an ECL Western blotting detection system.

Small Interfering RNA (siRNA) Transfection

Seventy percent confluent primary brown fat precursor cells were transfected with 25 nM siRNA targeting PFKL (sense, 5′-UGC ACC GCA UUA UGG AGG U(dTdT)-3′ and antisense, 5′-ACC UCC AUA AUG CGG UGC A(dTdT)-3′; Bioneer Corporation, Daejeon, South Korea) or nonsilencing control siRNA (Bioneer Corporation) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. After 24 h, the medium was replaced with fresh DMEM containing 10% bovine serum. For induction of adipocyte differentiation, the cells were incubated for two days, and replaced with the differentiation medium.

Immunohistochemistry

For immunohistochemical staining, deparaffinized subcutaneous adipose tissue sections were subjected to antigen retrieval and blocked with Ultra V Block (Lab Vision Corporation, Fremont, CA, U.S.A.) for 10 min at room temperature. Sections were incubated with primary antibodies against anti-UCP1 Antibody (diluted 1 : 500; Abcam), followed by horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) secondary antibodies (Lab Vision Corporation) according to the manufacturer’s instructions. Detection of the antibody complex was performed using the DAB+ substrate chromogen (Dako, Glostrup, Denmark).

Statistical Analyses

Data are expressed as the mean ± standard error of the mean (S.E.M.). Significant differences between groups were calculated using Student’s t-test for normally distributed data. In addition, one-way or two-way ANOVA followed by least significant difference (LSD) post hoc test. p < 0.05 was considered statistically significant.

RESULTS

Phenylbutyrate Ameliorates HFD-Induced Obesity

To see whether PBA has an effect on high-fat diet-induced body weight gain, 8-week-old mice were fed HFD (60% fat) and LFD (10% fat) for 8 weeks, and then PBA was administered for an additional 8 weeks with a vehicle control while being fed HFD and LFD. As expected, HFD dramatically induced body weight gain as mice aged, but PBA treatment strongly prevented and showed resistance to HFD-mediated obesity. In addition, body weight gains in the group fed LFD were also slightly but significantly reduced by PBA treatment (Fig. 1a), implying that PBA has a therapeutic effect on HFD-induced obesity. To determine which tissues have a dramatic change in weight, we first performed a computed tomography (CT) scan with a live mouse. As shown in Fig. 1b, the most dramatic change in tissue distribution was observed in fat tissue (blue color). The CT scan showed that the dramatic fat accumulation induced by HFD was evidently decreased by treatment with PBA (Fig. 1b). Further analysis of body composition showed that PBA treatment decreased fat mass by 9% (from 51.16 ± 0.67, N = 9 to 47.01 ± 0.94, N = 10), while lean body mass was increased by 8% (from 49.19 ± 0.79, N = 9 to 53.33 ± 1.9, N = 10) in the HFD group. Interestingly, PBA treatment to LFD group showed similar effect to that observed in HFD group (Fig. 1c).

Fig. 1. PBA Ameliorates HFD-Induced Obesity

(a) Mouse at the age of 8 weeks fed with LFD and HFD with or without PBA as depicted in the figure. During diet feeding, body weight was monitored in every weeks (left). A representative mouse is shown (right); n = 5 per LFD and n = 6–7 per HFD group. Values are expressed as mean ± S.E.M. Statistical analysis was performed by two-way ANOVA followed by LSD post hoc test. (b) CT scanning was performed and fat tissue was indicated as blue color. (c) Body composition analysis was performed to compare fat and lean mass between the HFD and HFD + PBA groups fed LFD or HFD; n = 8 per LFD, n = 9 per LFD + PBA, n = 9 HFD, and n = 10 per HFD + PBA group. Values are expressed as mean ± S.E.M. Statistical analysis was performed by one-way ANOVA followed by LSD post hoc test. (d) BAT, liver, sWAT and eWAT were compared. (e) Tissue sections of eWAT from mice fed LFD or HFD for 24 weeks with or without PBA treatment were subjected to hematoxylin and eosin (H&E) staining, and the arrows indicate crown-like structures (CLS). Representative pictures are presented. Magnification: ×400. * p < 0.05, ** p < 0.01 and *** p < 0.001. (Color figure can be accessed in the online version.)

Next, we tried to figure out whether PBA has an effect on other metabolically active organs such as BAT, liver and WAT. Interestingly, we found that the organ weight of brown adipose tissue was lower and the color was more brownish with PBA treatment than with vehicle in the HFD group, indicating that PBA decreases lipid accumulation by HFD in BAT. The color of the liver was also improved from pale to dark brown by PBA administration, which is similar to the normal phenotype and consistent with previous studies showing that PBA ameliorates liver steatosis.¹⁵⁾ As expected, there was smaller subcutaneous fat tissue (sWAT) in the group treated with PBA than in the group treated with vehicle. Unexpectedly, the size of the epididymal fat tissue (eWAT) was larger in the group treated with PBA than in the vehicle control in HFD group. However, the color of the eWAT was more yellowish in the group fed HFD than in the group fed HFD + PBA, indicating that there is a tissue dysfunction which is caused by accumulated fat with severe tissue inflammation and fibrosis by long-term HFD (Fig. 1d), which is consistent with previous report.²²⁾ This observation prompted us to examine tissue histology. Dissected eWAT tissue were subjected to hematoxylin and eosin (H&E) staining. Interestingly, we did not see a difference in the size of adipocytes between the HFD and HFD + PBA groups, but there was a dramatic increased number of crown-like structure (CLS), marked with a black arrow in eWAT in the group fed HFD with vehicle. In contrast, PBA treatment completely blocked the formation of CLS (Fig. 1e). As well as eWAT, HFD-induced lipid accumulation either in sWAT and liver was reduced by PBA treatment compare to vehicle treated HFD group (Supplementary Fig. S1a). According to UCP-1 expression in sWAT, PBA treatment seemed also increase browning of sWAT in LFD but not in HFD group (Supplementary Fig. S1b). Taken together, these results strongly suggested that PBA has beneficial effects on metabolically active organs.

PBA Increases BAT Activity

We hypothesized that the beneficial effect of PBA on HFD-induced mice may be due to the active disposal of glucose in blood and increased energy consumption. To confirm our hypothesis, we tested the effect of PBA on BAT activity, because BAT is well known as an active organ for taking up glucose from blood and converting it into heat and thereby controlling the energy level in the body. BAT from mice fed either LFD or HFD were subjected to H&E staining. As expected, HFD dramatically increased the formation of lipid droplets, but PBA treatment definitely decreased the fat amount in BAT (Fig. 2a). However, we could not make a decision whether the fat formation decreased by PBA is due to the increased BAT activity or other factors. To clarify which aspect is responsible, we measured in vivo glucose uptake using 18F-fluorodeoxyglucose (18F-FDG) PET/CT. Interestingly, the BAT activity decreased by HFD was significantly increased by PBA treatment (Fig. 2b). In line with these results, PBA significantly decreased fasting glucose levels (Fig. 2c) and improved glucose tolerance and insulin tolerance compared to either LFD or HFD with vehicle treated group (Fig. 2d, e). These results indicate that the decreased fat amount in BAT is due to the increased BAT activity by the PBA treatment, which thereby improves the whole body metabolic phenotype systematically.

Fig. 2. PBA Activates BAT

(a) Brown adipose tissue from mice fed LFD or HFD for 16 weeks were dissected, and H&E staining was performed. Magnification: ×400. (b) PET/CT using 18F-FDG was performed using mice fed HFD for 16 weeks (left), and a quantitative analysis was performed (right). n = 5 per each group. Values are expressed as mean ± S.E.M. Statistical analysis was performed by Student’s t-test. (c) Fasting blood glucose levels were measured in mice fasted for 16 h; n = 8–9 per LFD and n = 12–13 per HFD group. (d) GTT was conducted with mice fasted for 16 h. Glucose (1.5 g/kg) was administered by an i.p. injection. Blood glucose was measured at the indicated time points; n = 8–9 per LFD and n = 12–13 per HFD group. (e) ITT was performed with mice fasted for 6 h. Insulin (0.75 U/kg) was administered by an i.p. injection. Blood glucose was measured at the indicated time points; n = 6–8 per LFD and n = 11–12 per HFD group. Values are expressed as mean ± S.E.M. Statistical analysis was performed by one-way ANOVA followed by LSD post hoc test. * p < 0.05, ** p < 0.01 and *** p < 0.001. (Color figure can be accessed in the online version.)

Energy Expenditure Is Increased through PBA-Induced BAT Activation

It is previously reported that BAT activation is followed by increased whole body energy expenditure and it results in decreased body fat and obesity.^23,24) Our study demonstrates that PBA treatment decreased HFD-induced body weight gain and fat accumulation accompanying with BAT activation. Therefore, we tried to figure out whether PBA-induced BAT activation leads to energy expenditure augmentation. PBA treatment increased VO₂, VCO₂ and energy expenditure compared to vehicle treated group under LFD feeding, especially during dark cycle. HFD feeding decreased VO₂, VCO₂ and energy expenditure compared to corresponding LFD feeding group, but PBA treatment recovered all these parameters in both light and dark cycle (Figs. 3a–c). Physical activity, food consumption and water consumption were not altered among all experimental animal groups (Fig. 3d; Supplementary Fig. S2). It suggests that PBA improves metabolic processes without alteration in physical activity.

Fig. 3. PBA Increases Energy Expenditure

(a–d) The metabolic parameters of mice were determined by metabolic phenocages. VO₂ (a), VCO₂ (b), energy expenditure (c) and physical activity (d) were measured and it was represented by light cycle and dark cycle as separately. n = 5 per each group. Values are expressed as mean ± S.E.M. Statistical analysis was performed by one-way ANOVA followed by LSD post hoc test. * p < 0.05, ** p < 0.01 and *** p < 0.001.

PFKL Is Required for PBA Induced BAT Activation

As previously reported, PBA suppresses adipocyte differentiation¹²⁾ and increased differentiation can augment brown adipocyte activity with UCP-1 expression. To avoid any additional effect on PBA-induced brown adipocyte activity from different differentiation degree, we used only fully differentiated cells for the followed in vitro experiments to measure the PBA-induced brown adipocyte activity. Using immortalized brown adipocyte (IBA) cells, we tested whether PBA increased glucose uptake. As shown in Fig. 4a, PBA significantly increased glucose uptake measured with 18F-FDG (Fig. 4a). Next, we tried to measure the expression level of the marker genes of BAT activation such as Pparγ, Pgc1α, Prdm16, and Cidea. We found that PBA treatment significantly increased the transcript levels of Pgc1α, Prdm16, and Cidea but not Pparγ, indicating that PBA can increase BAT activity (Fig. 4b). To gain insight into the underlying mechanism by which PBA increases glucose uptake, we checked the protein expression levels involved in glycolytic pathways. The expression levels of most proteins were slightly decreased or not altered by long-term treatment with PBA, but PFKL showed a gradually increased expression pattern (Fig. 4c). Interestingly, the mRNA levels of Ucp1, Prdm16, Cidea, Pgc1α, and Pparγ were dramatically decreased when PFKL was knocked down using specific siRNA compared with control siRNA (Fig. 4d). Taken together, these results strongly indicate that PBA increases energy expenditure by increasing UCP1 through the induction of PFKL expression.

Fig. 4. PBA Increases Genes Related to BAT Activation and Glycolysis Related Enzyme, PFKL

(a) In vitro glucose uptake measurements using 18F-FDG were performed using differentiated immortalized brown adipocyte (IBA) cells. PBA was administered for 30 min before analysis. Insulin was used as a positive control. n = 3 per each group. (b) Genes related to mitochondrial biogenesis were measured in differentiated primary BAT cells after treatment with PBA for 12 h at a dose of 2 mM. n = 3 per each group. qPCR was performed with specific primers as described in Experimental. (c) The protein expression level of PFKL was examined by Western blot analysis with samples extracted from differentiated primary BAT cells after PBA treatment at the indicated times. PBA was administered at a dose of 2 mM. (d) Silencing of PFKL using siRNA decreased the expression level of genes related to mitochondrial activity in differentiated primary BAT cells. The mRNA levels of Ucp1, Prdm16, Cidea, Pgc1α, and Pparγ were measured by qPCR as described in Materials and Methods. Values are expressed as mean ± S.E.M. Statistical analysis was performed by Student’s t-test. * p < 0.05, ** p < 0.01 and *** p < 0.001.

DISCUSSION

In this study, we demonstrated that PBA could improve HFD-induced obesity by increasing the activities of BAT. PBA increased the expression level of PFKL, a rate-limiting enzyme in glycolysis, indicating that PBA could stimulate the glycolytic pathway. Moreover, PBA increased the expression of genes related to BAT activation such as Pgc1α, Prdm16, and Cidea transcripts, and UCP1, a key protein that dissipates excess energy as heat in mitochondria. Therefore, PBA could stimulate both the glycolytic pathway and BAT activity. Burning of excess glucose in BAT may give rise to an improved phenotype in other metabolically active organs such as liver and fat tissue. In fact, HFD significantly increased lipid accumulation in both liver and fat tissues, but PBA treatment dramatically decreased fat deposition. Even though the weight of eWAT was greater with PBA treatment than with control treatment in the HFD group, the histology analysis identified that crown-like structures (CLS) were significantly decreased in the group treated with PBA. This result indicates that PBA increases the storage capacity of adipocytes for excess energy by increasing fat size and mass without cellular stress. However, further analysis may need to address the unexpected observation that eWAT mass was increased in spite of the decrease in total fat mass by PBA through the comparing other major fat tissues such as retroperitoneal depots and the mesenteric depots.

BAT is one of the major organs that burns excess energy in mitochondria. UCP1 is a central key player in that process. Here, we found that PBA treatment increased UCP1 expression in primary cultured cells, strongly suggesting that PBA-mediated UCP1 induction is a major pathway to decrease the blood glucose level. It is likely that UCP1 expression is transcriptionally regulated by PBA, because the UCP1 mRNA level is highly induced by PBA treatment in primary cultured cells. Even though it is not clear how PBA could increase UCP1 mRNA expression in BAT and what is the role of PFKL in PBA mediated UCP1 induction, a recent study may explain a possible regulation of PBA on UCP1 expression via crosstalk between histone deacetylase (HDAC) and H3 lysine 27 residue²⁵⁾ since PBA has known as a HDAC inhibitor. Further studies needed to address PFKL involvement in PBA mediated UCP1 induction during BAT activation.

Phosphofructokinase 1 (PFK1) converts fructose 6-phosphate and ATP into fructose 1,6-bisphosphate and ADP. Since PFK1 induces ATP-dependent phosphorylation to convert fructose 6-phosphate during glycolysis, it is regarded as one of the rate-limiting enzymes in glycolysis. In this study, we found that the protein expression level of PFKL, one of the isoforms of PFK1, was dramatically increased by PBA treatment. These data strongly suggested that PBA may stimulate the rate-limiting step in glycolysis by upregulating the PFKL enzyme, thereby increasing glucose uptake. Increased PFKL expression by PBA may be occurring at the transcription level because PBA has an inhibitory effect on HDAC. However, further studies need to identify the detailed mechanisms. In addition, knockdown of PFKL in BAT cells suppressed BAT activation related genes. It demonstrated that increased PFKL expression by PBA could regulate BAT activation related genes. Somehow it is unclear whether PFKL induction is essential for PBA-induced BAT activation or it has just a role for that as partly.

In conclusion, we demonstrated that PBA evidently increases BAT activity and ameliorates HFD-induced obesity. Furthermore, PBA induced several genes related to BAT activation, such as Pgc1α, Prdm16, and Cidea as well as Ucp1, indicating that PBA stimulates. Taken together, our data suggest that PBA is a potential therapeutic agent for patients with obesity.

Acknowledgments

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A1A2055894, 2017R1D1A1B03030844, and 2017R1D1A1B03028340), by a Grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea. (Grant Number: HI16C1501), and by the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (2016M3A9B6026771, 2014M3A9D5A01073598).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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