Delta-Tocopherol Suppresses the Dysfunction of Thermogenesis due to Inflammatory Stimulation in Brown Adipocytes

Receptor γ （ PPAR γ ） and other transcriptional factors. Inflammatory signals have been reported to inhibit PGC-1 α activation and UCP1-mediated thermogenesis in brown adipocytes 5, 6 ） . Inflammation of brown adipocytes is mainly caused by cell hypertrophy and macrophage invasion due to obesity 5 − 7 ） . Invading macrophages secrete inflammatory cytokines, including TNF- α , IL1 β , and IL6, which suppress the thermogenesis and inhibit BAT functions. The decrease in thermo-Abstract: Brown adipose tissue (BAT) functions as a radiator for thermogenesis and helps maintain body temperature and regulate metabolism. Inflammatory signals have been reported to inhibit PGC-1α activation and UCP1-mediated thermogenesis in brown adipocytes. Inflammation is mainly caused by cell hypertrophy and macrophage invasion due to obesity, and invading macrophages secrete inflammatory cytokines, including TNF-α, IL1β, and IL6, which suppress the thermogenesis in BAT. Tocopherol is a lipid-soluble vitamin with anti-inflammatory effects is expected to contribute to the suppression of inflammation in adipose tissue. In this study, we investigated the protective effect of tocopherols, α-tocopherol (α-toc) and δ-tocopherol (δ-toc), against brown adipocyte inflammation and thermogenesis dysfunction. cytokine, significantly decreased the protein expression levels of UCP1 and PGC-1α in rat primary brown adipocytes. The pre-incubation of α-toc or δ-toc significantly suppressed the decrease in UCP1 and PGC-1α expression and lipid accumulation. Additionally, α-toc and δ-toc suppress the induction of ERK1/2 gene expression, implying that an anti-inflammatory effect is involved in this protective effect. We fed mice a high-fat diet for 16 weeks and investigated the effects of α-toc and δ-toc in the diet. Intake of α-toc and δ-toc significantly suppressed weight gain and hypertrophy of brown adipocytes. Our results suggest that α-toc and δ-toc suppress the dysfunction of thermogenesis in brown adipocytes due to inflammation and contribute to the treatment of obesity and obesity-related metabolic diseases.


Introduction
Adipose tissue is characterized as either white adipose tissue WAT or brown adipose tissue BAT . WAT serves as an energy storage site, while BAT acts as a radiator for thermogenesis. Classical brown adipocytes are derived from myf-5-positive, muscle-like cellular lineage. BAT has been recognized as an essential tissue for thermoregulation in infancy 1, 2 , but it has also been detected in the supraclavicular and neck region of adult humans using PET-CT 3 . There is a negative correlation between BAT mass and body mass index 4 , so it is gaining attention as a potential target for anti-obesity studies. Brown adipocytes possess large numbers of mitochondria, which contain the uncoupling protein 1 UCP1 , and activated UCP1 uncouples the genesis due to the dysfunction of brown adipocytes further reduces energy consumption, resulting in a vicious cycle. Maintaining the function of brown adipocytes is a critical issue in preventing obesity and metabolic diseases.
Tocopherols toc occur naturally in the form of four analogs α-, β-, γ-, and δ-, each differing in the number and position of the methyl groups on the chroman ring. We previously reported that α-tocopherol α-toc and δ-tocopherol δ-toc enhance thermogenesis in 3T3-L1 cells 8,9 . Treatment of α-toc or δ-toc significantly increases the expression level of UCP1 and the number of mitochondria in 3T3-L1 cells. It has been proposed that PGC-1α activation is involved in this mechanism, but it is unknown whether the anti-inflammatory effects of tocopherols are involved. There are several reports on the anti-inflammatory effect of tocopherol via the suppression of inflammatory signaling in adipocyte, liver, heart, etc 10 14 . In this study, we examined whether α-toc and δ-toc protect the function of brown adipocytes through anti-inflammatory effects.

Cell culture and differentiation
The rat primary brown adipocyte culture kit BAT10 was purchased from Cosmo Bio Co., Ltd. Cells were maintained at 37 with 5 CO 2 . Cells were seeded and cultured according to the manufacturer s instruction. In brief, cells were seeded in 12-well plates at a density of 8 10 4 cells per well and cultured in a growth medium BATGM, Cosmo Bio Co., Ltd. for four days postconfluency. Then, the cells were cultured in a differentiation medium BATDM, Cosmo Bio Co., Ltd. in order to induce differentiation into mature brown adipocytes. Vitamin E analogs or BHT 10 µM final concentration were added at the start of the differentiation induction, and the cells were cultured for two days. Mock cells were treated with equivalent DMSO 0.1 final concentration . Then, 10 ng/mL of TNF-α was treated to induce inflammation, and cells were collected 24 h later.

Immunoprecipitation and immunoblotting
Total protein was extracted from cells using RIPA buffer CST Japan K.K . Proteins were separated in a 4 -20 Mini-PROTEAN TGX Gel Bio-Rad Laboratories, Inc. before being transferred to polyvinylidene difluoride mem-branes. The membranes were blocked for 5 min using Ev-eryBlot blocking buffer Bio-Rad Laboratories, Inc. and then incubated overnight with primary antibodies. The concentrations used in immunofluorescence staining and Western blotting were in accordance with the recommended protocol for each antibody. After washing the membranes three times with 1 TTBS, they were incubated for 1 h at room temperature with the secondary antibody-conjugated HRP. The membranes were then washed three times with 1 TTBS, treated with reagents in Clarity Western ECL substrate Bio-Rad Laboratories, Inc , and detected with iBright FL1000 imaging system Thermo Fisher Scientific K.K. . PGC-1α protein was isolated from total protein using an immunoprecipitation IP kit BioVision Inc. according to the manufacturer s protocol. Total PGC-1α protein and phosphorylated PGC-1α were detected by Western blotting. The intensity of western blot bands was quantified using Image J quantification tool. The quantified data were normalized with α-tubulin.
2.4 Measurement of mRNA expression using real-time PCR Total RNA was extracted using Sepasol RNA II Nacalai Tesque Inc. . The RNA quantity and purity were determined based on the absorbance at 258/280 nm. Total RNA was reverse-transcribed into cDNA using the ABI high-capacity RNA-to-cDNA kit according to the manufacturer s protocol. The 7500 Fast Real-Time PCR system and realtime PCR kit ABI TaqMan ® Gene Expression Assays were used according to the manufacturer s instructions, and β-actin was used as an internal control. The assay IDs of the primer/probe mixtures in the TaqMan gene expression assays were as follows: b-actin Actb ; Mm00607939_m1, ERK1 Mapk3 ; Mm01973540_m1, ERK2 Mapk1 ; Mm00442479_m1, IL6 Il6 ; Mm00446190_m1.

Animals and diets
The breeding conditions for mice are the same as previously reported 10 . Male C57BL/6JJcl strain mice three weeks old, n 25 were purchased from CLEA Japan, Inc. and housed individually in plastic cages in an environment controlled at 23 2 and 55 5 humidity, with a 12 h light/dark cycle. Then, the mice were divided into four groups based on their average weight: the control group Cont, n 7 , the high-fat and high-sucrose group HFD, n 6 , the high-fat and high-sucrose diet α-tocopherol group HFD α-toc, n 6 , and the high-fat and high-sucrose diet δ-tocopherol group HFD δ-toc, n 6 . Table  S1 shows the composition of the experimental diet. The feed and water were supplied ad libitum for 16 weeks. After a 16-h fast, all mice were euthanized under isoflurane anesthesia, and BAT were collected for analysis. All animal experiments were conducted with the approval of the ethics committee and in accordance with the guidelines for the care and use of laboratory animals at Kanagawa Institute of Technology Kanagawa, Japan. Approval number; 0147, December 26, 2018. .

Pathological examination of the adipose tissue by
hematoxylin-eosin HE staining BAT specimens were paraffin-embedded by Sapporo General Pathology Laboratory Co. Ltd. Hokkaido, Japan . Sections were stained with HE and observed using an optical microscope Olympus Co., Tokyo, Japan .

Statistical analysis
All data were expressed as mean SD. Statistical analyses were performed using one-way ANOVA, followed by Bonferroni s post hoc test using Kaleida Graph v.4.5 Hulinks Inc., Tokyo, Japan . Differences were considered significant at p 0.05.

Results
3.1 α-and δ-tocopherol suppresses the reduction of thermogenic protein expression and inhibit lipid accumulation Figure 1A depicts the time course of cell culture. Primary brown adipocytes cultured under each condition were treated with TNF-α for 24 h and then analyzed. The morphology of differentiated rat primary brown adipocytes was shown in Fig. 1B.
The stimulation of TNF-α resulted in the enlargement of the lipid droplets, but the size of the lipid droplets did not change in cells supplemented with α-toc or δ-toc, com-pared with mock cells. This result indicates that α-toc and δ-toc may suppress the decrease in energy expenditure and inhibit lipid accumulation by TNF-α. TNF-α-induced inflammatory stimulation significantly reduced the protein expression levels of UCP1 and PGC-1α in brown adipocytes. The pre-incubation of α-toc or δ-toc significantly suppressed the decrease in UCP1 due to inflammation Fig.  2A . Also, δ-toc was effective in maintaining PGC-1α expression Fig. 2B . PGC-1 is a transcriptional cofactor activated by phosphorylation. As a result, proteins were extracted from cells stimulated with TNF-1α, and PGC-1α protein was isolated by immunoprecipitation to quantify the phosphorylation state. The addition of α-toc or δ-toc improved the phosphorylation of PGC-1α more than twice Fig. 2C .
3.2 α-and δ-tocopherol suppress the inflammatory signals in brown adipocytes ERK1/2 is a typical inflammatory signaling factor, which is activated downstream of TNF-α. ERK1/2 attenuates thermogenic function in brown adipocytes by suppressing the activation of the promoter region of UCP1 5, 6 . Here, the addition of TNF-α markedly induced the expression of ERK1/2 genes, Mapk3 and Mapk1, in primary cultured brown adipocytes. The addition of α-toc or δ-toc significantly suppressed the increased expression of these inflammatory signaling factors Figs. 3A, 3B . These results indicate that α-toc and δ-toc improve the UCP1 expression by suppressing inflammatory signaling. Interleukin-6 IL6 is an adipokine involved in inflammation and insulin resistance 15,16 . Transcription of IL6 is induced by activation of NFκB downstream of TNF-α signaling. The expression of IL6 tended to increase by TNF-α stimulation, but the gene expression was low level in primary brown adipocytes and there was no significant difference Fig. 3C .

Ingestion of tocopherols suppresses cell hypertrophy in mouse BAT
High-fat diet-induced obesity causes inflammation in brown adipocytes, resulting in cell hypertrophy. We fed mice a high-fat diet for 16 weeks and investigated the effects of α-toc and δ-toc in the diet. The weight of BAT did not differ between the groups Fig. 4A . The HDF group exhibited large lipid droplet accumulation and balloon-like hypertrophy, but normal morphology was ob-served in groups ingesting α-toc or δ-toc Fig. 4B . This result indicates that α-toc and δ-toc also protect BAT functions in vivo.

Discussion
In this study, we examined the effects of vitamin E analogs, α-toc and δ-toc, on inflammation and dysfunction in brown adipocytes. Macrophages invade and release inflammatory cytokines into BAT that has become hypertrop h i e d d u e t o o b e s i t y. I n p a r t i c u l a r, T N F -α o r IL-1β-mediated inflammatory signals inhibit heat produc- and PGC-1α. The cells were stimulated with TNF-α under each condition, and total protein was extracted. The expression level of UCP1 and PGC-1α was quantified by Western blotting. The figure on the right panel is the result of measuring signal intensity. C Protein expression of phosphorylated PGC-1α p-PGC-1α . The cells were stimulated with TNF-α under each condition, and total protein was extracted. PGC-α protein was purified by immunoprecipitation, and p-PGC-α was detected with an anti-pSer antibody. The figure on the right panel is the result of measuring signal intensity. The values represent the mean SD n 3 . Statistical analysis was performed using a one-way ANOVA, followed by Bonferroni s post hoc test *p 0.05 . tion by suppressing UCP1 transcription 5,6 . In this study, TNF-α-induced inflammatory stimulation significantly reduced the protein expression levels of UCP1 and PGC-1α in rat primary brown adipocytes. This result indicates that the decrease in thermogenic ability due to inflammation can be reproduced in an in vitro culture system. Brown adipocytes have a large number of mitochondria and rapidly convert energy into heat. Lipid droplets of brown adipocytes do not grow larger than those of white adipocytes. The enlargement of lipid droplets due to TNF-α indicates a decrease in energy consumption due to a decrease in thermogenic ability.
The pre-incubation of α-toc or δ-toc significantly suppressed the decrease in UCP1 and PGC-1α expression and inhibit lipid accumulation in rat primary brown adipocytes. As fat-soluble antioxidants, vitamin E analogs are well known to be involved in the protection of various cells. Interestingly, adding BHT, a lipid-soluble antioxidant similar to vitamin E analogs, had no protective effect on thermogenesis in brown adipocytes. These results indicate that tocopherols protect brown adipocytes via a pathway independent of antioxidative effects.
TNF-α binds to receptors and transmits inflammatory signals via ERK, JNK, NFκB and p38 MAPK. Inhibition of A, B Gene expression of ERK1/2 and IL6. The cells were stimulated with TNF-α under each condition, and total RNA was extracted. Real-time PCR was used to measure the expression of the ERK1 Mapk3 , ERK2 Mapk1 , and IL6 Il6 genes, which were normalized against that of β-actin. The values represent the mean SD n 3 . Statistical analysis was performed using a one-way ANOVA, followed by Bonferroni s post hoc test *p 0.05 . CREB by the MEK/ERK pathway suppresses cAMP-dependent transcription of UCP1 in adipocyte 17 . We showed that at the ERK pathway is suppressed by α-toc and δ-toc in this study. However, this result may only be part of the mechanism. JNK and NFκB suppresses the differentiation and function of adipocytes by suppressing the expression of PPARγ 18,19 . Reports that α-tocopherol and δ-tocopherol upregulate PPARγ expression suggest that maintenance of PPARγ expression is also involved in the mechanism 8,9,20 .
On the other hands, the role of p38 MAPK in adipocytes is complex. While p38 MAPK suppresses insulin signals 21 , it activates UCP1 transcription by promoting phosphorylation of PGC-1α 22 . Multiple inflammatory signals may indirectly be involved in UCP1 expression, further detailed studies of various signaling pathways are needed to elucidate the mechanism on the effect of tocopherol. Ingested tocopherols accumulate in the liver and adipose tissue 23,24 . Based on these findings, the role of vitamin E in suppressing inflammation in the liver and adipose tissue has been investigated for several years 10 14 . However, there are no reports on the function of vitamin E in BAT. In this study, the ingestion of α-toc and δ-toc suppressed fat accumulation and hypertrophy of brown adipocytes in mice. We previously reported that α-toc and δ-toc intake significantly suppressed weight gain in these mice Table  S2 10 . The results of this study suggest that the maintenance of thermogenesis and energy consumption in BAT is involved in the anti-obesity effect of α-toc and δ-toc ingestion. The effects of vitamin E analogs on macrophage invasion and inflammatory signals in vivo in BAT warrant further investigation. Recent studies have made it possible to induce differentiation from ES cells and iPS cells to brown adipocytes 25,26 . The effects of food ingredients on the function of human brown adipocytes will be further investigated in the future.

Conclusion
Conclusively, we suggest that α-tocopherol and δ-tocopherol suppress the decrease of thermogenic ability in brown adipocytes due to inflammation. Also, ingestion of α-tocopherol and δ-tocopherol suppresses brown adipocyte hypertrophy and weight gain in mice, so these vitamin E analogs may contribute to the treatment of obesity and obesity-related metabolic diseases.

Funding
This work was supported by JSPS KAKENHI Grant Number 18K17942.