Î±-Monoglucosyl Hesperidin but Not Hesperidin Induces Brown-Like

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Bioactive Constituents, Metabolites, and Functions

#-Monoglucosyl hesperidin but not hesperidin induces brown-like adipocyte formation and suppresses white adipose tissue accumulation in mice Sho Nishikawa, Takuma Hyodo, Tsubasa Nagao, Akihito Nakanishi, Mahamadou Tandia, and Takanori Tsuda J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06647 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 30, 2019

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Journal of Agricultural and Food Chemistry

α-Monoglucosyl hesperidin but not hesperidin induces brown-like adipocyte formation and suppresses white adipose tissue accumulation in mice

Sho Nishikawa†, Takuma Hyodo†, Tsubasa Nagao†, Akihito Nakanishi§, Mahamadou Tandia§, Takanori Tsuda†* † College

of Bioscience and Biotechnology, Chubu University, Kasugai,

Aichi 487-8501, Japan § Toyo

Sugar Refining Co., Ltd., Chuo-ku, Tokyo 103-0016, Japan

Correspondence: * To whom correspondence should be addressed (Takanori Tsuda) Address: College of Bioscience and Biotechnology, Chubu University, Matsumoto-cho, Kasugai, Aichi 487-8501, Japan TEL&FAX: +81-568-51-9659, E-mail: [email protected]

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Abstract

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Hesperidin (HES) is a flavanone glycoside found in citrus peel that contributes to its

3

bitter taste. It has low water solubility and poor oral bioavailability. To improve its solubility

4

and bioavailability, α-monoglucosyl HES (αGH) has been synthesized from HES by

5

transglucosylation using cyclodextrin glucanotransferase. Several reports indicate that αGH

6

significantly decreases body fat, but the underlying molecular mechanism remains unclear.

7

We hypothesized that the anti-obesity effects of αGH occur through the induced formation of

8

brown-like adipocytes. The present study verified that dietary αGH induces brown-like

9

adipocytes to form in mouse inguinal white adipose tissue (iWAT), thereby significantly

10

decreasing the weight of WAT. Furthermore, dietary αGH significantly induced

11

thermogenesis in iWAT. Dietary αGH also significantly suppressed high-fat-diet-induced

12

WAT accumulation in mice, which may be mediated by brown-like adipocyte formation.

13

These results indicate that dietary αGH induces increased energy expenditure by stimulating

14

the formation of brown-like adipocytes.


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Introduction

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Hesperidin (HES; Figure S1), a flavanone glycoside found in citrus peel, is

18

composed of the flavone hesperetin and the disaccharide rutinose, has been reported to have

19

several biological functions. For example, HES showed hypoglycemic effects in mice and

20

rats1,2, cardioprotective and hypolipidemic effects in rats3, and anti-hypertensive and

21

anti-inflammatory effects in patients with type 2 diabetes4. Several reports have shown that

22

HES has low solubility in water and poor oral bioavailability5,6. Accordingly, synthetic

23

α-monoglucosyl HES (αGH, Figure S1), which has higher water solubility and bioavailability,

24

can be obtained from HES via transglucosylation with cyclodextrin glucanotransferase7. At

25

present, αGH is an approved food additive in Japan8. The solubility of αGH is about 10,000

26

times greater than that of HES, and its bioavailability (determined as the area under the

27

concentration–time curve for hesperetin glucuronide in serum after αGH administration to

28

rats) is about 3.7-fold higher than that of HES9.

29

The improved water solubility and bioavailability has enabled applications in food

30

processing and the exploration of novel biological functions. For example, the

31

supplementation of αGH in canned mandarin syrup or orange juice significantly prevented

32

turbidity10. Additionally, a randomized, placebo-controlled, double blind, parallel group study

33

in humans showed that the administration of αGH (1 g/day, 12 weeks) significantly decreased

34

the accumulation of body fat11. Another study reported that the administration of αGH (500

35

mg/day) in combination with caffeine (50 or 75 mg/day) for 12 weeks significantly

36

suppressed body fat accumulation in human subjects12. The specific molecular mechanisms

37

underlying these biological effects however remain unclear.

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Recent research has shown that brown-like adipocytes, also called “brite” or “beige”

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cells, are "inducible" brown adipocytes that share a range of morphological and biochemical

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characteristics with classical brown adipocytes13 and occur within white adipose tissue

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(WAT)14. Brown-like adipocytes release excess energy through thermogenic uncoupling 3 ACS Paragon Plus Environment


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protein 1 (UCP1), a protein that shifts energy from ATP production to heat production by

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causing leakage of protons in mitochondria and uncoupling electron transport from ATP

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production15,16. Accordingly, the induction of brown-like adipocytes has potential as a

45

strategy for treating obesity and associated disorders, and many studies have investigated

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dietary factors associated with the development of brown-like adipocytes17. We previously

47

showed that a highly dispersible curcumin formulation with good bioavailability clearly

48

induced brown-like adipocyte formation in mouse inguinal WAT (iWAT)18. However, native

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curcumin, which has low aqueous solubility and poor bioavailability, did not induce

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brown-like adipocyte formation18. Thus, we hypothesized that αGH, but not HES, exerts

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anti-obesity effects by inducing brown-like adipocyte formation.

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Here, we demonstrated that dietary αGH, but not HES, can induce brown-like

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adipocyte formation within mouse iWAT and can significantly decrease WAT weight. In

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addition, dietary αGH significantly induced thermogenesis in iWAT. Dietary αGH also

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significantly suppressed the accumulation of high-fat diet (HFD)-induced WAT in mice,

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likely mediated by brown-like adipocyte formation.

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Materials and Methods

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Chemicals. Commercially available αGH powder (Lot No.TA2517400) and HES

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(Lot No.HE160401, purity > 92%) were obtained from Toyo Sugar Refining Co., Ltd.

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(Tokyo, Japan). The αGH powder consisted of 82% αGH, 10% HES, and 8% undisclosed

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components. Transglycosylation to HES and αGH purification were performed according to a

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previous study7. Purity was analyzed by high-performance liquid chromatography8. Rabbit

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polyclonal -actin (#4967), cytochrome c oxidase subunit IV (COXIV, #4844), total

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AMP-activated protein kinase α (t-AMPKα, #2532), and phospho-AMPKα (p-AMPKα,

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#2531) antibodies were obtained from Cell Signaling Technology Japan, K. K. (Tokyo, 4 ACS Paragon Plus Environment

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Japan). Rabbit polyclonal UCP1 (ab10983) antibody was obtained from Abcam (Tokyo,

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Japan).

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Animal experiments. The design and execution of all experiments involving animals

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was approved by the Animal Experiment Committee of Chubu University and conducted

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according to their guidelines (Permission Nos. 3010024, 2910006, and 2810005).

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Effect of dietary αGH on brown-like adipocyte and adipose tissue weight formation

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in mice. Four-week-old male C57BL/6J mice were obtained from Japan SLC (Hamamatsu,

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Japan) and maintained in an animal room under controlled conditions (23 ± 3°C; 12-h

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light:dark cycle, illuminated 08:00–20:00) with free access to water and a standard laboratory

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diet (CE-2, CLEA Japan Inc., Tokyo, Japan)18-20. After 1 week, mice were assigned to three

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different groups: the control diet group (AIN-93G, n = 8) 21, the 0.5 αGH group (control diet

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supplemented with αGH powder; 5 g of αGH powder/kg diet, i.e., 4.1 g of αGH/kg diet, n =

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8), or the 1.0 αGH group (control diet supplemented with αGH powder; 10 g of αGH

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powder/kg diet, i.e., 8.2 g of αGH/kg diet, n = 8) for 4 weeks. A summary of the diet

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compositions is provided in Table S1. Every 2 days, both the control and αGH diets were

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replaced. The αGH dose utilized in the present study was based on a preliminary experiment

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that confirmed the level of αGH supplementation had no effect on food intake. After 4 weeks

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of feeding, blood samples were collected from mice following -anesthesia with isoflurane

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with a syringe containing 1 mM EDTA disodium salt, after which plasma was isolated via

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centrifugation. At this time, iWAT, epididymal WAT (eWAT), brown adipose tissue (BAT),

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skeletal muscles, and livers were removed18-20. As per our previous study18,19, small adipose

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tissues samples were fixed for subsequent hematoxylin & eosin (H&E) staining and

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immunostaining for UCP1. The staining protocols employed were those used in our previous

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studies18,19. Further details are available in the Supporting Information. Tissue aliquots were

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homogenized and used for immunoblotting analysis of UCP1, COXIV, and β-actin proteins in 5 ACS Paragon Plus Environment


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BAT and iWAT samples as well as t-AMPKα and p-AMPKα (Thr172) proteins in the liver

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and skeletal muscle samples. Pierce Western Blotting Substrate (Thermo Fisher Scientific,

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Yokohama, Japan) was used to visualize immunoreactivity18-20,22.

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Effect of dietary HES on brown-like adipocyte formation in mice. Other 4-week-old

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male C57BL/6J mice were also divided into two groups, control (n = 8) or HES groups (8.1 g

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HES/kg control diet; n = 8), and received their respective experimental diets for 4 weeks. Diet

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compositions are summarized in Table S2. Adipose tissue and blood samples were collected

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as described above. The supplementary level of HES added to the diet was adjusted such that

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the molar amount of HES was the same in the HES and 1.0 αGH diets.

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Measurement of tissue temperature after αGH administration in mice. As described

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in the above section, other mice were divided into two groups, namely, the control or 1.0 αGH

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groups (n = 10 per group), and received the respective experimental diets for 4 weeks. On day

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28 (4 weeks), mice were deprived of food for 1 h, from 08:00 to 09:00, to avoid the effect of

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diet-induced thermogenesis and anesthetized with isoflurane. Mice were then placed on a

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precision heat-controlled pad (fixed at 37.0°C; SN-700H, Shinano Manufacturing. Co., Ltd.,

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Tokyo, Japan), and a needle-type temperature probe (MT-23/3 and 23/5, Physitemp

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Instruments, Inc., Clifton, NJ) or rectal temperature probe (RET-3, Physitemp Instruments,

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Inc.) connected to a digital microprobe thermometer (BAT-12R, Physitemp Instruments, Inc.)

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was inserted into iWAT, BAT, or the rectum. We confirmed that the probe was inserted into

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iWAT after measuring iWAT temperature in all mice by dissection. This analysis confirmed

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that the probe had been correctly inserted into iWAT and not muscle. The rectal temperature

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was maintained at [37.0°C for 10 min, and then mice received oral administration of vehicle

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(saline; control) or αGH (100 mg/kg body weight) by intragastric intubation. BAT, iWAT,

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and rectal temperatures were recorded at 1-min intervals. During the measurements, mice

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were covered with laboratory paper towels to prevent heat loss. 6 ACS Paragon Plus Environment

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Effect of dietary αGH on brown-like adipocyte formation and adipose tissue weight

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in mice fed an HFD. Male C57BL/6J mice were randomly assigned to two groups and fed an

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HFD based on AIN-93G containing 30% (w/w) lard (HFD group, n = 10) or an HFD

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supplemented with 1.0% αGH (HFD + αGH group, n = 10) for 13 weeks. Experimental diet

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compositions are summarized in Table S3. After 13 weeks of feeding, adipose tissue, skeletal

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muscle, and liver were dissected. As described above, small iWAT and BAT samples were

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then fixed and used for H&E staining and immunostaining. As described in our previous

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studies16,18,19,21, tissue aliquots were homogenized and used for immunoblotting analysis of

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COXIV, UCP1, and β-actin proteins in BAT and iWAT samples, while skeletal muscle and

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liver samples were analyzed for t-AMPKα and p-AMPKα (Thr172) proteins.

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Statistical analyses. Data were assessed using two-way analysis of variance

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(ANOVA) and a subsequent Bonferroni test to compare the differences in temperature

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changes at each time point (Figure 5). The differences among group means were analyzed by

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Tukey–Kramer or Student’s t-tests. All data are expressed as means ± standard error of the

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mean (SEM) values, with a threshold of P < 0.05 applied to determine statistical significance.

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Results

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αGH, but not HES, induced brown-like adipocyte formation in iWAT and suppressed

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body fat accumulation. We first investigated whether αGH induced brown-like adipocyte

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formation and suppressed body fat accumulation in mice. Overall weight gain as well as

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eWAT and iWAT weights significantly decreased in the 1.0α GH group relative to the control

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(Table 1). However, BAT weights and total food intake over the 4 weeks did not differ among

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the groups (Table 1). UCP1 immunostaining and H&E staining of iWAT from both the 0.5α

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GH and 1.0 αGH groups showed clear UCP1-immunopositive cells and multilocular

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adipocytes compared with the control group (Figure 1A). Conversely, eWAT samples from 7 ACS Paragon Plus Environment


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the αGH groups lacked both multilocular H&E-stained adipocytes and positive UCP1

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immunostaining (Figure 1B). H&E-stained and UCP1-immunostained BAT samples showed

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no difference among the three groups (Figure 1C).

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To confirm the results of immunohistochemical analysis shown in Figure 1, COXIV

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and UCP1 protein levels in both BAT and iWAT samples were examined. Expression levels

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of UCP1 and COXIV proteins were significantly increased in iWAT samples from mice fed

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1.0 αGH compared with the control (Figure 2A). UCP1 and COXIV levels in BAT samples

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showed no differences among the three groups (Figure 2B).

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The serine/threonine kinase AMPK is expressed in most eukaryotic cells; its

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activation induces glucose utilization in skeletal muscle and enhances fatty acid oxidation in

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the liver23. These metabolic changes result in decreased body fat accumulation22. We also

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examined another mechanism of αGH-induced reduction of WAT accumulation, the

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activation of AMPK in liver and skeletal muscle tissues. Dietary αGH did not significantly

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affect p-AMPKα or t-AMPKα protein levels or the p-AMPKα:t-AMPKα ratio in these tissues

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(Figure S2).

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Next, we examined whether HES induced brown-like adipocyte formation at the

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same dose (1.0 αGH) in mice. Food intake, overall weight gain, and adipose tissue weight

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(iWAT, eWAT, and BAT) did not differ between the HES and control groups (Table S4).

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H&E staining and UCP1 immunostaining of eWAT and iWAT samples from the HES group

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did not show a significant amount of multilocular adipocytes or UCP1-immunopositive cells

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(Figure 3A and B). In addition, UCP1-immunostained and H&E-stained BAT did not differ

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between these groups (Figure 3C). The expression of both COXIV and UCP1 proteins in

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BAT and iWAT samples from the HES and control groups did not differ (Figure 4A and B).

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αGH significantly induced thermogenesis in iWAT. As αGH was observed to induce

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brown-like adipocyte formation in iWAT samples, we investigated whether αGH induces the 8 ACS Paragon Plus Environment

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elevation of core body or tissue temperature. To examine thermogenesis, αGH was

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administered to mice for 4 weeks, and rectal, BAT, and iWAT temperatures were measured.

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Rectal and BAT temperatures of the groups were similar throughout the measurement period

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(Figure 5A and B). In contrast, changes in iWAT temperature seen in the αGH group were

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significantly elevated from 10 to 60 min compared with the control group after administration

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of vehicle or αGH (Figure 5C).

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Dietary αGH decreased adipose tissue weight in HFD-fed mice. Dietary αGH

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significantly induced the brown-like adipocytes formation, enhanced thermogenesis in iWAT,

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and suppressed the weight of WAT. We examined the overall effect of dietary αGH on

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obesity in HFC-fed mice. Overall weight gain and food intake throughout the experimental

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period was relatively consistent between the groups (Table 2). The weights of eWAT and

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iWAT from the HFD + αGH groups were significantly decreased relative to those of the HFD

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group (Table 2). Compared with the control, UCP1-immunopositive cells were detected in

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iWAT samples from the HFD + αGH group, but not in eWAT samples (Figure 6A and B). In

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addition, HFD-induced hypertrophy of adipocytes in iWAT and eWAT was suppressed in the

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HFD + αGH group (Figure 6A and B). UCP1-immunostained and H&E-stained BAT samples

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from the groups were relatively similar (Figure 6C). Average adipocyte areas of iWAT and

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eWAT samples from the HFD + αGH group were also significantly smaller than those of the

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control group (Figure S3). UCP1 and COXIV protein levels in iWAT samples from the HFD

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+ αGH group were significantly increased relative to those of the HFD group (Figure 7A).

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However, levels of expression for both proteins did not differ between BAT samples from the

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groups (Figure 7B).

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Furthermore, we examined AMPK activation in liver and skeletal muscle tissues.

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Dietary αGH had no significant effect on the p-AMPKα or t-AMPKα protein levels or the

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p-AMPKα:t-AMPKα ratio (Figure S4). 9 ACS Paragon Plus Environment


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Discussion

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HES is found in citrus peel and is the ingredient that confers the characteristic bitter

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taste. It is thought to be a functional food-derived factor with various health benefits. Several

196

reports show that HES has biological functions1-4; however, its low water solubility and oral

197

bioavailability impede its use. These deficiencies have delayed research into the biological

198

functions of HES and complicated its use in both food additives and dietary supplements. To

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address these problems, highly water soluble and bioavailable αGH has been developed7. This

200

has enabled studies of novel biological functions and potential applications as dietary

201

supplements. Some reports show that αGH intake leads to significant suppression of body fat

202

accumulation11,12, but the mechanism is not fully understood. These reports suggest that

203

water-soluble and bioavailable αGH, but not HES, can induce brown-like adipocyte formation

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and subsequent UCP1-related thermogenesis in WAT, resulting in reduced accumulation of

205

body fat. In addition, we previously demonstrated that highly dispersible and bioavailable

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curcumin, but not an equivalent native curcumin dose, significantly induces brown-like

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adipocyte formation in iWAT18.

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Based on this background, we first demonstrated that αGH taken for 4 weeks

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significantly induced brown-like adipocyte formation in mouse iWAT and reduced overall

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weight gain as well as WAT weight without significant changes in food intake compared with

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the control. This effect, however, was not induced by administering an equivalent HES dose.

212

These results demonstrate that αGH, but not HES, can significantly induce of brown-like

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adipocyte formation in mouse iWAT.

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Induction of brown-like adipocyte formation can enhance thermogenesis and

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contribute to suppressed body fat accumulation. In this study, BAT and rectal temperatures

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were similar across groups. In contrast, changes in iWAT temperature in the αGH group were 10 ACS Paragon Plus Environment

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significantly elevated over 4 weeks compared with the control group. These data indicate that

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αGH specifically induced thermogenesis in iWAT, and this was dependent on the induction of

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UCP1 expression.

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AMPK activation induces glucose utilization within skeletal muscle tissue and

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enhances fatty acid oxidation in liver tissue, and these metabolic changes result in decreased

222

accumulation of body fat23. However, dietary αGH did not significantly affect the activation

223

of AMPK in skeletal muscle or liver. These findings suggest that αGH induces brown-like

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adipocyte formation in iWAT, and this effect may have contributed to the significant

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suppression of body fat. In addition, HFD-induced WAT accumulation was both significantly

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suppressed by αGH intake and accompanied by significant induction of UCP1 and COXIV

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protein in iWAT, but not activation of AMPK in skeletal muscle or liver tissues. These results

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are consistent with αGH-induced brown-like adipocyte formation in iWAT being associated

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with the observed suppression of WAT accumulation. The molecular mechanism may be

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associated with αGH-induced reduction of WAT gain. However, a recent report showed that

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there are heat-producing brown-like adipocytes that are independent of UCP1 expression in

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eWAT24. The significant reduction of WAT weight by dietary αGH may also be associated

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with UCP1-independent energy expenditure.

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This study has raised further questions, and some limitations need to be addressed.

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First, no significant differences were observed UCP1 expression and tissue temperature

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changes in BAT between the control and αGH groups. While the exact reason remains

237

unclear, BAT is both more sensitive and dominant relative to WAT with respect to

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β3-adrenergic signaling activation25,26. At the very minimum, the induction of brown-like

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adipocyte formation by αGH is independent of the 3-adrenergic signaling pathway. Second,

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our findings failed to explain how αGH induced brown-like adipocyte formation in iWAT.

241

One

possible

molecular

mechanism

is

that

αGH


functions

as

a

peroxisome


242

proliferator-activated receptor γ (PPARγ) agonist with PRD1-BF-1-RIZ1 homologous

243

domain-containing protein-16 protein stabilization18,27. The absorbed αGH was metabolized,

244

and the main metabolite was found to be hesperetin, (the aglycone of αGH) glucuronide in

245

serum after a single oral administration of αGH in rats8. Another report showed that a high

246

dose (250 μM) of hesperetin glucuronide acts as a PPARγ agonist in vitro28. However, it is

247

unlikely that a high dose of hesperetin glucuronide is distributed in iWAT, incorporated into

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the cells, and then binds with PPARγ in vivo. Third, we did not demonstrate whether the

249

intact form, metabolites, or degradation products of αGH have the most bioactivity in vivo, so

250

it is necessary to investigate which products in particular contribute to induction of

251

brown-like adipocyte formation. Furthermore, identifying the primary molecular target or

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trigger of αGH -induced brown-like adipocyte formation, intact form, metabolites, or

253

degradation products, may reveal the underlying biochemical mechanism.

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In conclusion, highly water-soluble and bioavailable αGH, but not HES, significantly

255

induced brown-like adipocyte formation and enhanced thermogenesis in iWAT, which may

256

contribute to suppressed body fat accumulation. These results suggest that αGH increases

257

energy expended through brown-like adipocyte formation. Furthermore, our study showed

258

that αGH has significant in vivo bioactivity as a functional food factor.

259 260

Abbreviations used: AMP-activated protein kinase, AMPK; brown adipose tissue, BAT;

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cytochrome c oxidase subunit IV, COXIV; epididymal white adipose tissue, eWAT;

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hematoxylin & eosin, H&E; high fat diet, HFD; α-monoglucosyl hesperidin, αGH; inguinal

263

white adipose tissue, iWAT; peroxisome proliferator-activated receptor  PPAR phospho-,

264

p-; standard error of the mean, SEM; total-, t-; uncoupling protein 1, UCP1; white adipose

265

tissue; WAT.

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Acknowledgement We thank Hiroki Aoyama (Chubu University) for useful discussions and technical

268 269

advice.

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Funding sources

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This study was supported in part by Grants-in-Aid for Scientific Research (Nos.

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17K07804 and 18H02157 for T.T. and 18K14428 for S.N.) from the Japan Society for

274

Promotion of Science.

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Supporting Information Available Additional information (Supporting Materials and Methods, Table S1-S4 and Figure

277 278

S1-S4) as noted in the text.

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Jung, U. J.; Lee, M. K.; Jeong, K. S.; Choi, M. S. The hypoglycemic effects of hesperidin and naringin are partly mediated by hepatic glucose-regulating enzymes in C57BL/KsJ-db/db mice. J. Nutr. 2004, 134, 2499–2503.

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Akiyama, S.; Katsumata, S.; Suzuki, K.; Nakaya, Y.; Ishimi, Y.; Uehara, M. Hypoglycemic and hypolipidemic effects of hesperidin and cyclodextrin-clathrated hesperetin in Goto-Kakizaki rats with type 2 diabetes. Biosci. Biotechnol. Biochem. 2009, 73, 2779–2782.

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Selvaraj, P.; Pugalendi, K. V. Efficacy of hesperidin on plasma, heart and liver tissue lipids in rats subjected to isoproterenol-induced cardiotoxicity. Exp. Toxicol. Pathol. 2012, 64, 449– 452.

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Homayouni, F.; Haidari, F.; Hedayati, V.; Zakerkish, M.; Ahmadi, V. Blood pressure 13 ACS Paragon Plus Environment


lowering and anti-inflammatory effects of hesperidin in type 2 diabetes; a randomized double-blind controlled clinical trial. Phytother. Res. 2018, 32, 1073–1079. 5

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Demonty, I.; Lin, Y.; Zebregs, Y. E.; Vermeer, M. A.; van der Knaap, H. C.; Jäkel M.; Trautwein, E. A. The citrus flavonoids hesperidin and naringin do not affect serum cholesterol in moderately hypercholesterolemic men and women. J. Nutr. 2010, 140, 1615–1620.

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Kometani, T.; Terada, Y.; Nishimura, T.; Takii, H.; Okada, S. Transglycosylation to hesperidin by cyclodextrin glucanotransferase from an alkalophilic Bacillus Species in alkaline pH and properties of hesperidin glycosides. Biosci. Biotechnol. Biochem. 1994, 58, 1990–1994.

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The Ministry of Health, Labour and Welfare, Enzymatically Modified Hesperidin. In 9th Japan’s specifications and standards food additives. Japan Food Additives Association, Tokyo, Japan, 2018, pp 595–596.

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Yamada, M.; Tanabe, F.; Arai, N.; Mitsuzumi, H.; Miwa, Y.; Kubota, M.; Chaen, H.; Kibata, M. Bioavailability of glucosyl hesperidin in rats. Biosci. Biotechnol. Biochem. 2006, 70, 1386–1394.

10 Nishimura, T.; Kometani, T.; Okada, S.; Kobayashi, Y.; Fukumoto, S. Inhibitory effects of hesperidin glycosides on precipitation of hesperidin. J. Jpn. Soc. Food. Technol. 1998, 45, 186–191. 11 Hanawa, M.; Morimoto, Y.; Yokomizo, A.; Akaogi, I.; Mafune, E.; Tsunoda, K.; Azuma, M.; Nishitani, M.; Kajimoto, Y.; Kadowaki, T. Effect of long-term intake of the tablet containing glucosyl hesperidin on body weight and body fat. J. Nutr. Food 2008, 11, 1– 14 ACS Paragon Plus Environment

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17. 12 Ohara, T.; Muroyama, K.; Yamamoto, Y.; Murosaki, S. Oral intake of a combination of glucosyl hesperidin and caffeine elicits an anti-obesity effect in healthy, moderately obese subjects: a randomized double-blind placebo-controlled trial. Nutr. J. 2016, 15, 6. 13 Wu, J.; Boström, P.; Lauren, M.; Sparks, L. M.; Ye, L.; Choi, J. H.; Giang, A. H.; Khandekar, M.; Virtanen, K. A.; Nuutila, P.; Schaart, G.; Huang, K.; Tu, H.; van Marken Lichtenbelt, W. D.; Hoeks, J.; Enerbäck, S.; Schrauwen, P.; Spiegelman, B. M. Beige Adipocytes Are a Distinct Type of Thermogenic Fat Cell in Mouse and Human. Cell 2012, 150, 366–376. 14 Schoettl, T.; Fischer, I. P.; Ussar, S. Heterogeneity of adipose tissue in development and metabolic function. J. Exp. Biol. 2018, 221, jeb162958. 15 Frühbeck, G.; Becerril, S.; Sáinz, N.; Garrastachu, P.; García-Velloso, M. J. BAT: a new target for human obesity? Trends. Pharmacol. Sci. 2009, 8, 387–396. 16 Cannon, B.; Nedergaard, J. Brown adipose tissue: function and physiological significance, Physiol. Rev. 2004, 84, 277–359. 17 Okla, M.; Kim, J.; Koehler, K.; Chung, S. Dietary factors promoting brown and beige fat development and thermogenesis. Adv. Nutr. 2017, 8, 473–483. 18 Nishikawa, S.; Kamiya, M.; Aoyama, H.; Nomura, M.; Hyodo, T.; Ozeki, A.; Lee, H.; Takahashi, T.; Imaizumi, A.; Tsuda, T. Highly dispersible and bioavailable curcumin but not native curcumin induces brown-like adipocyte formation in Mice. Mol. Nutr. Food Res. 2018, 62, 1700731. 19 Nishikawa, S.; Aoyama, H.; Kamiya, M.; Higuchi, J.; Kato, A.; Soga, M.; Kawai, T.; Yoshimura, K.; Kumazawa, S.; Tsuda, T. Artepillin C, a typical Brazilian propolis-derived component, induces brown-like adipocyte formation in C3H10T1/2 cells, primary inguinal white adipose tissue-derived adipocytes, and Mice. PLoS One 2016, 11, 15 ACS Paragon Plus Environment


e0162512. 20 Iizuka, Y.; Ozeki, A.; Tani., T.; Tsuda, T. Blackcurrant extract ameliorates hyperglycemia in type 2 diabetic mice in association with increased basal secretion of glucagon-like peptide-1 and activation of AMP-activated protein kinase. J. Nutr. Sci. Vitaminol. 2018, 64, 258–264. 21 Reeves, P. G.; Nielsen, F. H.; Fahey Jr., G. C. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 1993, 123, 1939–1951. 22 Kurimoto, Y.; Shibayama, Y.; Inoue, S.; Soga, M.; Takikawa, M.; Ito, C.; Nanba, F.; Yoshida, T.; Yamashita, Y.; Ashida, H.; Tsuda, T. Black soybean seed coat extract ameliorates hyperglycemia and insulin sensitivity via the activation of AMP-activated protein kinase in diabetic mice. J. Agric. Food Chem. 2013, 61, 5558–5564. 23 Tsuda, T. Prevention and treatment of diabetes using polyphenols via activation of AMP-activated protein kinase and stimulation of glucagon-like peptide-1 secretion. In Recent Advances in Polyphenol Research; Yoshida, K., Cheynier, V., Quideau, S., Eds.; John Wiley & Sons: Hoboken, NJ, 2016, 5, pp 206–225. 24 Bertholet, A. M.; Kazak, L.; Chouchani, E. T.; Bogaczyńska, M. G.; Paranjpe, I.; Wainwright, G. L.; Bétourné, A.; Kajimura, S.; Spiegelman, B. M.; Kirichok, Y. Mitochondrial Patch Clamp of Beige Adipocytes Reveals UCP1-Positive and UCP1-Negative Cells Both Exhibiting Futile Creatine Cycling. Cell Metab. 2017, 25, 811–822. 25 Slavin, B. G.; Ballard, K. W. Morphological studies on the adrenergic innervation of white adipose tissue. Anat. Rec. 1987, 191, 377–389. 26 Trayhurn, P.; Ashwell, M. Control of white and brown adipose tissues by the autonomic nervous system. Proc. Nutr. Soc. 1987, 46, 135–142. 16 ACS Paragon Plus Environment

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27 Ohno, H.; Shinoda, K.; Spiegelman, B. M.; Kajimura, S. PPAR agonists Induce a White-to-Brown Fat Conversion through Stabilization of PRDM16 Protein. Cell Metab. 2012, 15, 395–404. 28 Gamo, K.; Miyachi, H.; Nakamura, K.; Matsuura, N. Hesperetin glucuronides induce adipocyte

differentiation

via

activation

and

expression

of

peroxisome

proliferator-activated receptor-γ. Biosci. Biotechnol. Biochem. 2014, 78, 1052–1059.

Figure captions Figure 1. Representative images of H&E and immunohistochemical staining of UCP1 in sections of different adipose tissues from mice fed control, 0.5 αGH, or 1.0α GH diets for 4 weeks. (A) iWAT, (B) eWAT, and (C) BAT.

Figure 2. Immunoblot analysis of UCP1, COXIV, and -actin in (A) iWAT and (B) BAT from mice fed control, 0.5 αGH, or 1.0 αGH diets for 4 weeks. Relative protein levels are expressed as fold-changes relative to the control (= 1) after normalization using -actin protein. Data are presented as mean ± SEM (n = 8). Mean values with different letters were significantly different (P < 0.05).

Figure 3. Representative images of H&E and immunohistochemical staining of UCP1 in sections of different adipose tissues from mice fed control or HES diet for 4 weeks. (A) iWAT, (B) eWAT, and (C) BAT.

Figure 4. Immunoblot analysis of UCP1, COXIV, and -actin in (A) iWAT and (B) BAT from mice fed control or HES diet for 4 weeks. Relative protein levels are expressed as fold-changes relative to the control (= 1) after normalization using -actin protein. Data are 17 ACS Paragon Plus Environment


presented as mean ± SEM values (n = 8).

Figure 5. Changes in (A) rectal, (B) BAT, and (C) iWAT temperatures in the control or 1.0α GH group. After being fed control or 1.0 αGH diet for 4 weeks, mice received oral administration of vehicle (saline control) or αGH (100 mg/kg body weight) by intragastric intubation. Next, rectal, BAT, and iWAT temperatures were recorded at 1-min intervals. Details of the measurements are described in the Materials & Methods section. Data are presented as mean ± SEM value (n = 10). (*) Significant differences were assessed at P < 0.05 compared with the control at each time point.

Figure 6. Representative images of H&E and immunohistochemical staining of UCP1 in sections of different adipose tissues from mice fed HFD or HFD + αGH diet for 13 weeks. (A) iWAT, (B) eWAT, and (C) BAT.

Figure 7. Immunoblot analysis of UCP1, COXIV, and -actin in (A) iWAT and (B) BAT from mice fed the HFD or HFD + αGH diet for 13 weeks. Relative protein levels are expressed as fold-changes relative to the control (= 1) after normalization using the -actin protein. Data are presented as mean ± SEM values (n = 9–10). (*) Mean values are significantly different from those of the control group (P < 0.05).


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Table 1. Body weight, food intake, and relative tissue weights in mice fed 1

Control, 0.5αGH or 1.0αGH diet for 4 weeks.

Control

0.5αGH a

Initial body weight, g

20.1 ± 0.5

Final body weight, g

26.3 ± 0.4

Food intake, g/(4 weeks ・ mouse)

120.7 ± 3.3

eWAT, g/100 g body

1.46 ± 0.05

iWAT, g/100 g body

1.13 ± 0.05

Interscapular BAT, g/100 g body

0.40 ± 0.05

a a

1.0αGH a

20.1 ± 0.5

ab

24.9 ± 0.6

a

111.5 ± 2.5

a

1.35 ± 0.10

a

0.98 ± 0.07

a

b

23.9 ± 0.3

a

113.7 ± 2.0

ab

1.17 ± 0.05

ab

0.91 ± 0.06

a

0.31 ± 0.02

1

a

20.1 ± 0.3

b b a

0.28 ± 0.02

Values are presented as means ± SEM (n = 8). Mean values in a row without a common letter were significantly different (P < 0.05).



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Table 2. Body weight, food intake, and relative tissue weights in mice fed 1

HFD or HFD + αGH diet for 13 weeks. Initial body weight, g Final body weight, g Food intake, g/(4 weeks ・ mouse) eWAT, g/100 g body iWAT, g/100 g body Interscapular BAT, g/100 g body

HFD

HFD + αGH

19.9 ± 0.3 34.0 ± 0.8 238.0 ± 2.9 4.45 ± 0.32 2.24 ± 0.16 0.53 ± 0.02

20.0 ± 0.3 32.1 ± 1.1 232.8 ± 5.4 3.41 ± 0.30* 1.68 ± 0.13* 0.47 ± 0.04

1

Values are presented as means ± SEM (n = 9-10). (∗ ) Significantly different (P < 0.05) compared with the HFD.


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Figure 1. (A)

(B) Control

0.5αGH

1.0αGH

Control

H&E

H&E

UCP1

UCP1

(C) Control

0.5αGH

1.0αGH

H&E

UCP1


0.5αGH

1.0αGH


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Figure 2. (A)

(B) Control

0.5 αGH

1.0 αGH

UCP1

Control

COXIV

COXIV

β-Actin

β-Actin

4.0

UCP1

a

COXIV

Relative protein level


4.0

ab

3.0

ab

a

2.0 b b

1.0

0

0.5 αGH

1.0 αGH

UCP1

Control

0.5αGH

COXIV

3.0

2.0

1.0

0

1.0αGH

UCP1

Control


0.5αGH

1.0αGH

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Figure 3. (A)

(B) Control

HES

Control

H&E

H&E

UCP1

UCP1

(C) Control

HES

H&E

UCP1


HES


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Figure 4. (A)

(B) Control

HES

Control

UCP1

UCP1

COXIV

COXIV

β-Actin

β-Actin

4.0

UCP1 COXIV



4.0

3.0

2.0

1.0

0

Control

HES

HES

UCP1 COXIV

3.0

2.0

1.0

0

Control


HES

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Figure. 5

(A)

(B) Control αGH

36.0

35.0

37.0

36.0

35.0

34.0

34.0 0

10 20 30 40 50 60

Time after administration (min)

0

10 20 30 40 50 60



Control αGH

38.0 iWAT temperature (°C)

37.0

Control αGH

38.0 BAT temperature (°C)

Rectal temperature (°C)

38.0

(C)

37.0

*

36.0

* ***

******

35.0

34.0 0

10 20 30 40 50 60



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Figure 6. (A)

(B) HFD

HFD

HFD + αGH

H&E

H&E

UCP1

UCP1

(C) HFD

HFD + αGH

H&E

UCP1


HFD + αGH

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Figure 7.

(A)

(B) HFD

HFD+αGH

HFD

UCP1

UCP1

COXIV

COXIV

β-Actin

β-Actin

UCP1 COXIV

4.0

* *

3.0

2.0

1.0

0

HFD



4.0

HFD+αGH

UCP1 COXIV

3.0

2.0

1.0

0

HFD+αGH

HFD


HFD+αGH


Page 28 of 28

Table of Contents

Hesperidin OH

α-Monoglucosyl hesperidin

OH OCH3

OO OO

O

OH

Glucose

OCH3

OO OO

OH OH OH

O

OH

OH

OH OH OH

OH

HO

O

OH O

H&E

OH

H&E

OH OH

O OH

Anti-UCP1

Anti-UCP1

α-Monoglucosyl hesperidin but not hesperidin induces brown-like adipocyte formation in inguinal white adipose tissue of mice.


OH

O

Î±-Monoglucosyl Hesperidin but Not Hesperidin Induces Brown-Like

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