Dietary Genistein Reduces Methylglyoxal and Advanced Glycation End Product Accumulation in Obese Mice Treated with High-Fat Diet

Article abstract of DOI:10.1021/acs.jafc.0c03286

Our previous study has found that dietary genistein could ameliorate high-fat diet (HFD)-induced obesity and especially lower methylglyoxal (MGO) and advanced glycation end product (AGE) accumulation in healthy mice exposed to genistein and HFD. However, it is still unclear whether dietary genistein intervention has a similar beneficial effect in obese mice. In this study, the mice were induced with obesity after being fed a HFD for nine weeks before being administered with two doses of genistein, 0.1percent (G 0.1) and 0.2percent (G 0.2), in the HFD for additional 19 weeks. After 19 week treatment, genistein supplementation reduced body and liver weights, plasma and liver MGO levels, and kidney AGE levels in mice. Mechanistically, genistein upregulated the expressions of glyoxalase I and II and aldose reductase to detoxify MGO, and genistein and its microbial metabolites, dihydrogenistein and 6′-hydroxy-O-demethylangolensin, were able to trap endogenous MGO via formation of MGO conjugates. Taken together, our results provide novel insights into the antiobesity and antiglycation roles of dietary genistein in obese subjects.

Full text of DOI:10.1021/acs.jafc.0c03286

Subscriber access provided by Macquarie University
Bioactive Constituents, Metabolites, and Functions
Dietary Genistein Reduces Methylglyoxal and Advanced Glycation
End Product Accumulation in Obese Mice Treated with High-Fat Diet
Yantao Zhao, Yingdong Zhu, Pei Wang, and Shengmin Sang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c03286 • Publication Date (Web): 23 Jun 2020
Downloaded from pubs.acs.org on June 23, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 32
Journal of Agricultural and Food Chemistry
Dietary Genistein Reduces Methylglyoxal and Advanced Glycation End Product
Accumulation in Obese Mice Treated with High-Fat Diet
Yantao Zhao, Yingdong Zhu, Pei Wang, Shengmin Sang*
Laboratory for Functional Foods and Human Health, Center for Excellence in Post-Harvest
Technologies, North Carolina Agricultural and Technical State University, North Carolina
Research Campus, 500 Laureate Way, Kannapolis, North Carolina 28081, United States
*Corresponding author: Tel: 704-250-5710; Email: ssang@ncat.edu or
shengminsang@yahoo.com
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 32
1
2
ABSTRACT
Our previous study has found that dietary genistein could ameliorate high-fat diet (HFD)-induced
obesity and especially lower methylglyoxal (MGO) and advanced glycation end products (AGEs)
accumulation in healthy mice exposed to genistein and HFD. However, it is still unclear whether
dietary genistein intervention has a similar beneficial effect in obese mice. In this study, the mice
were induced with obesity after being fed a HFD for nine weeks before being administered with
two doses of genistein, 0.1% (G 0.1) and 0.2% (G 0.2), in the HFD for additional 19 weeks,
respectively. After 19-week treatment, genistein supplementation reduced body and liver weights,
plasma and liver MGO levels, and kidney AGEs levels in mice. Mechanistically, genistein
upregulated the expressions of glyoxalase I and II, and aldose reductase to detoxify MGO, and
genistein and its microbial metabolites, dihydrogenistein and 6'-hydroxy-O-demethylangolensin,
were able to trap endogenous MGO via formation of MGO conjugates. Taken together, our results
provide novel insights into the anti-obesity and anti-glycation roles of dietary genistein in obese
subjects.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
KEYWORDS: genistein, methylglyoxal, advanced glycation end products, detoxification enzyme,
trapping, obesity
ACS Paragon Plus Environment

Page 3 of 32
Journal of Agricultural and Food Chemistry
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
INTRODUCTION
Methylglyoxal (MGO), a highly reactive dicarbonyl compound and potent glycating agent, is an
ubiquitous metabolite that spontaneously reacts with proteins and DNA forming advanced
glycation end products (AGEs). As the major precursor of AGEs, MGO can be generated
endogenously as a byproduct of glycolysis and several other metabolic pathways even under
physiological circumstances.1-3 The glyoxalase system, the most important system in the
metabolism of MGO, and other enzymes play an important role in regulating MGO levels.2, 4-5
Moreover, MGO is also present in many food products, such as cookie, bread, cheese, and
carbonated soft drinks, and in cigarette smoke as exogenous sources.6-8 MGO and MGO-derived
AGEs can afflict organs and tissues affecting their functions and structure. Accumulating evidence
highlights that the formation and accumulation of MGO and AGEs are strongly associated with
aging-related diseases, including cancer, neurodegenerative diseases, diabetes, and obesity.2-4, 9-13
Therefore, lowering MGO levels would be an effective approach to inhibit the formation of AGEs
and ameliorate the associated conditions of diseases.
Genistein (4',5,7-trihydroxyisoflavone), the principal soy isoflavone, is widely used as a
dietary supplement in the United States.14-15 Many studies have found that genistein has many
biological activities, such as anti-diabetes, anti-obesity, anti-cancer, antioxidant, anti-
inflammation, and inhibition of tyrosine-specific protein kinases.14, 16-22 As a selected, effective,
and safe natural bioactive compound for the prevention of diabetes and obesity, our previous
studies have shown that genistein can effectively trap MGO and inhibit AGE formation in vitro
and in vivo.7, 23-24 Importantly, dietary genistein intervention alleviates the very-high fat diet
(VHFD, 60% energy from fat) and HFD (45% energy from fat) plus exogenous MGO-induced
metabolic syndrome (MetS) and lowers the MGO and MGO-induced AGE accumulation via
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 32
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
trapping MGO and mediating the detoxification pathways of MGO.7 Started with young healthy
mice, co-treatment with HFD, dietary genistein has shown the potential beneficial effects on
lowering of MGO and AGEs accumulation in mice, suggesting genistein has the potential to
prevent the MGO and AGE accumulation in healthy individuals eating fat-enriched foods.
However, it is still unclear whether dietary genistein intervention has the same or similar beneficial
effects in overweight and obese individuals. To mimic this situation in the current study, young
healthy mice were fed a HFD for nine weeks to induce obesity before administering genistein, and
then the obese mice were given two doses of dietary genistein for the following 19 weeks. The
objective of this study is to elucidate the impact of genistein on the accumulations of MGO and
AGEs in obese mice and its underlying mechanisms to detoxify MGO.
MATERIALS AND METHODS
Chemicals and Reagents
Methylglyoxal solution (40% in water) and other chemicals were obtained from Sigma (St. Louis,
MO, USA). Genistein was purchased from LC Laboratories (Woburn, MA). The antibodies were
purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and Cell Signaling (Danvers, MA,
USA). HPLC-grade and LC/MS-grade solvents and other reagents were obtained from VWR
International (South Plainfield, NJ, USA) and Thermo Fisher Scientific (Pittsburgh, PA, USA).
Animal treatment
Five weeks old male C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor,
ME, USA). All animal experiments were performed according to the experimental protocol
approved by the Institutional Animal Care and Use Committee of the North Carolina Research
Campus (Protocol number 15-010).
ACS Paragon Plus Environment

Page 5 of 32
Journal of Agricultural and Food Chemistry
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
After one week of acclimation, all mice were fed the high-fat diet (HFD, 45% energy from
fat, D12451) and water ad libitum for nine weeks to induce moderate obesity with 20% - 25%
greater body weight than control mice. Then, all the obese mice were randomly divided into three
groups (n=10 in each group) given the following treatments for 19 weeks: (1) high-fat (HF) group,
given HF diet; (2) 0.1% genistein group (G 0.1), given 0.1% (w/w) genistein in HF diet; (3) 0.2%
genistein group (G 0.2), given 0.2% (w/w) genistein in HF diet. All the special diets were prepared
by and ordered from Research Diets (New Brunswick, NJ). The exact nutrient composition of the
diets is shown in Table 1.
The body weight (BW) and food intake were recorded weekly. The 24-h fecal samples were
collected using a metabolic cage and then frozen at -80 °C until further analysis. The mice were
sacrificed at the end of the experiment and the samples of blood, liver, kidney, and adipose tissues
(epididymal (Ep), retroperitoneal (Rp), and mesenteric fat) were harvested, weighed, frozen
rapidly in liquid nitrogen, and stored at -80 °C until further analysis. The outline of the study design
was shown in Fig. 1.
Biochemical analyses of plasma and liver samples
Blood samples were collected by cardiac puncture and plasma was obtained by centrifuging the
blood at 2,000 g for 15 min at 4 °C. The activities of alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) in plasma and levels of cholesterol and triglyceride (TG) in plasma and
liver were measured as described previously.7, 25 Fasting blood glucose levels (6 h fasting) were
measured at week 18 following the previous procedure.25
Measurement of MGO and AGEs levels
Plasma MGO levels were measured using the LC-MS method described previously.7 1, 2-
diaminobenzene was employed to incubate with the supernatant of the deproteinized plasma
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 32
87
88
sample for 3 h at 37 °C in the dark to form the specific quinoxaline derivate, 2-methylquinoxaline
(2-MQ). Then, 2-MQ and the internal standard 2, 3-dimethylquinoxaline (DMQ) were detected by
LC-MS.
89
90
The total AGEs levels in plasma, liver, and kidney tissues were measured based on the
fluorescence absorption as described previously.7, 25
91
92
Western blot assay
93
Liver and kidney tissues were prepared in RIPA buffer as whole protein lysates and the expressions
of MGO detoxification related enzymes (glyoxalase I/II (GLO I/II), glutamate-cysteine ligase
catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), and aldose
reductase (AR)) and receptor of AGEs (RAGE) were investigated by western blot as described
previously.7, 25
94
95
96
97
98
Fecal sample preparation
99
Dried feces (~50 mg) from each group (HF, G 0.1, and G 0.2) were separately ground and soaked
into 0.5 mL methanol, and subsequently homogenized for 60 s (10 cycles) by a Bead Ruptor
Homogenizer (Omni International, Kennesaw, GA), and then sonicated for 30 min. The suspension
was centrifuged at 16000 g for 10 min. The supernatant was removed. The process of sonication
was repeated once with the addition of another 0.5 mL MeOH. The supernatants were combined
and evaporated under a gentle stream of nitrogen. The residue was reconstituted into 50 μL 95%
MeOH (0.1% FA). After centrifugation at 16000 g for another 10 min, the supernatant was
removed for LC-MS analysis.
100
101
102
103
104
105
106
107
108
109
HPLC-MS analysis
Chromatographic separation of major MGO conjugates of genistein was achieved on a Gemini
C18 column (3.0 i.d. × 150 mm, 5 µm) (Phenomenex, Torrance) using a Vanquish Ultra-High
ACS Paragon Plus Environment

Page 7 of 32
Journal of Agricultural and Food Chemistry
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
Pressure Liquid Chromatography (UHPLC) (Thermo Scientific, San Jose). The binary mobile
phase system consisted of water with 0.1% formic acid (FA) (phase A) and acetonitrile with 0.1%
FA (phase B). The flow rate was 0.2 mL/min, and the injection volume was 5 μL. The column was
eluted with a gradient program (0% B from 0 to 2 min, 0 to 25% B from 2 to 15 min, 25 to 88% B
from 15 to 25 min, 88 to 100% B from 25 to 26 min, maintaining 100% B for 3 min and back to
0% B in 1 min, and then re-equilibrated with 0% B for another 3 min). The identification of major
MGO conjugates was conducted with a Thermo Scientific Q Exactive Plus mass spectrometer
system (Thermo Scientific, San Jose) equipped with a heated electrospray ionization source
(HESI-II). The negative ion polarity mode was set for an ESI ion source with the voltage on the
ESI interface maintained at approximately 2.50 kV. Temperatures of the HESI-II auxiliary gas
heater and capillary were set as 250 °C and 300 °C. Sheath, auxiliary, and sweep gases were
operated at 45, 10 and 2 AU. The S-lens RF level was set at 65. The scanning duration (from 10
to 27min) was determined under Full Mass mode with a mass range of m/z 100 to 1000. The
resolution for Full Scan was acquired at 70,000 FWHM (full width at half maximum) with an
automatic gain control (AGC) of 3 × 106 and a maximum injection time (IT) of 100 msec. The
MS1 data of the potential MGO conjugates were transferred into inclusion list with a specific NCE
of 40 eV for the parallel reaction monitoring (PRM) analysis. MS2 data in PRM mode were
acquired with a resolution at 35,000, AGC target at 3 × 106, maximum IT at 120 ms, isolation
window at 2.0 m/z, and stepped NCE at 15, 35, and 60 eV. The mass range was measured from
m/z 50 to 800. Data were acquired in time-scheduled PRM events using the Thermo Fisher
Scientific Xcalibur Quan Browser (Version 4.1.31.9).
Synthesis of authentic standards
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 32
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
6'-Hydroxy-O-demethylangolensin (6'-OH-DMA). 6'-OH-DMA was synthesized in house by
following the reported method.26 In detail, to a stirred slurry of lithium aluminium hydride (77 mg,
5.5 equv.) in reflux with THF (5 mL) was added to a solution of genistein (100 mg, 1.0 equv.) in
THF (10 mL) over 30 min. After further refluxing for 2.5 h, the reaction mixture was cooled and
poured into saturated NH4Cl aqueous solution at 0 oC. The mixture was neutralized with 2 M HCl
and extracted with ethyl acetate (EA). The EA layer was dried over Na2SO4 and filtered. The
filtration was evaporated in vacuo. The residue was loaded onto a column silica gel column (H/E
= 10:1, 5:1, 2:1, 1:1, and 1:2) and followed by repeated Pre-TLC (C/M = 8:1), giving rise to the
1
target compound (47 mg, yield: 47%) as a light brown solid. H-NMR (600 MHz, MeOD-d4) δ
5.23 (1H, q, J = 7.0 Hz, H-2), 1.40 (3H, d, J = 7.0 Hz, H-3), 5.78 (2H, s, H-3'/5'), 7.12 (2H, d, J =
8.5 Hz, H-2''/6''), and 6.69 (2H, d, J = 8.5 Hz, H-3''/5''); 13C-NMR (100 MHz, MeOD-d4) δ 209.1
(s, C-1), 50.8 (d, C-2), 20.7 (q, C-3), 106.0 (s, C-1'), 166.6 (s, C-2'/6'), 96.7 (d, C-3'/5'), 166.8 (s,
C-4'), 135.7 (s, C-1''), 131.1 (d, C-2''/6''), 116.8 (d, C-3''/5''), and 157.8 (s, C-4'').
MGO conjugates of genistein (MGO-GEN) and 6'-OH-DMA (MGO-6'-OH-DMA). In situ
synthesis of MGO-GEN and MGO-6'-OH-DMA was carried out using our previous method with
some modifications.27 In detail, a mixture of MGO (0.5 mM) and each flavonoid (genistein or 6'-
OH-DMA, 1.5 mM) in PBS (100 mM, pH 7.4) was incubated at 37 oC for 3 h. Then, acetic acid
(3 µL) was added to stop the reaction. The reaction medium was subsequently diluted 1000 times
for MGO-GEN or 10,000 times for MGO-6'-OH-DMA with 95% MeOH (0.1% FA) for LC-MS
analysis. The extracted ions were used as the according authentic references.
NMR Analysis
1H- and 13C-NMR spectra were obtained on a Bruker AVANCE 600 MHz spectrometer (Bruker,
Silberstreifen, Rheinstetten, Germany). All compounds were analyzed in MeOD-d4. Multiplicities
ACS Paragon Plus Environment

Page 9 of 32
Journal of Agricultural and Food Chemistry
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
are indicated by s (singlet), d (doublet), t (triplet), q (quartet), and br (broad). The 13C-NMR spectra
are proton-decoupled.
Statistical analysis
All values are expressed as means ± standard deviation (SD). One-way ANOVA with Tukey’s test
was performed to analyze the difference using GraphPad Prism 8. A p-value < 0.05 was considered
significant.
RESULTS
Dietary genistein inhibited HFD-induced body weight gain in obese mice
After the mice were fed a HFD for nine weeks to induce obesity, the BW was increased from 21.87
± 1.80 g to 36.98 ± 3.58 g, which is consistent with our previous data as a moderate obesity.7, 28-29
Dietary genistein supplementation at the dose of 0.2% significantly reduced the BW starting from
week 2 (by 9.7%) to week 19 (by 12.3%) compared with the mice in the HF group, and 0.1%
genistein treatment also decreased the BW from week 14 by 4.4% to week 19 by 3.0%, but was
not statistically significant compared with HF mice due to the large individual variation (Fig. 2A).
In Fig. 2A, the BW of the mice in the HF group at week 9 dropped down suddenly due to an
accident with the water bottle leaking, but the mice recovered quickly after solving the problem.
Also, the slight drop in BW at week 19 was due to the fasting of mice for 6 h to measure blood
glucose levels during week 18. Additionally, both doses of genistein slightly reduced the food
intake (3% for G 0.1 and 5.8% for G 0.2) compared with the HF group (Fig. 2B).
There was no change in the weights of the kidney and total fat after administering dietary
genistein (Fig. 2C). Also, the weights of Rp fat and mesenteric fat were decreased by
approximately 19.5% and 19.3% by 0.2% genistein treatment, respectively, compared with HF
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 32
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
mice, but not statistically significant due to the large individual variation. (Fig. 2C). Importantly,
genistein at 0.2% significantly decreased the liver weight by 36.2% compared with HF mice (Fig.
2C, 1.85 ± 0.63 g vs 2.90 ± 0.42 g). However, genistein treatment at 0.2% significantly increased
the weight of Ep fat (2.15 ± 0.28 g) compared with HF (1.73 ± 0.54 g) and G 0.1 (1.65 ± 0.18 g)
mice (Fig. 2C).
Effects of dietary genistein on biochemical parameters in obese mice
After 18 weeks of dietary genistein consumption, the fasting glucose concentration was
significantly decreased in the G 0.1 group (by 10%, 150.4 ± 9.08 mg/dL), but not in the G 0.2
group (166.6± 12.18 mg/dL), compared with HF mice (165.7 ± 13.82 mg/dL, Fig. 3). As shown
in Fig. 3, dietary genistein intervention did not affect the activity of AST and liver TG level, but
slightly decreased the ALT levels by 25% (G 0.1, 10.48 ± 4.29 U/L) and 16.7% (G 0.2, 11.64 ±
4.79 U/L) respectively, but not statistically significant compared with HF mice (13.97 ± 2.13 U/L,
Fig. 3) due to the large individual variation. In addition, plasma TG and cholesterol levels were
slightly reduced by 7.98% (21.49 ± 3.09 mg/dL) and 18.2% (114.98 ± 42.26 mg/dL) in genistein
at 0.2% treatment, respectively, but not significantly compared with HF mice (TG, 29.47 ± 10.35
mg/dL; cholesterol, 140.5 ± 10.35 mg/dL, Fig. 3). However, plasma TG concentration was
significantly lowered by 10.7% in G 0.1 group (18.78 ± 6.58 mg/dL, Fig. 3).
Effects of dietary genistein on MGO and AGEs levels in obese mice
Dietary genistein supplementation at 0.2% significantly decreased the MGO levels in the plasma
and liver by 43.9% (122.2 ± 48.76 nM) and 30.4% (5.93 ± 1.31 nmol/g) compared with HF mice
(217.92 ± 18 nM and 8.53 ± 1.89 nmol/g, respectively, Fig. 4). In addition, the kidney MGO level
was slightly decreased by 12.5% in G 0.2 group, but not statistically significant compared with the
HF group (Fig. 4).
ACS Paragon Plus Environment

Page 11 of 32
Journal of Agricultural and Food Chemistry
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
Furthermore, the kidney AGE level was significantly lowered by 48.3% in dietary genistein
treatment at 0.2% and decreased by 26% in G 0.1 group, but not statistically significant due to
large individual variation (Fig. 4). However, there was no significant change of AGE levels in
plasma and liver after dietary genistein treatments.
Dietary genistein activated MGO detoxification systems and blocked AGE/RAGE pathway
in obese mice
The expressions of GLO I/II, GCLC, and GCLM, the important enzymes in the MGO
detoxification glyoxalase system, were determined in kidney and liver samples. In the kidney, the
expressions of GLO I and GLO II were significantly increased by dietary genistein treatment at
0.2% (by 1.29-fold and 1.32-fold, respectively, Fig. 5), but not by 0.1% genistein treatment.
Similarly, the expressions of GCLC and GCLM were also elevated by genistein treatment at 0.2%
(by 1.24-fold and 1.20-fold) compared with HF mice (Fig. 5). Importantly, the expression of AR,
another MGO detoxification enzyme, in the kidney was dramatically increased over 3-fold by 0.2%
genistein treatment compared with HF mice (Fig. 5). Moreover, dietary genistein treatment at 0.2%
significantly lowered the RAGE expression by 25% in the kidney compared with HF mice (Fig.
5), indicating that the AGE/RAGE pathway was interrupted.
Similarly, the expressions of GLO I and GLO II in the liver were significantly elevated by
1.25- fold and 3-fold in dietary genistein treatment at 0.2%, respectively (Fig. 6). Meanwhile, the
expressions of GCLC and GCLM were also increased by 1.27-fold and 1.32-fold in the G 0.2
group compared with HF mice (Fig. 6). However, dietary genistein treatments did not significantly
alter the expressions of AR in the liver compared with HF mice. Moreover, the RAGE expression
in the liver was decreased significantly by 40% with genistein treatment at 0.2% (Fig. 6).
Dietary genistein trapped endogenous MGO in obese mice
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 32
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
Our previous studies showed that genistein and its microbial metabolites, such as dihydrogenistein
(DHGEN) and 6'-OH-DMA, are able to trap MGO via formation of MGO conjugates in mice.7, 24
In this work, MGO-GEN and MGO-6'-OH-DMA were synthesized in situ and used as references.
The excretion of MGO-GEN (m/z 341.0667 [M−H]−) was characterized by the predominant MS2
ion at m/z 269.0458 [M−MGO−H]− (Fig. 7A). Typical MS2 fragments at m/z 327.0879
[M−H2O−H]−, 312.0644 [M−H2O−Me−H]−, 299.0929 [M−H2O−CO−H]−, and 273.0771
[M−MGO−H]− confirmed the identity of MGO-6'-OH-DMA (Fig. 7B). In addition, the daughter
ion at m/z 165.0196 [M−MGO−C7H6O−H]−, corresponding to the formation of 4,6-
dihydrooxybenzofuran-3-one and as evidence for the DHGEN moiety,30 suggested the presence of
MGO-DHGEN (Fig. 7C). As shown in Fig. 7, three MGO conjugates, including MGO-GEN,
MGO-6'-OH-DMA, and MGO-DHGEN, appeared in mouse feces after genistein intake in a dose-
dependent manner, but not in HF group.
DISCUSSION
The current study demonstrated for the first time that dietary genistein at 0.2% significantly
decreased the accumulations of MGO and AGEs in obese mice. Further mechanistic studies
showed that dietary genistein reduced the accumulations of MGO and AGEs via two different
pathways, activation of MGO detoxification pathways and directly trapping MGO to form the
corresponding MGO conjugates. The similar beneficial effects of dietary genistein (0.25% and 0.2%
in the HFD, respectively) on inhibiting MGO and AGEs accumulations have also been found in
our previous preventive study in either VHF (VHF, 60 kcal% fat) or HF (45 kcal% fat) plus
exogenous MGO-induced MetS in young healthy mice. 7 Therefore, it suggests that genistein may
ACS Paragon Plus Environment

Page 13 of 32
Journal of Agricultural and Food Chemistry
246
247
be a candidate agent to prevent unhealthy food-induced MetS and MGO-AGEs associated chronic
diseases in both healthy and obese individuals.
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
It has been reported that overexpression of GLO I, the major MGO detoxification enzyme, and
GLO I inducer could reduce the MGO level and AGE formation in vivo.31-32 Even though genistein
has not been reported as a GLO I inducer, it was found to have the capacity to upregulate the
expressions of GLO I and II, 7 and normalize the high-fructose diet-induced reductions of GLO I
and II activity in rats.33 In this study, both GLO I and GLO II were significantly upregulated by
0.2% genistein treatment in the liver and kidney of obese mice, which are consistent with the
findings in our previous genistein studies.7 Also, the two GSH synthase enzymes (GCLC/GCLM)
were upregulated by 0.2% genistein treatment in obese mice, indicating that the activation of the
glyoxalase system may lower the concentrations of endogenous MGO. Moreover, our results
demonstrated that the expression of AR, another important enzyme for MGO detoxification, was
significantly upregulated by 0.2% genistein treatment in the kidney, which is consistent with our
7
previous genistein study started with young healthy mice . However, the AR expression in the
liver was not altered by genistein in obese mice, which is different from our previous genistein
studies started with young healthy mice 7. Therefore, dietary genistein has the capacity to decrease
MGO accumulation via activating the glyoxalase system and upregulating the AR expression in
HFD-treated mice that were initially either healthy or obese. Similar to our previous experiments,
the significantly down-regulated RAGE expressions in the kidney and liver indicated that the
AGE/RAGE pathway was interrupted by dietary genistein at 0.2%.
In this study, besides the mono-MGO conjugate of genistein observed in our previous studies,
the mono-MGO adducts of the microbial metabolites of genistein, such as DHGEN and 6'-OH-
DMA, were also found in genistein treated mice, indicating that the MGO trapping mechanism
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 32
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
plays a role in lowering MGO levels in both healthy and obsese mice and gut microbiota may
impact the in vivo trapping efficacy of genistein. Taken together, genistein can inhibit HFD
induced MGO and AGEs accumulations through both trapping of MGO (direct effect) and
activation of MGO detoxification systems (indirect effect).
The beneficial effects of genistein against obesity and diabetes associated MetS have been
reported in numerous studies.16, 34-44 In most of these studies, young healthy mice were used to
start the co-treatment of HFD and genistein.7, 40-42, 44-46 There is only one very recent study that
began testing the effect of genistein against HFD-induced MetS after the mice were induced with
obesity. In that study, a single dose of genistein (0.02% in HFD (40% fat)) was administered to
the obese female ICR mice and the HFD induced BW gain was significantly decreased after 4
weeks of treatment,47 which is contradictory to the results of genistein at 0.1% started with obese
mice in the current study and genistein treatment at 0.067% in healthy mice that were fed a HFD
plus MGO in our previous study.7 In the current and previous studies, we only observed that high
doses of genistein (0.2% and 0.25%) significantly decreased HFD-induced BW gain. This may
due to different gender (male vs female) and strain (C57 BL/6J vs ICR) of mice that were used in
our studies vs the recently reported study.
In this study, we observed that the inhibitory effects of genistein on HFD-induced BW gain
and elevated biomarkers related to MetS in obese mice were not as strong as those in the young
healthy mice given the same treatment from the beginning. We observed that genistein treatment
at 0.25% in the VHF model and 0.2% in the HF plus MGO model significantly inhibited BW gain
by 88% and 75%. However, the BW gain was inhibited by 41.2% by 0.2% genistein treatment in
obese mice. Moreover, genistein treatment at 0.25% in the VHF model and 0.2% in the HF plus
MGO model significantly decreased the levels of plasma glucose, cholesterol, ALT, and AST and
ACS Paragon Plus Environment

Page 15 of 32
Journal of Agricultural and Food Chemistry
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
liver TG induced by VHFD and HFD plus MGO treatment, respectively. However, none of these
markers were significantly decreased by 0.2% genistein treatment in this study. Both the current
and our previous studies found that 0.2% and 0.25% genistein significantly decreased liver weight
comparing to HFD treated mice, which is also consistent with previous reports.7, 40, 42, 44-45
Interestingly, the weight of Ep fat was increased in G 0.2 group mice compared with the HF group.
The similar effect of the increase of Ep fat was also found at the doses ranging from 50 µg/kg/d to
50, 000 µg/kg/d (50 mg/kg/d) and in dietary genistein at 800 ppm (0.08% in diet) in the short-term
genistein administration studies in mice,48 which are different to most other genistein obesity
studies.
Altogether, dietary genistein exerted certain beneficial effects in the HFD pre-induced obese
mice, albeit less potent to HFD treated young healthy mice. This is potentially due to it is always
easier to prevent the development of a disease than to treat an existing diease. To healthy mice,
genistein can efficiently inhibit HFD-induced MGO and AGEs accumulation and body weight
gain at the early stage. While to obese mice, MGO and AGEs have already been accumulated,
whichh will be much more difficult to be reversed by genistein treatment. In addition, our previous
studies started with 6-week old mice for a 16-week treatment and this study stared genistein
treatment with 15-week old mice for a 19-week treatment. Therefore, age could be a tributing
factor to the observed differences between this study and our previous studies.
In conclusion, the present study demonstrated that dietary genistein intervention in obese mice
significantly decreased HFD-induced body weight gain, inhibited the accumulation of MGO and
AGEs via trapping MGO and activating MGO detoxification pathways, and blocked the
AGE/RAGE pathway, albeit the beneficial effects of genistein in obese mice are not as significant
as those in the young healthy mice. Furthermore, the findings from our present and previous studies
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 32
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
suggest that genistein may be a candidate agent for alternative or complementary treatment in
unhealthy food-induced MetS. Therefore, it is worthwhile to further study the beneficial effects of
genistein in human trials.
ACKNOWLEDGMENT
The authors wish to thank Mr. Hunter Snooks who assisted in the proofreading of the manuscript.
AUTHOR INFORMATION
Corresponding Author
*Email: ssang@ncat.edu or shengminsang@yahoo.com
ORCID
Shengmin Sang: 0000-0002-5005-3616
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
AGEs, advanced glycation end products; ALT, alanine aminotransferase; AR, aldose reductase;
AST, aspartate aminotransferase; Ep fat: epididymal fat; FFA: free fatty acid; GCLC, glutamate-
cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase modifier subunit; GLO,
glyoxalase; HDL, high-density lipoproteins; HFD, high-fat diet; LDL, low-density lipoprotein;
MGO, methylglyoxal; MetS, metabolic syndrome; RAGE, the receptor for AGEs; Rp fat:
retroperitoneal fat; SIM, selective ion monitoring; VHFD, very-high-fat diet.
ACS Paragon Plus Environment

Page 17 of 32
Journal of Agricultural and Food Chemistry
337
REFERENCES
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
1.
Understanding methylglyoxal metabolism. IUBMB Life 2014, 66 (10), 667-678.
2. Kold-Christensen, R.; Johannsen, M., Methylglyoxal Metabolism and Aging-Related Disease:
Moving from Correlation toward Causation. Trends Endocrinol. Metab. 2019, 31 (2), 81-92.
3. Schalkwijk, C.; Stehouwer, C., Methylglyoxal, a Highly Reactive Dicarbonyl Compound, in
Chakraborty, S.; Karmakar, K.; Chakravortty, D., Cells producing their own nemesis:
Diabetes, Its Vascular Complications, and Other Age-Related Diseases. Physiol. Rev. 2020, 100 (1), 407-
461.
4.
system in diabetes and other age-related diseases. Clin. Sci. 2015, 128 (12), 839-61.
5. Vander Jagt, D. L.; Hunsaker, L. A., Methylglyoxal metabolism and diabetic complications: roles
Maessen, D. E.; Stehouwer, C. D.; Schalkwijk, C. G., The role of methylglyoxal and the glyoxalase
of aldose reductase, glyoxalase-I, betaine aldehyde dehydrogenase and 2-oxoaldehyde dehydrogenase.
Chem. Biol. Interact. 2003, 143-144, 341-351.
6.
Nutr. Food Res. 2006, 50 (12), 1105-1117.
7. Zhao, Y.; Wang, P.; Sang, S., Dietary genistein inhibits methylglyoxal-induced advanced glycation
end product formation in mice fed a high-fat diet. J. Nutr. 2019, 149 (5), 776-787.
8. Fujioka, K.; Shibamoto, T., Determination of toxic carbonyl compounds in cigarette smoke.
Environ. Toxicol. 2006, 21 (1), 47-54.
9. Matafome, P.; Rodrigues, T.; Sena, C.; Seica, R., Methylglyoxal in Metabolic Disorders: Facts,
Myths, and Promises. Med. Res. Rev. 2017, 37 (2), 368-403.
10. Allaman, I.; Bélanger, M.; Magistretti, P. J., Methylglyoxal, the dark side of glycolysis. Front.
Neurosci. 2015, 9 (23), doi: 10.3389/fnins.2015.00023.
11. Masania, J.; Malczewska-Malec, M.; Razny, U.; Goralska, J.; Zdzienicka, A.; Kiec-Wilk, B.; Gruca,
Nemet, I.; Varga‐Defterdarović, L.; Turk, Z., Methylglyoxal in food and living organisms. Mol.
A.; Stancel-Mozwillo, J.; Dembinska-Kiec, A.; Rabbani, N., Dicarbonyl stress in clinical obesity.
Glycoconjugate J. 2016, 33 (4), 581-589.
12.
Matafome, P.; Sena, C.; Seiça, R., Methylglyoxal, obesity, and diabetes. Endocrine 2013, 43 (3),
472-484.
13.
Antognelli, C.; Moretti, S.; Frosini, R.; Puxeddu, E.; Sidoni, A.; Talesa, V. N., Methylglyoxal Acts
as a Tumor-Promoting Factor in Anaplastic Thyroid Cancer. Cells 2019, 8 (6), 547.
14.
data. Acta Pol. Pharm. 2000, 57 (2), 135-155.
15. Braxas, H.; Rafraf, M.; Hasanabad, S. K.; Jafarabadi, M. A., Effectiveness of genistein
Polkowski, K.; Mazurek, A. P., Biological properties of genistein. A review of in vitro and in vivo
supplementation on metabolic factors and antioxidant status in postmenopausal women with type 2 diabetes
mellitus. Can. J. Diabetes 2019, 43 (7), 490-497.
16.
Eur. J. Pharmacol. 2013, 698 (1-3), 31-38.
17. Ji, G.; Yang, Q.; Hao, J.; Guo, L.; Chen, X.; Hu, J.; Leng, L.; Jiang, Z., Anti-inflammatory effect
Behloul, N.; Wu, G., Genistein: a promising therapeutic agent for obesity and diabetes treatment.
of genistein on non-alcoholic steatohepatitis rats induced by high fat diet and its potential mechanisms. Int.
Immunopharmacol. 2011, 11 (6), 762-768.
18.
soybean isoflavone genistein. Proc. Soc. Exp. Biol. Med. 1995, 208 (1), 124-130.
19. Taylor, C. K.; Levy, R. M.; Elliott, J. C.; Burnett, B. P., The effect of genistein aglycone on cancer
and cancer risk: a review of in vitro, preclinical, and clinical studies. Nutr. Rev. 2009, 67 (7), 398-415.
20. Tuli, H. S.; Tuorkey, M. J.; Thakral, F.; Sak, K.; Kumar, M.; Sharma, A. K.; Sharma, U.; Jain, A.;
Wei, H.; Bowen, R.; Cai, Q.; Barnes, S.; Wang, Y., Antioxidant and antipromotional effects of the
Aggarwal, V.; Bishayee, A., Molecular Mechanisms of Action of Genistein in Cancer: Recent Advances.
Front. Pharmacol. 2019, 10, 1336. doi: 10.3389/fphar.2019.01336.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 32
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
21.
Ganai, A. A.; Farooqi, H., Bioactivity of genistein: A review of in vitro and in vivo studies. Biomed.
Pharmacother. 2015, 76, 30-38.
22.
183.
23.
formation by trapping methylglyoxal. Chem. Res. Toxicol. 2011, 24 (4), 579-586.
24. Wang, P.; Chen, H.; Sang, S., Trapping methylglyoxal by genistein and its metabolites in mice.
Chem. Res. Toxicol. 2016, 29 (3), 406-414.
25. Zhao, Y.; Sedighi, R.; Wang, P.; Chen, H.; Zhu, Y.; Sang, S., Carnosic acid as a major bioactive
component in rosemary extract ameliorates high-fat-diet-induced obesity and metabolic syndrome in mice.
J. Agric. Food. Chem. 2015, 63 (19), 4843-4852.
26.
isoflavonoid phytoestrogens daidzein and genistein. Proc. Soc. Exp. Biol. Med. 1998, 217 (3), 293-9.
27. Huang, Q.; Zhu, Y.; Lv, L.; Sang, S., Translating In Vitro Acrolein-Trapping Capacities of Tea
Polyphenol and Soy Genistein to In Vivo Situation is Mediated by the Bioavailability and
Biotransformation of Individual Polyphenols. Mol. Nutr. Food Res. 2020, 64 (1), e1900274.
28.
(2), 270-99.
29.
Ajay, G.; Bhatia, A., Genistein: a multipurpose isoflavone. Int. J. Green Pharm. 2009, 3 (3), 176-
Lv, L.; Shao, X.; Chen, H.; Ho, C.-T.; Sang, S., Genistein inhibits advanced glycation end product
Wähälä, K.; Salakka, A.; Adlercreutz, H., Synthesis of novel mammalian metabolites of the
Hariri, N.; Thibault, L., High-fat diet-induced obesity in animal models. Nutr. Res. Rev. 2010, 23
Thibault, L., Chapter 13 - Animal Models of Dietary-Induced Obesity. In Animal Models for the
Study of Human Disease, Conn, P. M., Ed. Academic Press: Boston, 2013; pp 277-303.
30.
Polyphenol and Soy Genistein to In Vivo Situation is Mediated by the Bioavailability and
Biotransformation of Individual Polyphenols. Mol Nutr Food Res 2020, 64 (1), e1900274.
Huang, Q.; Zhu, Y.; Lv, L.; Sang, S., Translating In Vitro Acrolein-Trapping Capacities of Tea
31.
Brouwers, O.; Niessen, P. M.; Ferreira, I.; Miyata, T.; Scheffer, P. G.; Teerlink, T.; Schrauwen, P.;
Brownlee, M.; Stehouwer, C. D.; Schalkwijk, C. G., Overexpression of glyoxalase-I reduces
hyperglycemia-induced levels of advanced glycation end products and oxidative stress in diabetic rats. J.
Biol. Chem. 2011, 286 (2), 1374-80.
32.
Rabbani, N.; Thornalley, P. J., Glyoxalase 1 Modulation in Obesity and Diabetes. Antioxid. Redox
Signaling 2019, 30 (3), 354-374.
33.
Genistein improves liver function and attenuates non-alcoholic fatty liver disease in a rat model of insulin
resistance. J. Diabetes 2009, 1 (4), 278-87.
Mohamed Salih, S.; Nallasamy, P.; Muniyandi, P.; Periyasami, V.; Carani Venkatraman, A.,
34.
Shen, H.-H.; Huang, S.-Y.; Kung, C.-W.; Chen, S.-Y.; Chen, Y.-F.; Cheng, P.-Y.; Lam, K.-K.; Lee,
Y.-M., Genistein ameliorated obesity accompanied with adipose tissue browning and attenuation of hepatic
lipogenesis in ovariectomized rats with high-fat diet. J. Nutr. Biochem. 2019, 67, 111-122.
35.
Genistein ameliorates cardiac inflammation and oxidative stress in streptozotocin-induced diabetic
cardiomyopathy in rats. Mol. Cell. Biochem. 2015, 408 (1-2), 63-72.
36.
Gupta, S. K.; Dongare, S.; Mathur, R.; Mohanty, I. R.; Srivastava, S.; Mathur, S.; Nag, T. C.,
Tang, C.; Zhang, K.; Zhao, Q.; Zhang, J. J. J. o. a.; chemistry, f., Effects of dietary genistein on
plasma and liver lipids, hepatic gene expression, and plasma metabolic profiles of hamsters with diet-
induced hyperlipidemia. J. Agric. Food. Chem. 2015, 63 (36), 7929-7936.
37.
reaction in nonalcoholic steatohepatitis rats. Biomed. Pharmacother. 2019, 111, 1290-1296.
38. Fu, Z.; Liu, D., Long-term exposure to genistein improves insulin secretory function of pancreatic
β-cells. Eur. J. Pharmacol. 2009, 616 (1-3), 321-327.
39. Fu, Z.; Gilbert, E. R.; Pfeiffer, L.; Zhang, Y.; Fu, Y.; Liu, D., Genistein ameliorates hyperglycemia
in a mouse model of nongenetic type 2 diabetes. Appl. Physiol., Nutr., Metab. 2012, 37 (3), 480-488.
40. Kim, S.; Sohn, I.; Lee, Y. S.; Lee, Y. S., Hepatic gene expression profiles are altered by genistein
Yin, Y.; Liu, H.; Zheng, Z.; Lu, R.; Jiang, Z., Genistein can ameliorate hepatic inflammatory
supplementation in mice with diet-induced obesity. J. Nutr. 2005, 135 (1), 33-41.
ACS Paragon Plus Environment

Page 19 of 32
Journal of Agricultural and Food Chemistry
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
41.
Kim, H.-K.; Nelson-Dooley, C.; Della-Fera, M. A.; Yang, J.-Y.; Zhang, W.; Duan, J.; Hartzell, D.
L.; Hamrick, M. W.; Baile, C. A., Genistein decreases food intake, body weight, and fat pad weight and
causes adipose tissue apoptosis in ovariectomized female mice. J. Nutr. 2006, 136 (2), 409-414.
42.
on hepatic lipid metabolism and mitochondrial function in mice fed high-fat diets. Nutrition 2006, 22 (9),
956-964.
43.
lipid parameters and obesity in C57Bl/6J mice fed a high-fat diet. J. Med. Food 2006, 9 (4), 459-467.
44. Wang, W.; Chen, J.; Mao, J.; Li, H.; Wang, M.; Zhang, H.; Li, H.; Chen, W., Genistein Ameliorates
Non-alcoholic Fatty Liver Disease by Targeting the Thromboxane A2 Pathway. J. Agric. Food. Chem. 2018,
66 (23), 5853-5859.
Lee, Y. M.; Choi, J. S.; Kim, M. H.; Jung, M. H.; Lee, Y. S.; Song, J., Effects of dietary genistein
Yang, J. Y.; Lee, S. J.; Park, H. W.; Cha, Y. S., Effect of genistein with carnitine administration on
45.
López, P.; Sánchez, M.; Perez‐Cruz, C.; Velázquez‐Villegas, L. A.; Syeda, T.; Aguilar‐López, M.;
Rocha‐Viggiano, A. K.; del Carmen Silva‐Lucero, M.; Torre‐Villalvazo, I.; Noriega, L. G., Long‐Term
Genistein Consumption Modifies Gut Microbiota, Improving Glucose Metabolism, Metabolic
Endotoxemia, and Cognitive Function in Mice Fed a High‐Fat Diet. Mol. Nutr. Food Res. 2018, 62 (16),
1800313. doi: 10.1002/mnfr.201800313.
46.
Lu, Y.; Zhao, A.; Wu, Y.; Zhao, Y.; Yang, X., Soybean soluble polysaccharides enhance
bioavailability of genistein and its prevention against obesity and metabolic syndrome of mice with chronic
high fat consumption. Food Funct. 2019, 10 (7), 4153-4165.
47.
X.; Zhang, S.; Zhu, L., Genistein inhibits high fat diet-induced obesity through miR-222 by targeting BTG2
and adipor1. Food Funct. 2020, 11 (3), 2418-2426.
48.
Magdalena, P.; Nadal, A.; Ottobrini, L., Genistein affects adipose tissue deposition in a dose-dependent and
gender-specific manner. Endocrinology 2006, 147 (12), 5740-5751.
49.
of intragastrically administered Lactobacillus plantarum No. 14 in the gut of mice fed a high-fat diet. J.
Nutr. 2010, 140 (11), 1963-9.
Gan, M.; Shen, L.; Wang, S.; Guo, Z.; Zheng, T.; Tan, Y.; Fan, Y.; Liu, L.; Chen, L.; Jiang, A.; Li,
Penza, M.; Montani, C.; Romani, A.; Vignolini, P.; Pampaloni, B.; Tanini, A.; Brandi, M.; Alonso-
Takemura, N.; Hagio, M.; Ishizuka, S.; Ito, H.; Morita, T.; Sonoyama, K., Inulin prolongs survival
461
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 32
462
Table 1. Nutrient composition of HF diet, 0.1% and 0.2% genistein in HF diet1
HF(D12451)
0.1% G (G 0.1) 0.2% G (G 0.2)
Macronutrients 2
Protein
20
35
20
35
20
35
Carbohydrate
Fat
45
45
45
Total
100
100
100
Ingredient 3
Casein/80 mesh
L-cystine
200
3
200
3
200
3
Corn starch
Maltodextrin 10
Sucrose
72.8
100
172.8
50
72.8
100
172.8
50
72.8
100
172.8
50
Cellulose, BW200
Soybean oil
Lard
25
25
25
177.5
10
177.5
10
177.5
10
Mineral mix4, S10026
DiCalcium Phosphate
13
13
13
Calcium carbonate
5.5
5.5
5.5
Potassium citrate, 1H2O
16.5
16.5
16.5
Vitamin mix4, V10001
10
10
10
Choline Bitartrate
Genistein
2
0
2
2
0.86
0.05
1.72
0.05
Dye (different color)
0.05
Total
858.15
859.01
859.87
Genistein (%)
0
0.1
0.2
ACS Paragon Plus Environment

Page 21 of 32
Journal of Agricultural and Food Chemistry
1 Diets were prepared by Research Diets (New Brunswick, NJ).
2 Values are presented as the percent of energy in the diet (kcal%).
3 Values are expressed as g/kg of diet.
463
4 Complete vitamin and mineral mixture compositions are described previously 49.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 32
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
FIGURE LEGENDS
Figure 1. Outline of study design. Six-week-old male C57BL/6J mice were fed with a high-fat
diet (HFD, 45% energy from fat) for 9 weeks to induce obesity. Then the obese mice were divided
into three groups (WK 0) and given the special diets of HF, G 0.1% in HFD, and G 0.2% in HFD
for 19 weeks. At the end of the study (WK 19), the mice were sacrificed, plasma and tissue samples
were collected for later analysis. G 0.1, 0.1% genistein in HFD; G 0.2, 0.2% genistein in HFD;
HFD, high-fat diet; WK, week.
Figure 2. Effects of genistein supplementation on body weight, food intake, and tissue weights of
mice fed the HFD, HFD supplemented with 0.1% (G 0.1), and HFD supplemented with 0.2%
genistein (G 0.2) for 19 weeks. The body weight was monitored every week (A) and the cumulative
daily food consumption was calculated at the termination of experiment (B). Major organs were
weighed after mice were sacrificed (C). Data are shown as means ± SD, n=10. Labeled by different
letters are significantly different, p < 0.05. Ep fat: epididymal fat; G 0.1, 0.1% genistein in HFD;
G 0.2, 0.2% genistein in HFD; HFD, high-fat diet; Rp fat: retroperitoneal fat.
Figure 3. Effects of genistein supplementation on fasting blood glucose, plasma AST and ALT,
plasma TG and cholesterol, and liver TG of mice fed the HFD, HFD supplemented with 0.1% (G
0.1), HFD supplemented with and 0.2% genistein (G 0.2) for 19 weeks. Data are shown as means
± SD, n=10. Labeled by different letters are significantly different, p < 0.05. ALT, alanine
aminotransferase; AST, aspartate aminotransferase; G 0.1, 0.1% genistein in HFD; G 0.2, 0.2%
genistein in HFD; HFD, high-fat diet; TG, triglyceride.
Figure 4. Effects of genistein supplementation on the MGO and AGEs concentrations in plasma,
liver, and kidney of mice fed the HFD, HFD supplemented with 0.1% (G 0.1), HFD supplemented
with and 0.2% genistein (G 0.2) for 19 weeks. Data are shown as means ± SD, n=10. Labeled by
ACS Paragon Plus Environment

Page 23 of 32
Journal of Agricultural and Food Chemistry
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
different letters are significantly different, p < 0.05. AGEs, advanced glycation end products; G
0.1, 0.1% genistein in HFD; G 0.2, 0.2% genistein in HFD; HFD, high-fat diet; MGO,
methylglyoxal.
Figure 5. Protein expressions of GLO I and II, GCLC and GCLM, AR, and RAGE in the kidney
of mice fed the HFD, HFD supplemented with 0.1% (G 0.1), HFD supplemented with and 0.2%
genistein (G 0.2) for 19 weeks (A). The immunoblot bands were quantified by densitometry
analysis by ImageJ, and the ratio of β-actin was calculated by setting the value of HF as 1 (B).
Data are shown as means ± SD. Labeled by different letters are significantly different, p < 0.05.
AR, aldose reductase; G 0.1, 0.1% genistein in HFD; G 0.2, 0.2% genistein in HFD; GCLC,
glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase modifier subunit;
GLO, glyoxalase; HFD, high-fat diet; RAGE, the receptor for AGEs.
Figure 6. Protein expressions of GLO I and II, GCLC and GCLM, AR, and RAGE in the liver of
mice fed the HFD, HFD supplemented with 0.1% (G 0.1), HFD supplemented with and 0.2%
genistein (G 0.2) for 19 weeks (A). The immunoblot bands were quantified by densitometry
analysis by ImageJ, and the ratio of β-actin was calculated by setting the value of HF as 1 (B).
Data are shown as means ± SD. Labeled by different letters are significantly different, p < 0.05.
AR, aldose reductase; G 0.1, 0.1% genistein in HFD; G 0.2, 0.2% genistein in HFD; GCLC,
glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase modifier subunit;
GLO, glyoxalase; HFD, high-fat diet; RAGE, the receptor for AGEs.
Figure 7. The LC chromatograms under selected ion monitoring mode and the MS2 spectra of
MGO-GEN (A), MGO-6-OH-DMA (B) and MGO-DHGEN (C) in mouse feces after feeding with
HF diet, HFD supplemented with 0.1% (G 0.1), HFD supplemented with and 0.2% genistein (G
0.2) for 19 weeks, as well as respective references obtained by a negative ESI-MS interface. MGO-
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 32
510
511
512
513
514
GEN, mono-methylglyoxal adducts of genistein. MGO-6-OH-DMA, mono-methylglyoxal
adducts of 6'-hydroxy-O-demethylangolensin. MGO-DHGEN, mono-methylglyoxal adducts of
dihydrogenistein. HF diet, high-fat diet. HF+G 0.1, HF diet supplemented with 0.1% genistein.
HF+G 0.2, HF diet supplemented with 0.2% genistein. SIM, selected ion monitoring. ESI,
electrospray ionization.
ACS Paragon Plus Environment

Page 25 of 32
Journal of Agricultural and Food Chemistry
C57BL/6J mice
(n=30, male, 6-wk old)
HFD
(45 kcal% fat)
9 weeks
Obese mice
Grouping
WK 0
HF
n=10
G 0.1
n=10
G 0.2
n=10
Feeding
Feeding
0.1% G in HFD
Feeding
0.2% G in HFD
HFD
WK 19
Endpoint (sample collections and following assays)
Figure 1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 32
Figure 2
ACS Paragon Plus Environment

Page 27 of 32
Journal of Agricultural and Food Chemistry
Figure 3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 32
Figure 4
ACS Paragon Plus Environment

Page 29 of 32
Journal of Agricultural and Food Chemistry
Figure 5
ACS Paragon Plus Environment