pubs.acs.

org/JAFC Article

Role of Thioredoxin-Interacting Protein in Diabetic Fatty Kidney

Induced by Advanced Glycation End-Products
Hong Sun,*,⊥ Juan Chen,⊥ Lili Sun, Bimin Shi, and Jianzhong Li*

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ABSTRACT: Advanced glycation end-products (AGEs) have been identified as the etiological factors associated with the fatty
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kidney. Thioredoxin-interacting protein (TXNIP) might be a mediator involved in AGE-induced fatty kidney. This study focused on
investigating how TXNIP affected the AGE-mediated renal lipid deposition. In an in vivo experiment, the db/db mice injected with
the lentiviral vector encoding shRNA targeting TXNIP were given the AIN-76 basal or the high-AGE diet. TXNIP-targeting siRNA-
transfected human renal proximal tubular epithelial (HK-2) cells were exposed to AGE-BSA in a study in vitro. The results showed
that the silencing of TXNIP reduced tubular lipid droplets and intracellular cholesterol content, as well as upregulated Insig-1 and
downregulated HMGCoAR, LDLr, nSREBP-2, and SCAP in the kidneys of the db/db mice, the high-AGE-diet-fed db/db mice, and
AGE-BSA-treated HK-2 cells. Furthermore, AGE-BSA enhanced SCAP−SREBP-2 complex formation while promoting their
transportation to the Golgi apparatus. However, these could be inhibited by TXNIP silencing in the HK-2 cells. The above findings
indicated that TXNIP knockdown mitigated the accumulation of renal tubular lipids in diabetes through the regulation of SCAP,
thereby inhibiting the SCAP−SREBP-2 signaling pathway, resulting in reduced cholesterol uptake and synthesis. Therefore, TXNIP
might be a potential therapeutic target to treat a diabetic fatty kidney.
KEYWORDS: type 2 diabetes mellitus, fatty kidney, advanced glycation end-products, thioredoxin-interacting protein

■ INTRODUCTION
The latest findings presented by the International Diabetes
that renal tubular lesions occur earlier than glomerular lesions,
and the severity of the renal tubular injury has become an
Federation (IDF) show that the incidence of diabetes mellitus important indicator of the progression and prognostic outcome
(DM) has been increasing rapidly worldwide, and the number of kidney function of DM patients.6,7 Among the pathogenic
of diabetes patients is expected to exceed 435 million by 2030.1 factors associated with DM, an excess of advanced glycation
DM and its associated organ damage seriously affect people’s end-products (AGEs) have been recognized as the critical
health and quality of life. In recent years, the diabetes-related driving factor for renal tubular injury in patients with T2DM.
AGEs can be produced during the nonenzymatic reaction of
fatty kidney has been reported increasingly frequently. The
protein amino groups with reducing sugars, which have key
concept of the fatty kidney was first proposed as early as the
functions in the pathogenic mechanisms of DM as well as the
1900s. However, it was not until John Moorhead proposed the
related complications. Our previous studies have shown that
hypothesis of renal lipid toxicity in 1982 that fatty kidney
excessive AGEs can disrupt intracellular cholesterol feedback
began to attract the attention of experts.2 Lipometabolic
regulation, thereby leading to the accumulation of intracellular
disturbance induces lipid ectopic deposits in the kidney and
lipids within the renal proximal tubular epithelial cells (HK-2
caused the development of fatty kidney, which is a common
cells) and promoting the development of a fatty kidney.8,9
phenomenon in type 2 diabetes mellitus (T2DM). Evidence
Sterol regulatory element-binding protein (SREBP)-cleavage
suggests that the renal lipid contents among T2DM patients
activating protein (SCAP) modulates intracellular cholesterol
detected through magnetic resonance imaging (MRI) remark-
feedback. SCAP is not only an intracellular cholesterol sensor
ably increased relative to those among non-T2DM patients.3,4
but also SREBP-2’s molecular chaperone. Upon the demand
Moreover, histological staining indicated abnormal deposition
for cholesterol by cells, SCAP separates with insulin-induced
of lipid droplets in renal glomeruli, mesangia, interstitium, and
gene-1 (Insig-1), the endoplasmic reticulum (ER) protein,
especially, renal tubules in T2DM patients,2,5 which not only
while delivering SREBP-2 within ER into Golgi apparatus and
represents the formation of fatty kidney but is also associated
with impairment of renal morphology and function. Therefore,
it is urgently important to investigate the pathogenic Received: June 15, 2021
mechanism by which fatty kidney develops in association Revised: September 22, 2021
with T2DM. Accepted: September 23, 2021
Previously, glomerular injury has a central effect on the
pathogenic mechanism of diabetic kidney disease (DKD).
Nonetheless, an increasing number of studies have reported

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activating it through proteolytic cleavage. After cleavage, the
N-terminal in SREBP-2 (nSREBP-2) experiences nuclear
translocation to activate transcriptional factors including 3-
hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR,
a key enzyme involved in cholesterol production) as well as
low-density lipoprotein receptor (LDLr, a cholesterol uptake
channel), leading to increased synthesis and uptake of
cholesterol. By contrast, Insig-1−SCAP−SREBP complexes
are reserved within ER once there is enough cholesterol within
cells, which prevents cholesterol overloading in cells under
physiological conditions.10,11 However, the specific mechanism
underlying AGE-induced abnormal cholesterol feedback
regulation is still elusive and so further research is warranted.
Thioredoxin-interacting protein (TXNIP), referred to as
thioredoxin-binding protein-2 (TBP-2) or vitamin D3
upregulated protein 1 (VDUP1), is closely related to metabolic
disorders like diabetes, fatty liver, obesity, and atheroscle-
rosis.12−14 Recently, some clinical studies have shown that the
level of TXNIP gene in the peripheral blood cells of T2DM
patients is positively correlated with serum AGEs levels,15 and
another animal study has shown that AGEs could upregulate
the expression of TXNIP in the kidneys of BALB/c mice,16
indicating AGEs might be stimulators that regulate the
expression of TXNIP. Additionally, Chang Hyun Byon et al.
reported that knocking out the TXNIP gene reduces lipid
deposition in the arterial intima of ApoE−/− mice, thereby
alleviating atherosclerosis.17 Other researchers found that
TXNIP deletion ameliorates HFD-induced hepatic steatosis
in C57Bl/6J wild-type mice.18 Furthermore, Wang W et al.
demonstrated that reducing the expression of TXNIP can
lower SREBP-2-mediated liver cholesterol synthesis, resulting
in the alleviation of fatty liver disease in type 1 diabetic rats.19
Hence, we suspect that TXNIP might be involved in the
abnormal feedback regulation of cholesterol, which may be
attributed to tubular foam cell formation. Based on the findings
described above, this work focused on investigating how
TXNIP affected the AGE-mediated renal lipid accumulation,
especially cholesterol synthesis and uptake, in diabetic mice fed
high-AGE diets and subjected to TXNIP silencing. We also
attempted to elucidate the exact mechanism of TXNIP in
regulating lipid metabolism within the AGE-mediated human
HK-2 cells at the molecular level.
■
MATERIALS AND METHODS
Animal Experimental Design. This study acquired male db/m
mice together with the C57BL/KsJ db/db mice at the National Model
Animal Center of Nanjing University (Nanjing, China). The study
protocols gained approval from the Ethics Committee of Soochow
University in line with the Declaration of Helsinki and in compliance
with the ARRIVE guidelines. Animals were raised within the
polypropylene cages under the following conditions, light/dark
cycle of 12/12 h, room temperature of 22 ± 2 °C, and relative
humidity (RH) of 60 ± 5%. Each mouse was adaptively fed for 2
weeks, then alcohol was used to wipe their tails for tail vein expansion
for subsequent injection. Later, each animal was given a slow injection
of 100 μl of a lentiviral vector encoding shRNA targeting TXNIP
(Genepharma, Shanghai, China) at 1 × 107 TU/ml by the 0.5 ml
syringe (injection velocity, 0.2 μL/min) via tail vein, and the sequence
of sh-TXNIP was established as GTCTCTGCTCGAATTGACA (5′-
3′). Equal volumes of the negative plasmid (sh-NC) or saline were
injected into the control groups. To ensure that the downregulation of
TXNIP was induced, injection was administered at weeks 1, 3, 5, and
7.20,21 One group of 8-week-old db/db mice was given the AIN-76
basal diet (Xietong Biology Co., Ltd., Nanjing, China), and another
group of these mice was given a high-AGE diet, which was prepared
B https://doi.org/10.1021/acs.jafc.1c03559
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by subjecting the AIN-76 basal diet to 10 min of heat exposure at 90
°C for generating the diet-mediated AGEs.22 Then, all animals were
classified into six groups, with five animals each. Group 1: control db/
m mice were exposed to the AIN-76 basal diet (db/m); Group 2: db/
db mice were exposed to the AIN-76 basal diet (db/db); Group 3:
db/db mice were exposed to sh-NC and the AIN-76 basal diet (db/
db + sh-NC); Group 4: db/db mice injected with sh-TXNIP and fed
an AIN-76 basal diet (db/db + sh-TXNIP); Group 5: db/db mice fed
a high-AGE diet (db/db + AGEs); and Group 5: db/db mice were
exposed to sh-TXNIP and fed high-AGE diet (db/db + AGEs + sh-
TXNIP). The animals were raised in separate metabolic cages to
collect the 24 h urine. When the experiment was completed, the blood
was sampled to conduct biochemical tests, and renal tissues were
employed to assess the histology.
Biochemical Tests. Each animal was killed upon the completion
of the experiment. Blood was sampled in the right ventricle to carry
out biochemical tests, like serum creatinine (SCR), fasting blood
glucose (FBG), total cholesterol (TC), total triglycerides (TG), and
blood urea nitrogen (BUN) by the use of automatic analyzers
(Hitachi). Serum AGEs and markers related to renal tubular injuries,
such as urinary neutrophil gelatinase-associated lipocalin (u-NGAL)
and β2-microglobulin (β2-MG), were detected by ELISA (CUSA-
BIO, Wuhan, China). In addition, Coomassie Brilliant Blue protein
assay (Jiancheng Bioengineering Institute, Nanjing, Jiangsu) was
adopted for measuring the 24 h urine proteins.
Renal Morphology. Renal cortical tissue was prepared into
paraffin-embedded sections in sequence. Then, each cross section
(measuring 3 μm) was put in the gelatin-coated slides and subjected
to hematoxylin-eosin (HE), periodic acid Schiff (PAS), and Masson
and immuohistochemical (IHC) staining.
Cell Culture. This study acquired HK-2 cells at the American
Type Culture Collection (Manassas, VA), cultivated them within the
RPMI-1640 medium HyClone (Logan, Utah) that contained 10%
fetal bovine serum (FBS, HyClone; GE Healthcare Life Sciences,
Logan, UT), 1% streptomycin/penicillin (Merck KGaA; Sigma-
Aldrich), and 2 mM L-glutamine solution, and incubated them under
37 °C, 5% CO2 and 95% humidity conditions for a period of 24 h.
Each experiment was carried out within the serum-free RPMI-1640
medium supplemented with 0.2% bovine serum albumin (BSA;
Sigma-Aldrich; Merck KGaA). AGE-BSA was purchased from Abcam
(Cambridge, U.K.). We cultured HK-2 cells within the 200 μg/ml
AGE-BSA-containing medium or the experimental medium for 48 h.
Small Interfering (si) RNA Transfection. This study cultured
HK-2 cells (5 × 105 cells/well) into six-well plates and transiently
transfected them with TXNIP siRNA (sense: 5′-CTCCCUG-
CUAUAUGGAUGUtt-3′; antisense: 5′-ACAUCCAUAUAGCAGG-
GAGtt-3′) or empty vector siRNA (Enhancer Biotechnology Co.,
Ltd., Nanjing, China) with Lipofectamine 3000 (Invitrogen; Thermo
Fisher Scientific, Inc., Waltham, MA) in line with specific protocols,
which was performed as previously described.23
Observation of Lipid Accumulation. Renal lipid deposition
within HK-2 cells and the db/db mice was evaluated by Oil Red O
staining. Briefly, 4% paraformaldehyde was used to fix samples,
followed by 30 min of Oil Red O staining. Subsequently, hematoxylin
was adopted to counterstain samples for a period of 5 min. A light
microscope (Carl Zeiss, Hertfordshire, U.K.) was employed for the
result examination.
Quantification of Intracellular Cholesterol. Free cholesterol
(FC) and intracellular TC levels were quantitatively measured in vivo
and in vitro by enzymatic assays (Applygen Technologies Inc., Beijing,
China). In brief, after sample collection, lipids were extracted with 1
mL of chloroform/methanol (2:1). Afterward, the samples were
sonicated and centrifuged to obtain the lipid phase. Thereafter, we
treated samples according to specific instructions. The standard curve
was utilized to analyze FC and TC contents in each sample.
Cholesterol ester (CE) level was measured by subtracting TC by FC.
Protein Extraction and Western Blot (WB) Analysis. The total
cellular and nuclear proteins were collected, followed by denaturation
and 6%−15% SDS-PAGE, which was performed as previously
described.24 Later, proteins were transferred onto poly(vinylidene
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Table 1. Biochemical Characteristics of the Micea

parameters db/m db/db db/db + sh-NC db/db + sh-TXNIP db/db + AGEs db/db + AGEs + sh-TXNIP
FBG (mmol/L) 6.33 ± 0.25 18.83 ± 1.48b 17.35 ± 0.67 11.78 ± 1.40c 29.18 ± 1.36c 22.43 ± 1.83d
AGEs (ng/mL) 1.22 ± 0.14 3.97 ± 0.19b 3.56 ± 0.21 3.57 ± 0.26 5.94 ± 0.25c 5.54 ± 0.42
TG (mmol/L) 1.22 ± 0.09 2.43 ± 0.18b 2.57 ± 0.23 2.37 ± 0.16 2.66 ± 0.16 2.60 ± 0.26
TC (mmol/L) 2.27 ± 0.04 3.46 ± 0.16b 3.29 ± 0.18 3.02 ± 0.21 3.36 ± 0.16 3.30 ± 0.13
body weight (g) 21.55 ± 0.52 50.95 ± 0.36b 49.78 ± 0.62 50.88 ± 2.22 52.85 ± 1.57 49.78 ± 1.11
food intake (g/day) 5.48 ± 0.62 6.35 ± 0.96 5.92 ± 1.14 6.58 ± 1.32 6.86 ± 1.42 6.04 ± 1.10
water intake (mL/day) 5.60 ± 0.78 22.53 ± 3.21b 20.66 ± 2.98 14.52 ± 2.12c 28.00 ± 3.42c 16.04 ± 2.78d
a
The levels of FBG, AGEs, TG, TC, the body weight, the food intake, and the water intake of the mice. Values are expressed as the mean ± S.E.M.
of five mice in each group. bP < 0.01 versus the db/m group. cP < 0.01 versus the db/db group. dP < 0.01 versus the AGEs + db/db group.

fluoride) (PVDF) membranes (GE Healthcare, Buckinghamshire,
U.K.), followed by 1 h of blocking using 5% BSA within Tris-buffered
saline supplemented with 0.05% Tween 20 (TBST) under ambient
temperature. After washing, we incubated the blots with 5% BSA
within TBST that contained diluted antibodies against TXNIP, SCAP,
SREBP-2, LDLr, HMGCoAR, Insig-1, Lamin A, and GAPDH
(Abcam, Cambridge, U.K.) under 4 °C overnight. After rinsing with
TBST thrice, the secondary antibody diluted at 1:5000 with TBST
that contained 1% BSA (Santa Cruz Biotechnology) was used to
further incubate the membranes under ambient temperature for 1 h,
followed by rinsing by TBST thrice again. Thereafter, the
chemiluminescence reaction (Pierce, Rockford, IL) was conducted
and blots were exposed to X-ray film for a suitable period of time to
detect bands. Later, LabWorks software (UVP Laboratory Products,
Upland, CA) was utilized for densitometrical detection and evaluation
of bands, and the bands were normalized with the Lamin A or
GAPDH bands.
Confocal Microscopy. The human SCAP cDNA was ligated at
BstE-XbaI sites in pEGFP-C1 vector (Genechem Co. Ltd., Shanghai,
China) to prepare the green fluorescent protein (GFP)-SCAP
expression construct, which was performed as previously described.8
Effectene Transfection Reagent (Invitrogen, Paisley, U.K.) was
utilized to transfect pEGFP-SCAP into cells in accordance with
specific instructions. Subsequently, we placed cells onto chamber
slides to incubate them within the growth medium for a period of 48
h, followed by 30 min of fixation with 5% formalin solution, 15 min of
permeabilization using 0.25% Triton X-100, and 2 h of staining using
Golgin-97 antibodies (Abcam, Cambridge, U.K.) under ambient
temperature. Later, cells were rinsed and counterstained with the
secondary fluorescent antibody for an additional one hour. The
resulting cells were examined by confocal microscopy using Zeiss
LSM 510 Meta (Carl Zeiss, Hertfordshire, U.K.).
Coimmunoprecipitation. The interactions between SCAP and
SREBP-2, TXNIP, or Insig-1 proteins in HK-2 cells were analyzed by
coimmunoprecipitation (Co-IP). In brief, the cell IP lysis buffer
(Thermo Scientific Pierce) was adopted for extracting total protein.
Cell lysates were cleared by centrifugation, and supernatants were
immunoprecipitated with the appropriate antibodies using protein A/
G-agarose beads. Next, the samples were subjected to immunoblot-
ting analysis with the indicated antibodies.
Statistical Analysis. Results were analyzed with SPSS20.0 and
expressed in the form of mean ± S.E.M. One-way ANOVA and then
Bonferroni’s multiple comparison tests were adopted for multiple
comparisons. P values <0.05 indicated statistical significance.
of FBG, compared with the db/db mice or db/db mice
exposed to high-AGEs diet, while AGEs, TG, and TC exhibited
no changes. In addition, compared with that in the db/m mice,
polydipsia was observed in the db/db mice, especially in the
db/db mice exposed to high-AGE diets, and silencing of
TXNIP decreased the water intake in the db/db mice exposed
to basal diet and those exposed to high-AGE diet. However,
the difference in food intake was not significant among mice.
Silencing of TXNIP Reduced Renal Tubular Lipid
Accumulation In Vivo and In Vitro. For exploring the
involvement of TXNIP in regulating in vivo abnormal lipid
deposition within diabetic kidneys, this study analyzed renal
lipid droplet deposition in diabetic mice transfected with sh-
TXNIP or a control plasmid. This work first verified TXNIP
depletion within the kidneys of the db/db mice through IHC
staining and Western blot analysis (Figure 1A,B). Then, Oil
Red O staining was conducted. Strong positive areas were
found within renal tubules from the db/db mice, and nearly no
stain was observed in the db/m mice (Figure 2A). Intracellular
cholesterol level was quantified, which verified the increase in
the level of renal cholesterol ester deposition in the db/db
mice (Figure 2B). Moreover, significantly increased intra-
cellular cholesterol contents and Oil Red O staining were
observed among the db/db mice fed with high-AGE diet
compared with those fed with basal diet. However, silencing of
TXNIP reduced intracellular cholesterol and tubular lipid
droplet contents in the db/db mice exposed to high-AGE and
basal diets (Figure 2A,B).
To further analyze the function of TXNIP in vitro, we
transfected HK-2 cells with control vectors or TXNIP siRNA
plasmids, followed by 48 h of AGE-BSA treatment. TXNIP
deletion within HK-2 cells was confirmed through Western
blot analysis (Figure 1C). Then, we conducted Oil Red O
staining. The lipid droplet accumulation degree in the AGE-
BSA-treated HK-2 cells increased relative to control (Figure
2C). This result was confirmed using the results of the
cholesterol quantitative assay (Figure 2D). However, the
silencing of TXNIP reduced the intracellular cholesterol and
lipid droplet contents within HK-2 cells exposed to AGE-BSA

■
RESULTS
Biochemical Features of the Mice. The basic mouse
features when the experiment is completed are presented in
Table 1. As a result, the diabetic mice showed higher FBG and
the db/db mice had increased body weight, AGEs, TG, and
TC levels compared with the db/m mice. Relative to those in
the db/db mice, the levels of FBG and AGEs in the db/db
mice exposed to high-AGE diets were significantly elevated.
However, silencing of TXNIP exhibited a remarkable decrease
C https://doi.org/10.1021/acs.jafc.1c03559
treatment (Figure 2C,D).
Silencing of TXNIP Reduced HMGCoAR, LDLr,
nSREBP-2, and SCAP Expression In Vivo and In Vitro.
To explore the mechanism by which TXNIP regulated the
tubular cholesterol metabolism at the molecular level, we
analyzed LDLr, HMGCoAR, SCAP, SREBP-2, and nSREBP-2
levels within HK-2 cells and the db/db mouse kidneys.
According to Figure 3A, the db/db mice had increased LDLr,
HMGCoAR, SCAP, SREBP-2, and nSREBP-2 protein levels
within the kidneys relative to those in the db/m mice. The
renal levels of these proteins in the db/db mice exposed to
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Figure 1. AGEs increased TXNIP expression in the kidneys of
diabetic mice and HK-2 cells. The expression of TXNIP protein in the
kidneys of mice (A, B) and in HK-2 cells (C) was determined by IHC
staining and Western blotting. Image J was used to quantify the
relative levels of proteins. GAPDH was used as an internal control.
Values are expressed as the mean ± S.E.M. of five independent
experiments. **P < 0.01 versus the db/m group or the control cells
(Ctr). #P < 0.05 versus db/db group, and ##P < 0.01 versus AGE-BSA-
treated cells.▲▲P < 0.01 versus AGEs + db/db group.
high-AGE diet markedly increased relative to those exposed to
the basal diet. Nonetheless, silencing of TXNIP clearly reduced
the expression levels of HMGCoAR, LDLr, nSREBP-2, and
SCAP but did not reduce SREBP-2 expression in the db/db
mice exposed to high-AGE and basal diets, respectively. In the
in vitro study, the HMGCoAR, LDLr, SREBP-2, nSREBP-2,
and SCAP protein expressions within AGE-BSA-treated HK-2
cells considerably increased relative to control. These proteins
can be suppressed by silencing TXNIP, other than SREBP-2
(Figure 3B). These results suggested that the upregulation of
SCAP and SREBP-2 induced by AGEs may promote SCAP−
SREBP-2 complexes to transport to Golgi apparatus, where
SREBP-2 was hydrolyzed. Its active fragment (nSREBP-2)
entered the nucleus and increased the expression levels of
HMGCoAR and LDLr. However, TXNIP silencing decreased
nSREBP-2 expression without affecting the SREBP-2 level,
D https://doi.org/10.1021/acs.jafc.1c03559
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indicating that the reduced expression of nSREBP-2 might be
due to the attenuated ER-to-Golgi apparatus transport of the
SCAP−SREBP-2 complexes.
Silencing of the TXNIP Reduced the Translocation of
SCAP−SREBP-2 Complexes In Vitro. To explore the above
speculation, this study conducted confocal microscopy to
analyze the translocation of SCAP from ER-to-Golgi apparatus
within HK-2 cells. As a result, loading with AGE-BSA
promoted the deposition of SCAP within the Golgi apparatus,
but this effect was notably inhibited by the silencing of TXNIP
(Figure 4A). However, the results of Co-IP showed a lack of
interaction between TXNIP and SCAP−SREBP-2 protein
complexes, although the number of SCAP−SREBP-2 protein
complexes was significantly higher in the AGE-BSA-treated
HK-2 cells (Figure 4B). Based on the above findings, TXNIP
does not affect the transport of SCAP−SREBP-2 protein
complexes by directly binding to the complexes. The enhanced
transport of the complexes might be related to the increased
number of bonds between SREBP-2 and SCAP after
upregulating SCAP and SREBP-2. However, silencing of
TXNIP weakened the transport of SCAP (Figure 4A) and
reduced the interaction between SCAP and SREBP-2 (Figure
4C), indicating that TXNIP silencing attenuated the ER-to-
Golgi apparatus transport of SCAP−SREBP-2 protein
complexes, which reduced the activation of nSREBP-2,
followed by the downregulation of HMGCoAR and LDLr
proteins.
Silencing of TXNIP Increased Insig-1 Expression In
Vivo and In Vitro. Given that Insig-1 has an important role in
the transport of SCAP−SREBP-2 complexes, we further
evaluated the expression of Insig-1 within HK-2 cells and
kidneys of the db/db mice. It was found that the protein level
of Insig-1 decreased within the kidneys of the db/db mice
relative to those in the db/m mice. The protein levels of Insig-
1 in the kidneys of the high-AGE-diet-fed db/db mice
markedly declined compared with those fed with basal diet.
Nonetheless, the silencing of TXNIP clearly upregulated Insig-
1 protein of both groups (Figure 5A). Furthermore, we
observed the low Insig-1 protein level within the AGE-BSA-
treated HK-2 cells and the silencing of TXNIP upregulated
Insig-1 (Figure 5B). Additionally, Insig-1 physically interacted
with SCAP−SREBP-2 protein complexes in the HK-2 cells,
and AGE-BSA inhibited the interaction between Insig-1 and
SCAP−SREBP-2 protein complexes (Figure 5C). The
inhibitory effect may be ascribed to the low expression of
Insig-1. However, silencing of TXNIP did not obviously
change the interaction between SCAP and Insig-1 (Figure 5D),
suggesting that silencing of TXNIP reduced the ER-to-Golgi
apparatus transport of the SCAP−SREBP-2 complexes but did
not increase their retention in the ER in spite of the
upregulation of Insig-1, which might be due to the down-
regulation of SCAP.
Silencing of TXNIP Improved the Renal Morphology
and Function in the Diabetic Mice. We also examined the
role of TXNIP on the structure and function of the kidneys of
diabetic mice. First, the results of HE staining indicated no
obvious pathological change in the glomeruli and tubules in
the db/m mice. Vacuolar degeneration of renal tubular
epithelial cells was observed within the db/db mouse kidneys.
Such an alteration was pronounced in the db/db mice exposed
to high-AGE diet relative to those fed with basal diet.
However, the silencing of TXNIP improved the morphology of
the renal tubules (Figure 6A). The results of PAS staining
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Figure 2. Silencing of TXNIP reduced renal tubular lipid accumulation in vivo and in vitro. Renal Oil Red O staining and the semiquantitative
analysis for the percent of the positive areas in each group (A). The intracellular cholesterol contents in the kidneys of mice (B). Oil Red O staining
and the positive percentage of HK-2 cells (C). The intracellular cholesterol content in HK-2 cells (D). Oil Red O staining was observed under a
light microscope (400×). The percentage of positive staining areas in the kidneys of mice was semiquantitative analyzed by Image J. The positive
percentage of HK-2 cells was counted from five experiments. Values of intracellular cholesterol content are expressed as the mean ± S.E.M. of five
independent experiments. **P < 0.01 versus the db/m group or the control cells (Ctr). #P < 0.05 and ##P < 0.01 versus db/db group or AGE-BSA-
treated cells. ▲P < 0.05 and ▲▲P < 0.01 versus the AGEs + db/db group.

showed that the glomerular vascular loop was clear in the db/
m mice, along with a normal basement membrane. Mesangial
expansion in the renal glomeruli was detected among the db/
db mice, and this change was more significant within the db/
db mouse kidneys fed with high-AGE diet compared with
those fed with basal diet. However, TXNIP silencing
significantly alleviated these pathological changes (Figure
6B). As revealed by Masson staining, collagen fibers
significantly accumulated within tubular interstitium in the
db/db mice (stained blue), especially in the tubular
interstitium of high-AGE diet-fed db/db mice, while TXNIP
silencing significantly reduced the deposition of collagen fibers
(Figure 6C). Furthermore, compared with the db/m mice,
SCR (Figure 6D), BUN (Figure 6E), u-NGAL (Figure 6F),
β2-MG (Figure 6G), and 24 h urine protein (Figure 6H) levels
E https://doi.org/10.1021/acs.jafc.1c03559
among the db/db mice were elevated. Additionally, for the db/
db mice fed on a high-AGEs diet, the above parameters
showed significantly elevated levels relative to those in the db/
db mice, while no obvious change in BUN was observed in
both groups. However, TXNIP silencing remarkably decreased
these parameters compared with those in the db/db mice or
those fed with high-AGE diet. These results suggested that
AGEs caused the morphological and functional injuries of renal
glomeruli and tubules in diabetic mice. However, TXNIP
silencing greatly improved these changes. This effect might be
related to the decreased level of lipid deposition in the kidney,
although other mechanisms may be involved given that TXNIP
deletion can improve renal inflammatory response and
oxidative stress.25,26
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Figure 3. Silencing of TXNIP reduced HMGCoAR, LDLr, nSREBP-2, and SCAP expression in the kidneys of diabetic mice and HK-2 cells. The
protein expression of HMGCoAR, LDLr, SREBP-2, nSREBP-2, and SCAP in the kidneys of mice (A) and in HK-2 cells (B) was determined by
Western blotting. Image J was used to quantify the relative levels of proteins. GAPDH or Lamin A was used as an internal control. Values are
expressed as the mean ± S.E.M. of five independent experiments. *P < 0.05 and **P < 0.01 versus the db/m group or the control cells (Ctr). #P <
0.05 and ##P < 0.01, versus db/db group or AGE-BSA-treated cells. ▲P < 0.05 and ▲▲P < 0.01 versus the AGEs + db/db group. ns = not
significant, versus db/db group, AGEs + db/db group or AGE-BSA-treated cells.

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Journal of Agricultural and Food Chemistry
Figure 4. Interactions among SCAP, SREBP-2, and TXNIP proteins.
The translocation of GFP-SCAP from the ER to the Golgi was
investigated using confocal microscopy after staining with an anti-
Golgi antibody. Silencing of TXNIP inhibited the translocation of
GFP-SCAP from the ER to the Golgi in HK-2 cells (A). The
interaction between SCAP and SREBP-2 and TXNIP proteins was
investigated using Co-IP. AGE-BSA increased the interactions of
SCAP and SREBP-2 in HK-2 cells, whereas TXNIP protein was not
detected (B). Silencing of TXNIP reduced the interaction between
SCAP and SREBP-2 (C).
■
DISCUSSION
The fatty kidney is an important risk factor for renal injury.27
Therefore, reducing renal lipid accumulation is considered an
effective means of preventing the development of DKD. In a
previous study, we showed that AGEs can cause abnormal
cholesterol metabolism in the renal tubules of T2DM rats. The
current study first examined the role of TXNIP in the
accumulation of lipids in the kidneys of high-AGE diet-fed
diabetic mice. A functional study was conducted on AGE-
treated HK-2 cells. As a result, TXNIP accounts for an
important regulator for lipid deposition within renal tubular
cells under high-AGE conditions. TXNIP silencing remarkably
inhibited AGE-induced cholesterol accumulation by down-
regulating the expression of SCAP, thus inhibiting the ER-to-
Golgi apparatus transport of SCAP−SREBP-2 complexes.
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pubs.acs.org/JAFC Article
Figure 5. Expression of Insig-1 proteins and the interactions among
Insig-1, SREBP-2, and SCAP proteins. The protein expression of
Insig-1 in the kidneys of mice (A) and in HK-2 cells (B) was
determined by Western blotting. Image J was used to quantify the
relative levels of proteins. GAPDH was used as an internal control.
Values are expressed as the mean ± S.E.M. of five independent
experiments. **P < 0.01 versus the db/m group or the control cells
(Ctr). #P < 0.05 and ##P < 0.01 versus db/db group or AGE-BSA-
treated cells. ▲P < 0.05 versus the AGEs + db/db group. Interactions
among Insig-1, SREBP-2, and SCAP proteins were investigated using
Co-IP. AGE-BSA increased the interactions of SCAP−SREBP-2
protein complexes in HK-2 cells and reduced the interactions between
SCAP and Insig-1 (C). Silencing of TXNIP did not change the
interaction between SCAP and Insig-1 (D).
Then, SREBP-2 hydrolysis was inhibited in the Golgi
apparatus, followed by reduced cholesterol production
regulated by HMGCoAR and cholesterol absorption regulated
by LDLr.
As suggested by more and more studies, TXNIP has a key
function in lipid metabolism.28 Recently, Chunyang Du et al.
reported that TXNIP deficiency alleviates renal fatty acid
metabolism in mice with streptozotocin-induced type 1
diabetes.29 However, to date, only few studies have analyzed
the TXNIP’s function in the metabolism of cholesterol in the
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Journal of Agricultural and Food Chemistry
Figure 6. Silencing of TXNIP improved renal morphology and
function of the diabetic mice. HE (A), PAS (B), and Masson (C)
staining in the kidneys of mice was observed under a light microscope
(200×). The mesangial area was expressed quantitatively by
calculating the percentage of the total glomerular area that was PAS
positive. Fifteen glomerular tufts per animal were chosen randomly for
analysis. The histogram represents the mesangial matrix index,
H https://doi.org/10.1021/acs.jafc.1c03559
pubs.acs.org/JAFC Article
Figure 6. continued
expressed as the mean ± S.E.M. of five mice in each group. Positive
fibrotic tissue stained by Masson staining was quantified by image
analysis using Image J software, presented as the percentage of the
total area. The histogram is expressed as the mean ± S.E.M. of five
mice in each group. The levels of SCR (D), BUN (E), u-NGAL (F),
β2-MG (G), and 24 h urine protein (H) in mice. Values are expressed
as the mean ± S.E.M. of five independent experiments. *P < 0.05 and
**P < 0.01 versus the db/m group. #P < 0.05 and ##P < 0.01 versus
db/db group. ▲P < 0.05 and ▲▲P < 0.01 versus the AGEs + db/db
group. ns = not significant, versus db/db group.
kidney. The present work suggested the role of TXNIP as a key
regulatory factor for the metabolism of cholesterol within the
kidneys of T2DM mice as well as within the cultured HK-2
cells exposed to high-AGE conditions. Renal TXNIP level was
dramatically upregulated in the diabetic mice relative to that in
the control mice, and the amounts of lipid droplets and
intracellular cholesterol levels in the kidneys increased.
Moreover, renal pathological changes and decreased renal
function were observed, especially in the renal tubules. These
effects were more pronounced in the high-AGE-diet-fed
diabetic mice. TXNIP silencing remarkably decreased these
variables. Meanwhile, the TXNIP protein level was upregulated
by AGEs in the HK-2 cells, and lipid droplet accumulation
resulted from AGEs. However, TXNIP silencing remarkably
suppressed the above process within the HK-2 cells. Based on
the above findings, TXNIP was related to cholesterol
deposition within the renal tubules of diabetic mice.
To further investigate the mechanism governing the effect of
TXNIP on the fatty kidney, we investigated the effect of
TXNIP on the feedback regulation of cholesterol in the HK-2
cells and kidneys of T2DM mice. We observed that the renal
protein levels of HMGCoAR, LDLr, SCAP, nSREBP-2, and
SREBP-2 increased in diabetic mice, especially in the kidneys
of the high-AGE-diet-fed diabetic mice. Similar results were
found in the AGE-treated HK-2 cells. However, TXNIP
silencing clearly reduced HMGCoAR, LDLr, nSREBP-2, and
SCAP protein expression, in addition to SREBP-2, in the
kidneys of diabetic mice and high-AGE-diet-fed diabetic mice,
as well as AGE-treated HK-2 cells. Further studies showed that
loading with AGE-BSA accelerated SCAP−SREBP-2 complex
formation while promoting their transportation to the Golgi
apparatus. However, these could be inhibited by TXNIP
silencing in the HK-2 cells. The above results suggested that
AGEs facilitated the ER-to-Golgi apparatus transport of
SCAP−SREBP-2 complexes by upregulating SREBP-2 and
SCAP expressions. Thus, SREBP-2 hydrolysis was promoted.
Then, nSREBP-2 fragments that entered the nucleus increased
in number and were bound to the promoters of HMGCoAR
and LDLr, thus enhancing the transcription and translation of
HMGCoAR and LDLr, leading to increased cholesterol
synthesis and uptake. However, TXNIP silencing decreased
the expression of SCAP and attenuated ER-to-Golgi apparatus
translocation of SCAP−SREBP-2 complexes, thus down-
regulating the expression of HMGCoAR and LDLr and
ultimately reducing renal tubular cholesterol synthesis and
uptake. Until now, there is no report on the relationship
between TXNIP and SCAP. Previous studies have shown that
glycosylation of SCAP prolongs its half-life and enhances the
stability of SCAP protein.30 Therefore, we suspect that
silencing TXNIP may affect the glycosylation of SCAP,
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Journal of Agricultural and Food Chemistry
resulting in the accelerated degradation of SCAP. However,
further research is warranted to verify the above speculation.
Insig-1 is a membrane protein that resides within ER, which
has an important function in regulating SREBP proteolytic
processing.31 It is ubiquitinated and degraded rapidly by
proteasomes when cholesterol is depleted in the cells under
physiological conditions. Conversely, the ubiquitination of
Insig-1 is blocked, and the protein is stabilized when
intracellular cholesterol is accumulated in cells.32 However,
cells come round the SREBP cholesterol suppression
mechanism by modulating Insig-1 protein expression upon
ER stress or hypotonic shock. In the current study, we found
that the renal protein level of Insig-1 decreased in the diabetic
mice, especially in high-AGE-diet-fed diabetic mice. Similar
results were found in the AGE-treated HK-2 cells.
Furthermore, the results of Co-IP showed that AGE-BSA
inhibited the interaction between Insig-1 and SCAP−SREBP-2
protein complexes. These results implied that AGEs facilitated
the ER-to-Golgi apparatus transport of SCAP−SREBP-2
complexes by downregulating the expression of Insig-1 but
upregulating that of SREBP-2 and SCAP, which enhanced ER-
to-Golgi apparatus delivery of the SCAP−SREBP-2 complexes.
Hence, the transcriptional rates of SREBP-2 target genes
increased, thereby leading to an increase in cholesterol
synthesis and uptake. However, TXNIP silencing did not
increase SCAP−SREBP-2 complex retention within ER in HK-
2 cells, despite the clearly elevated expression of the Insig-1
protein, suggesting that the effect of TXNIP on the
transportation of complexes was mainly due to its regulation
of SCAP rather than Insig-1. In addition, previous studies have
shown that binding of HMGCoAR to Insig-1 proteins leads to
the ubiquitination and degradation of HMGCoAR,33 indicat-
ing that the upregulation of Insig-1 induced by TXNIP
silencing may further promote the combination of Insig-1 and
HMGCoAR, which promotes the degradation of HMGCoAR
and inhibits cholesterol synthesis.
To sum up, this study suggests the key function of TXNIP in
regulating lipid accumulation in the kidneys of diabetic mice
fed with high-AGE diet as well as in HK-2 cells exposed to
AGE treatment. The AGEs can promote SREBP-2 and SCAP
levels while downregulating Insig-1 expression, thus enhancing
the ER-to-Golgi apparatus translocation of SCAP−SREBP-2
complexes, where SREBP-2 is hydrolyzed. The active frag-
ments of SREBP-2 (nSREBP-2) enter the nucleus, thereby
upregulating HMGCoAR and LDLr expression levels and
ultimately increasing the cholesterol synthesis and uptake.
However, TXNIP silencing alleviates diabetic renal tubular
lipid accumulation by inhibiting cholesterol absorption
induced by LDLr and cholesterol production regulated by
HMGCoAR through the downregulation of SCAP expression
and subsequent inhibited ER-to-Golgi apparatus translocation
of SCAP−SREBP-2 complexes. Therefore, TXNIP is a
potential therapeutic target for the diabetic fatty kidney.
■ AUTHOR INFORMATION
Corresponding Authors
Hong Sun − Department of Endocrinology and Metabolism,
The First Affiliated Hospital of Soochow University, Suzhou,
Jiangsu 215000, China; orcid.org/0000-0003-4335-
2789; Email: sunhong_611@126.com
Jianzhong Li − Department of Nephrology, The First Affiliated
Hospital of Soochow University, Suzhou, Jiangsu 215000,
China; Email: ljzsnk@hotmail.com
I https://doi.org/10.1021/acs.jafc.1c03559
pubs.acs.org/JAFC Article
Authors
Juan Chen − Department of Endocrinology, Jiangsu Province
Hospital of Chinese Medicine, Affiliated Hospital of Nanjing
University of Chinese Medicine, Nanjing 210000, China
Lili Sun − Department of Endocrinology and Metabolism, The
First Affiliated Hospital of Soochow University, Suzhou,
Jiangsu 215000, China
Bimin Shi − Department of Endocrinology and Metabolism,
The First Affiliated Hospital of Soochow University, Suzhou,
Jiangsu 215000, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.1c03559
Author Contributions
⊥
H.S. and J.C. contributed equally to this work. H.S. designed
the experiments. J.L., B.S., J.C., and L.S. performed the
experiments. H.S., J.C., and J.L. revised the manuscript. All
authors read and approved the final manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by grants from the National Natural
Science Foundation of China (grant no. 81700632 to H.S.),
the Natural Science Foundation of Jiangsu Province (grant no.
BK20170366 to H.S.), People’s Livelihood Science and
Technology of Suzhou (grant no. SYS2020104 to H.S., grant
no. SS202008 to B.S.), and China Postdoctoral Science
Foundation (2020M671559 to J.C.).
■
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