Full-Text PDF - Hindawi Publishing Corporationdownloads.hindawi.com/journals/jdr/2017/3417306.pdf · Research Article Quercetin Improves Glucose and Lipid Metabolism of Diabetic Rats:

Research ArticleQuercetin Improves Glucose and Lipid Metabolism ofDiabetic Rats: Involvement of Akt Signaling and SIRT1

Jing Peng,1 Qingde Li,1 Keye Li,2 Li Zhu,3 Xiaoding Lin,3 Xiaohong Lin,2 Qianhui Shen,2

Guoping Li,4 and Xi Xie2,3,4

1Yue Bei People’s Hospital, Shaoguan 512026, China2State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou 570228, China3Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, College of Marine Science, Hainan University,Haikou 570228, China4Key Laboratory of Tropical Biological Resources of Ministry of Education, Hainan University, Haikou 570228, China

Correspondence should be addressed to Xi Xie; [email protected]

Received 9 June 2017; Accepted 25 September 2017; Published 12 December 2017

Academic Editor: Raffaella Mastrocola

Copyright © 2017 Jing Peng et al. This is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Glucose and lipid metabolism disorder in diabetes mellitus often causes damage to multiple tissues and organs. Diabetes mellitus isbeneficially affected by quercetin. However, its concrete mechanisms are yet to be fully elucidated. In our study, diabetes wasinduced in Sprague-Dawley rats by STZ injection. The rats were randomly divided into normal control, diabetic model, low-dose quercetin treatment, high-dose quercetin treatment, and pioglitazone treatment groups. Fasting blood glucose was collectedto evaluate diabetes. Immunohistochemistry and fluorometric assay were performed to explore SIRT1. Akt levels were measuredthrough immunoprecipitation and Western blot. After 12 weeks of quercetin treatment, the biochemical parameters of glucoseand lipid metabolism improved to varying degrees. Hepatic histomorphological injury was alleviated, and hepatic glycogencontent was increased. The expression and activity of hepatic SIRT1 were enhanced, and Akt was activated by phosphorylationand deacetylation. These results suggested that the beneficial effects of quercetin on glucose and lipid metabolism disorder areprobably associated with the upregulated activity and protein level of SIRT1 and its influence on Akt signaling pathway. Hence,quercetin shows potential for the treatment of glucose and lipid metabolism disorder in diabetes mellitus.

1. Introduction

Flavonoids beneficially affect human and animal health. As atypical flavonol-type flavonoid, quercetin is ubiquitouslypresent in fruits and vegetables and is considered a strongantioxidant because of its ability to scavenge free radicalsand bind to transition metal ions; as such, this flavonoid par-ticipates in multiple pharmacological activities, includinganti-inflammation, platelet aggregation prevention, and anti-tumor effects [1–5]. Quercetin protects organisms againstseveral types of liver diseases [6, 7] and elicits beneficialeffects on glucose and lipid metabolism [8]. Euonymus alatusas a folk medicine in China has been clinically used to treattype 2 diabetes and to alleviate hyperglycemia in animalswith diabetes. Interestingly, quercetin is one of the active

components of E. alatus. This flavonoid can effectively atten-uate hyperglycemia in alloxan-induced diabetic mice [9];however, its concrete mechanisms remain unclear. Consider-ing that glucose and lipid metabolism disorder is the leadingcause of diabetes-related complications, we aimed to explorethe protective effect of quercetin against glucose and lipidmetabolism disorder in diabetes and further clarify theunderlying molecular mechanisms.

Akt is a serine/threonine protein kinase, which is alsoknown as PKB. Its phosphorylation is closely associated withglucose and lipid metabolism. After metformin treatment isadministered, Akt phosphorylation is restored in the pan-creas and visceral adipose tissues [10]. Hepatic Dapper1overexpression increases the phosphorylated Akt levels byfourfold and suppresses gluconeogenesis and lipogenesis in

HindawiJournal of Diabetes ResearchVolume 2017, Article ID 3417306, 10 pageshttps://doi.org/10.1155/2017/3417306

https://doi.org/10.1155/2017/3417306

db/db mouse livers [11]. MiR-423-5p promotes hepaticgluconeogenesis and lipid deposition and induces glucoseintolerance, insulin resistance, and hyperglycemia by inhibit-ing the FAM3A-ATP-Akt pathway in the liver [12]. Dysfunc-tional Akt causes glucose and lipid metabolism disorderbecause this form of Akt changes the functions of down-stream kinases that regulate glucose and lipid metabolismor other related signal molecules [13].

Sirtuin type 1 (SIRT1), a nicotinamide adeninedinucleotide-positive-dependent protein deacetylase belong-ing to the mammalian sirtuin family, regulates gene tran-scription and chromosome instability and targets proteinfunctions and activities by deacetylating histone, transcrip-tion factors, transcriptional regulators, and lysine residuesin intracellular functional proteins [14]. Therefore, SIRT1participates in a series of energy metabolism and pathologicaland physiological processes, such as gluconeogenesis andlipid accumulation, aging, development, inflammation, andtumorigenesis [15–17]. SIRT1 gene mutation may inducetype 1 diabetes mellitus [18]. In SIRT1 transgenic mice,hepatic insulin sensitivity is increased, body energy demandsare decreased, and insulin resistance and diabetes are furtheralleviated [19]. SIRT1 also protects pancreatic β-cells againstcytokine toxicity [20]. In our previous studies, SIRT1 elicitsprotective effects on diabetic nephropathy [21]. Recent stud-ies also showed that SIRT1 was involved in the regulation ofAkt activity [22, 23]. SIRT1-induced deacetylation of K14and K20 of Akt is necessary to promote the binding of Aktto PIP3 and cause membrane localization and activation[23]. Lysine acetylation inhibits IRS2 activity, and SIRT1-dependent deacetylation increases its activity, therebyenhancing Akt activity [24].

This work aimed to determine whether quercetin canimprove the glucose and lipid metabolism of rats with diabe-tes and to explore its underlying mechanisms, especiallythose associated with Akt and SIRT1.

2. Materials and Methods

2.1. Experimental Design. Male Sprague-Dawley (SD) rats(n = 40, 6w, 130± 15 g) were purchased from the LaboratoryAnimal Center, Sun Yat-sen University, Guangzhou, China.(Animal quality certificate number: 0112395). The rats weremaintained under standard conditions with free access towater and chow. All experimental procedures conformed tothe China Animal Welfare Legislation and approved by theEthics Committee on the Care and Use of Laboratory Ani-mals of Sun Yat-sen University. After high-fat and sugar diet(contained 0.25% bile salt, 1% cholesterol, 10% lard, and 20%sucrose) for 4 weeks, SD rats received a low dose of strepto-zocin (STZ) intraperitoneal injection (30mg/kg, Sigma-Aldrich, USA) to induce diabetes. Diabetes was regarded assuccessfully induced in rats with fasting blood glucose(FBG) levels above 11.1mM. Diabetic rats were randomlyassigned to STZ-induced diabetic group (n = 8), low-dosequercetin treatment (QU-low, 10mg/kg, n = 8, gavage,purity> 98%, HPLC; Zelang, Nanjing, China), high-dosequercetin treatment (QU-high, 50mg/kg, n = 8, gavage),and pioglitazone treatment (5mg/kg, n = 8, gavage, Takeda,

Japan, subpackaged by Tianjin Takeda) groups. The dosesof quercetin chosen were based on other previous preclin-ical experiment [25–27]. Both normal control rats andSTZ -induced diabetic rats were also fed equal volumes of0.5% (w/v) CMC-Na. Rats in all group were administeredby gavage 6 days every week at 9:00–10:00 am for 12 weeks.

2.2. Biochemical Analysis. At the age of 16 weeks, rats werefasted for 12 h and killed after anesthesia. FBG levels wereanalyzed by a OneTouch glucometer (Johnson and Johnson,USA). Glycosylated hemoglobin (HbA1c) detection was car-ried out following the manufacturer’s instruction (Jiancheng,Nanjing, China). Glycosylated serum protein (GSP), triglyc-eride (TG), total cholesterol (TC), low-density lipoproteincholesterol (LDL-C), and high-density lipoprotein choles-terol (HDL-C) in serum were measured by commercial avail-able kits purchased from Nanjing Jiancheng BioengineeringInstitute (Nanjing, China). Liver tissues were stored at−80°C until analysis. Liver samples were rinsed with physio-logical saline, dried, and immediately weighed for hepaticglycogen measurement (Jiancheng, Nanjing, China).

2.3. Liver Histological Examination. Paraffin-embedded livertissue samples were cut into 5μm thick sections for periodicacid-Schiff pas staining, and the sections were then examinedby light microscopy. Rat livers were fixed with 10% formalin,cut into 10μm sections with a cryostat for Sudan IV staining,and then examined by light microscopy.

2.4. Immunohistochemistry. Liver specimens fixed in 10%buffered formalin were embedded in paraffin blocks. Sections(5μm thick) of the liver were processed using a standardimmunostaining protocol. After routine deparaffinization,hydration, and blockage of endogenous peroxidase, sectionswere pretreated by microwave for 20min in 10mmol/Lsodium citrate buffer (pH6.0) for antigen retrieval andfollowed by incubation sequentially with blocking agent,rabbit anti-SIRT1 antibody (1 : 100, Abcam, Cambridge, MA,USA) and secondary antibody (1 : 200, Promega, Madison,WI, USA).

Slides were counterstained with hematoxylin after 3minof diaminobenzidine reaction, and cover slipped usingVectashield (Vector Labs, Burlingame, CA, USA), thenphotographed and converted to a digital image using lightmicroscopy equipped with a camera (Olympus CX31, NY,USA). Negative control was carried out by omitting the pri-mary antibody. Positive staining (dark brown) for SIRT1 inlivers was quantified by two investigators in a blinded man-ner at a magnification of 400x using image analysis softwareImage-Pro Plus, and the results were expressed as the ratioof the mean of normal SD rats. Three slides were chosenat random in each animal for quantification (total 24 slidesfor each group).

2.5. Western Blotting. Liver tissues were lysed and proteinwas extracted as previously described [28]. Equal amountsof supernatant of liver lysates were separated on 8% SDS-PAGE gels and transferred onto a PVDF or a NC membrane.After washing, the membranes were blocked with 5% nonfatmilk at room temperature and then incubated overnight at

2 Journal of Diabetes Research

4°Cwith primary antibodies. After further washing, themem-branes were exposed to the secondary antibodies (anti-rabbitIgG or anti-mouse IgG, 1 : 10,000; Promega, USA) for 2 h atroom temperature before visualizing by enhanced chemilu-minescence (ECL) (Thermo Fisher Scientific, Rockford, IL,USA). The signals were visualized by GE ImageQuant LAS4000 mini quantified by densitometry using Gel Doc XRSystem (Bio-Rad Laboratories, Hercules, CA, USA). Theprimary antibodies used were phospho-Akt (Ser473) (rabbit,1 : 1000 dilution, Cell Signaling Technology), phospho-Akt(Thr308) (rabbit, 1 : 1000 dilution, Cell Signaling Technol-ogy), phospho-GSK-3β (Ser9) (rabbit, 1 : 1000 dilution, CellSignaling Technology), GSK-3 β (rabbit, 1 : 1000 dilution,Cell Signaling Technology), GCK (rabbit, 1 : 1000 dilution,AbcamCompany), SREBP-1 (rabbit, 1 : 1000 dilution, AbcamCompany), LDLR (rabbit, 1 : 1000 dilution, Abcam Com-pany), SIRT1 (rabbit, 1 : 1000 dilution, Abcam Company),Akt (rabbit, 1 : 1000 dilution, Cell Signaling Technology),acetylated-lysine (rabbit, 1 : 1000 dilution, Cell SignalingTechnology), and tubulin (mouse, 1 : 10000 dilution, Sigma).The secondary antibodies used were anti-rabbit IgG (H+L)(1 : 1000 dilution, Cell Signaling Technology) and anti-mouseIgG (H+L) (1 : 1000 dilution, Cell Signaling Technology).

2.6. Deacetylase Activity Assay. The deacetylase activity ofSIRT1 was determined with a fluorometric assay kit (CycLex,Ina, Nagano, Japan). Liver nuclear extracts (100mg) wereimmunoprecipitated with rabbit anti-SIRT1 polyclonalantibody (Sigma, USA). Precipitates (15mL) were mixedwith a reaction mixture (35mL) containing 50mM Tris-HCl (pH8.8), 0.25 mAU·mL-1 lysyl-endopeptidase, 1mMtrichostatin A, 800mM NAD, and 20mM fluorosubstratepeptide and followed by incubation for 30min at room tem-perature. The fluorescence intensity was measured using amicrotiter plate fluorimeter with excitation at 490± 10 nmand emission at 530± 10nm and normalized to the proteinconcentration. All tests were performed in triplicate.

2.7. Immunoprecipitation. Immunoprecipitation was per-formed by incubating 0.5mg liver tissue lysate protein whichwas determined by bicinchoninic acid assay (BCA) accordingto the manufacturer’ s instructions (Thermo Fisher Scientific,Rockford, IL) with 1μg of corresponding antibody and pro-tein G/A agarose bead (Merk, Darmstadt, Germany) at 4°Covernight. Western blotting was performed as previouslydescribed [28].

2.8. Statistical Analysis. All experiments were performed atleast in triplicate. Values were expressed as means± S.D.All data were assessed by SPSS 11.5. Statistical analysesof data were performed by one-way ANOVA using posthoc multiple comparisons. P < 0 05 was considered to bestatistically significant.

3. Results

3.1. Biochemical Parameters of STZ-Induced Diabetic Ratsafter Quercetin Treatment. In Table 1, the biochemicalparameters were notably deteriorated in the STZ-induceddiabetic rats compared with those in the normal controlgroup at the end of the experimental period (P < 0 01). Quer-cetin (10 and 50mg/kg) and pioglitazone treatments evi-dently ameliorated the biochemical parameters comparedwith those of the STZ-induced diabetic rats (P < 0 05). Noevident difference in HDL-C levels was observed betweenthese groups.

3.2. Quercetin Ameliorated the Hepatic HistomorphologicalInjury and Enhanced the Hepatic Glycogen Content in theSTZ-Induced Diabetic Rats. The liver is an important organfor glucose and lipid metabolism. Excessive lipid accumula-tion in the liver may cause liver dysfunction and hepatic insu-lin resistance that affect gluconeogenesis, thereby promotingthe conversion of glycogen to glucose in this organ. In ourstudy, the structure of the hepatic lobule from the controlgroup was normal (Figure 1(a)). The hepatocytes showed adistinct outline, distributed evenly, and arranged radicallyaround the central vein in a streak. The structure of thehepatic tissues from the model group was significantly dis-rupted compared with that from the control group. However,quercetin and pioglitazone evidently alleviated the morpho-logical damage to the hepatocytes of the diabetic rats. Theseresults suggested that quercetin elicited protective effectsagainst liver injury in STZ-induced diabetic rats. The hepaticglycogen content in STZ-induced diabetic rats largelydecreased compared with that in the control group (P <0 05). By contrast, the hepatic glycogen content in the STZ-induced diabetic rats remarkably increased in the quercetin-and pioglitazone-treated groups (Figure 1(b), P < 0 05).

3.3. Quercetin Treatment Enhanced the Expression andActivity of Hepatic SIRT1 in STZ-Induced Diabetic Rats.SIRT1 activation shows therapeutic potential for type 2

Table 1: Blood biochemical parameters of experimental diabetic rats after 12 weeks of quercetin treatment (n = 8).

GroupFBG

(mmol·L−1)GSP

(mmol·L−1)HbA1c

(OD·10 g−1)TC

(mmol·L−1)TG

(mmol·L−1)LDL-C

(mmol·L−1)HDL-C

(mmol·L−1)Control 5.27± 0.31 1.61± 0.13 18.937± 3.83 1.41± 0.25 0.69± 0.13 0.31± 0.08 0.62± 0.12Diabetes 25.26± 3.45bb 4.68± 0.77bb 38.83± 8.11bb 4.33± 1.29bb 4.61± 1.33bb 5.35± 1.68b 0.59± 0.20QU-low 22.98± 2.16abb 2.56± 0.67abb 26.251± 5.23aa 2.12± 0.29aa 1.03± 0.63aa 0.98± 0.37aa 0.70± 0.19QU-high 22.76± 3.61aabb 2.23± 0.46aa 25.97± 5.13aab 1.49± 0.41aa 0.98± 0.49aa 0.84± 0.14a 0.66± 0.22Pioglitazone 16.71± 0.81abb 1.98± 0.33aa 29.16± 4.22aab 1.46± 0.28aa 0.96± 0.56aa 0.635± 0.26a 0.68± 0.17Data are represented as mean ± S.D. FBG: fasting blood glucose; GSP: glycosylated serum protein; HbA1c: glycosylated hemoglobin A1c; TC: serum totalcholesterol; TG: triglycerides; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol. bP < 0 05 versus control, bbP < 0 01versus control, aP < 0 05 versus diabetes, and aaP < 0 01 versus diabetes.

3Journal of Diabetes Research

Control

QU-low

Pioglitazone

QU-high

Diabetes

50 �휇m 50 �휇m

50 �휇m

50 �휇m

50 �휇m

(a)

Hep

atic

gly

coge

n (m

g/g)

Con

trol

20

⁎

⁎⁎

#

##

15

10

5

0

Dia

bete

s

QU

-low

QU

-hig

h

Piog

litaz

one

(b)

Figure 1: Quercetin ameliorated the hepatic histomorphological injury and enhanced hepatic glycogen content levels in STZ-induceddiabetic rats. (a) Images of hepatic histopathological analysis by hematoxylin-eosin (HE) staining display representative hepatocytes andstructures of the hepatic lobule of HE-stained sections in control, diabetes, QU-low, QU-high, and pioglitazone-treated rats at an originalmagnification of 200x. (b) Hepatic glycogen content of control, diabetes, QU-low, QU-high, and pioglitazone-treated rats was evaluated byanthrone assay. Data are means± SD, n = 8. ∗P < 0 05 versus control group and #P < 0 05 versus diabetic group by ANOVA.


Negative

QU-highQU-low Pioglitazone

Control Diabetes

20 �휇m 20 �휇m 20 �휇m

20 �휇m 20 �휇m 20 �휇m

(a)

100

#

#

#

⁎

⁎

⁎

80

60

40

0

Con

trol

Dia

bete

s

QU

-low

QU

-hig

h

Piog

litaz

one

Relat

ive S

IRT1

cont

ent

(% o

f con

trol)

20

(b)

# #

#

⁎

⁎

120

�훼-Tubulin

SIRT1

100

80

60

40

20

0

Con

trol

Dia

bete

s

QU

-low

QU

-hig

h

Piog

litaz

one

SIRT

1/�훼

-tubu

lin(%

to co

ntro

l)

(c)

#

#

#

⁎

⁎

120

100

80

60

40

20

0

Con

trol

Dia

bete

s

QU

-low

QU

-hig

h

Piog

litaz

one

Rela

tive S

IRT1

activ

ity(%

of c

ontro

l)

(d)

Figure 2: Quercetin treatment enhanced hepatic SIRT1 protein expression and deacetylase activity in STZ-induced diabetic rats. The effectsof quercetin on hepatic SIRT1 expression levels in the livers of control, diabetes, QU-low, QU-high, and pioglitazone-treated rats weredetected through immunohistochemistry. Staining without SIRT1 antibody was used as a negative control. (a and b) Originalmagnification of 400x, solid arrow indicates nuclear SIRT1, and hollow arrow corresponds to cytoplasmic SIRT1. (c) Western blot.Immunohistochemistry showed SIRT1 (dark brown) was decreased in diabetes compared with control and was partially increased by thequercetin treatment. (d) Hepatic SIRT1 deacetylase activity in rat livers was detected using a fluorometric assay kit. Data are means± SD,n = 8. ∗P < 0 05 versus control group and #P < 0 05 versus diabetic group by ANOVA.


diabetes and obesity [15]. Immunohistochemistry was con-ducted to investigate SIRT1 expression, which is involved inglucose and lipid metabolism in diabetes, in the livers ofdiabetic rats (Figure 2(a)). The results showed that SIRT1was expressed in the nuclei and cytoplasm of the hepatocytesfrom the control group. Conversely, the positively stainedSIRT1 was markedly reduced by fivefold in the hepatocytes,especially in the cytoplasm of the diabetic group comparedwith that of the control group (P < 0 05). The hepatic SIRT1expression substantially increased by threefold in thequercetin-treated group compared with that in the diabeticgroup (P < 0 05, Figure 2(b)). The hepatic SIRT1 expressionin the pioglitazone-treated group increased to a certainextent, but this increase was not as evident as that in thequercetin-treated group. No visible staining was also foundin the negative group. We further confirmed the SIRT1 pro-tein expression in the livers through Western blot(Figure 2(c), P < 0 05). Our results showed that the proteinlevel of SIRT1 severely decreased in the diabetic group com-pared with that in the control group. The SIRT1 protein inthe pioglitazone-treated group was slightly upregulated com-pared with that in the diabetic group. However, the SIRT1expression was increased in the quercetin-treated diabeticrats. Immunohistochemistry and Western blot results

suggested that the protective effect of quercetin on glucoseand lipid metabolism dysfunction in diabetes might partlydepend on the upregulated SIRT1 protein level. We alsoexplored the deacetylase activity of SIRT1 in the livers ofthe diabetic rats (Figure 2(d), P < 0 05). The deacetylaseactivity of SIRT1 in the diabetic group was severely inhibitedcompared with that in the control group. Conversely, thisactivity was slightly enhanced by pioglitazone and was signif-icantly improved by high-dose quercetin treatment.

3.4. Quercetin-Activated Hepatic Akt Signaling in STZ-Induced Diabetic Rats. Akt activity is related to phosphoryla-tion and deacetylation. The phosphorylated Akt expressionlevels in the livers were examined through Western blot,and our results showed that these levels significantlydecreased in the livers of the diabetic rats. Pioglitazone andquercetin markedly enhanced the expression of phosphory-lated Akt (Figure 3(a), P < 0 05). Akt as a substrate of SITR1can be deacetylated by SIRT1, thereby upregulating Aktactivity [22]. The acetylated Akt levels were also exploredthrough immunoprecipitation and Western blot. Our find-ings demonstrated that acetylated Akt levels were enhancedin the livers of the diabetic rats but were slightly reduced bypioglitazone. By comparison, its levels were markedly

p-Akt (T308)

p-Akt (S473)

p-Akt (T308)p-Akt (S473)

t-Akt

p-A

kt/t-

Akt

(% o

f con

trol)

120

100

⁎⁎

⁎

##

#

#

⁎

#⁎80

60

40

20

0

Con

trol

Dia

bete

s

QU

-low

QU

-hig

h

Piog

litaz

one

(a)

Ac-Lys (Ac-Akt)IgG

IP : Akt

Ac-L

ys (A

c-A

kt)/

Akt

(% to

cont

rol)

Akt

Ac-Lys (Ac-Akt)

Akt

300

#

#

⁎

⁎

⁎

250

200

150

100

50

0

5% input

Con

trol

Dia

bete

s

QU

-low

QU

-hig

h

Piog

litaz

one

Con

trol

Dia

bete

s

QU

-low

QU

-hig

h

Piog

litaz

one

(b)

Figure 3: Effects of quercetin on Akt. The effects of quercetin on (a) Akt phosphorylation levels and (b) Akt deacetylation levels in the livers ofcontrol, diabetes, QU-low, QU-high, and pioglitazone-treated rats were determined through immunoprecipitation andWestern blot. Data aremeans± SD, n = 8. ∗P < 0 05 versus control group and #P < 0 05 versus diabetic group by ANOVA.


attenuated after quercetin was administered (Figure 3(b),P < 0 05). These observations implied that deacetylated Aktlevels corresponded to hepatic SIRT1 protein levels.

3.5. Effects of Quercetin on the Related Downstream EffectorProtein Levels in the Livers of Diabetic Rats. Western blotresults showed that the GSK-3β phosphorylation level was

markedly reduced in the diabetic rats, compared with thatin the normal control (Figure 4(a), P < 0 05). No evidentdifference in the GSK-3β phosphorylation was detectedbetween the pioglitazone group and the diabetic group(Figure 4(a), P < 0 05). The GSK-3β phosphorylation levelin the quercetin-treated diabetic rats significantly increased(Figure 4(a), P < 0 05). The hepatic GCK protein expression

p-GSK3�훽

p-G

SK3�훽

/t-G

SK(%

of c

ontro

l)

t-GSK

120

#

#

⁎ ⁎

100

80

60

40

20

0

Con

trol

Dia

bete

s

QU

-low

QU

-hig

h

Piog

litaz

one

(a)

�훼-Tubulin

#

#

#

⁎

⁎

GCK

/�훼-tu

bulin

(% to

cont

rol)

140

120

100

80

60

40

20

0

GCK

Con

trol

Dia

bete

s

QU

-low

QU

-hig

h

Piog

litaz

one

(b)

�훼-Tubulin

##

⁎ ⁎

140

SREB

P-1c

/�훼-tu

bulin

(% to

cont

rol)

120

100

80

60

40

20

0

SREBP-1c

Con

trol

Dia

bete

s

QU

-low

QU

-hig

h

Piog

litaz

one

(c)

�훼-Tubulin

##

⁎⁎

LDLR

/�훼-tu

bulin

(% to

cont

rol)

120

100

80

60

40

20

0

LDLRC

ontro

l

Dia

bete

s

QU

-low

QU

-hig

h

Piog

litaz

one

(d)

Figure 4: Effects of quercetin on (a) GSK-3β, (b) GCK, (c) SREBP-1c, and (d) LDLR expression. The effects of quercetin on (a) GSK-3β, (b)GCK, (c) SREBP-1c, and (d) LDLR expression in the livers of control, diabetes, QU-low, QU-high, and pioglitazone-treated rats wereexamined throughWestern blot. Data are means± SD, n = 8. ∗P < 0 05 versus control group and #P < 0 05 versus diabetic group by ANOVA.


was markedly downregulated in the diabetic rats comparedwith that in the normal control (Figure 4(b), P < 0 05). Bycomparison, the protein expression of GCK in thepioglitazone-treated group was upregulated (Figure 4(b),P < 0 05). The protein expression of GCK was remarkablyincreased by quercetin (Figure 4(b), P < 0 05). Likewise, theprotein expression of SREBP-1c in the liver was increasedin the diabetic group, compared with that in the controlgroup (Figure 4(c), P < 0 05). Quercetin and pioglitazonetreatment evidently attenuated SREBP-1c protein level(Figure 4(c), P < 0 05). The hepatic LDLR expression in thediabetic group was markedly downregulated compared withthat in the control group (Figure 4(d), P < 0 05) but wasevidently upregulated after quercetin and pioglitazonetreatments were administered (Figure 4(d), P < 0 05).

4. Discussion

Most patients with type 2 diabetes suffer not only fromhyperglycemia but also from lipid metabolism dysfunction.Pioglitazone is a prescription drug belonging to the thiazoli-dinedione class with a hypoglycemic action to treat diabetes.It modulates the transcription of genes involved in the con-trol of glucose and lipid metabolism in the muscles, adiposetissues, and livers [29, 30]. In clinical trials, pioglitazone asmonotherapy or in combination with other oral antidiabeticdrugs or insulin enhances blood glucose levels, long-termglucose control, and lipid profiles [31]. Patients with predia-betes or T2DM treated with pioglitazone experience aremarkable improvement in hepatic steatosis, and steatohe-patitis can be cured within 6 months [32]. Therefore, pioglit-azone was chosen as a positive control drug in the analysis ofglucose and lipid metabolism in our research. Our resultsrevealed that FBG, HbA1c, GSP, TC, TG, and LDL-C levelssignificantly increased in STZ-induced diabetic rats. Thesebiochemical parameters were also improved in diabeticrats treated with quercetin and pioglitazone, thereby suggest-ing that quercetin ameliorated the dysfunction of glucoseand lipid metabolism in STZ-induced diabetic rats. How-ever, the exact mechanism remains unclear and needsfurther investigations.

The hypoglycemic effect mediated by quercetin in dia-betic rats is closely related to changes in hepatic Akt activity.Quercetin and pioglitazone effectively increase Akt phos-phorylation levels at sites T308 and S473, which are pivotalsites for activity Akt [33], in the livers of diabetic rats, andthus contribute to the improvement of glucose metabolism.

GSK-3β is a downstream factor regulated by Akt andAkt-induced GSK-3β phosphorylation at the site of serine 9which inhibits GSK-3β [13]. Increased GSK-3β activityalso prevents IRS activity under diabetic conditions, therebyleading to dysfunction in insulin-induced signaling pathwayand consequently aggravating damage to the Akt signalingpathway [34]. In our study, the downregulation of phosphor-ylated GSK-3β (Ser9) in the diabetic liver was significantlyincreased by quercetin. However, no significant changes wereobserved between the pioglitazone-treated group and thediabetic group. Based on our data, our conclusion is thatquercetin could effectively prevent the abnormal increase

in GSK-3β activity by regulating Akt in the livers ofdiabetic rats, relieve the inhibitory effect on downstreamglycogen synthase, and consequently promote the synthesisof liver glycogen.

Glucokinase (GCK), also known as hexokinase type IVexpressed mainly in pancreatic β-cells and hepatocytes andaffected by Akt, is an important regulatory factor in glucosemetabolism [35, 36]. GCK serves as a glucose sensor to pre-dict the variability of blood glucose concentrations and playsa crucial role in the regulation of insulin secretion in pancre-atic β-cells [13]. Glucose is initially transported into cells byglucose transporters in the liver, subsequently converted toglucose-6-phosphate (G6P) by GCK and stored as glycogen.Consistent with previous findings [13], our results indicatedthat the GCK protein expression and activity in the livers ofthe diabetic rats significantly decreased. However, quercetinand pioglitazone promoted the GCK protein expression inthe livers of the diabetic rats, suggesting that quercetin andpioglitazone enhanced the synthetic function of hepaticglycogen and alleviated the glucose metabolism disorderby regulating GCK.

Akt activation is involved not only in the regulation ofglucose metabolism but also in other processes. For example,Akt controls sterol regulatory element-binding proteins(SREBPs), which are the main transcriptional regulators oflipid metabolism [37]. Akt also regulates lipogenesis by mod-ulating LDLR expression [13]. Thus, Akt signaling pathway isclosely implicated in the control of lipid metabolism. On thebasis of our data, we concluded that quercetin and pioglita-zone improved the blood lipid parameters of diabetic ratsby activating Akt and upregulating SREBPs and LDLR.

In addition to phosphorylation activation, reversibleacetylation is a posttranslational modification that regulatesAkt activity. Under basal conditions, Akt is acetylated in var-ious tissues, and this modification suppresses Akt activity.SIRT1, as a deacetylase, can activate Akt by inducing theAkt deacetylation [38]. Akt acetylated by SIRT1 at lysine res-idues in the pH domain can upregulate Akt activation to acertain extent, play a crucial role in the binding of Akt toPIP3, and consequently trigger cascades of downstream reac-tions [22, 23, 38]. Thus, whether quercetin improves glucoseand lipid metabolism disorder in STZ-induced diabetic ratsthrough the SIRT1/Akt pathway should be further examined.In our study, the level of lysine acetylation of Akt increased,but quercetin induced the lysine deacetylation of Akt in thelivers of diabetic rats to a certain degree. As such, whetherthis effect results from quercetin-induced SIRT1 activationshould be elucidated. To confirm this effect, we observedthe expression of SIRT1 in the livers of diabetic rats andfound that the reduced SIRT1 protein level and deacetylaseactivity in the livers of the diabetic rats were upregulated inthe quercetin-treated group. One of the mechanisms toachieve SIRT1 activation in vivo is to raise intracellular levelsof NAD+ either by increased synthesis or diminished degra-dation [39, 40]. Quercetin has been shown to activate SIRT1in vivo by enhancing NAD+ level as an inhibitor of NAD+

nucleosidaase CD38 [41]. The change of SIRT1 in diabeticliver is consistent with the variation tendency of deacetyla-tion on the Akt lysine site, suggesting that quercetin might


induce Akt deacetylation and further activate Akt by increas-ing SIRT1 protein levels and activities in the livers of diabeticrats. Thus, this flavonoid might improve the glucose and lipidmetabolism disorder in diabetic rats. Furthermore, SIRT1interacted with the Akt in cytoplasm and then induced thedeacetylation of Akt, promoting Akt membrane locationand binding to PIP3 [23]. So, it is possible that the changeof SIRT1 in the cytoplasm is more important than in thenuclei for Akt activation. Compared with quercetin treat-ment, pioglitazone treatment did not significantly affect theSIRT1/Akt pathway, and this finding reveals the advantageof quercetin in the treatment of glucose and lipid metabolismdisorder to some degree. However, further in vitro studiesshould be performed to verify this hypothesis.

In our study, quercetin effectively alleviated the glucoseand lipid metabolism disorder in STZ-induced diabetic ratmodel, and this observation was correlated with hepaticAkt activation in diabetic rats. Quercetin promoted hepaticglycogen synthesis and reduced blood glucose by increasingAkt phosphorylation, GSK-3 phosphorylation, and GCKprotein expression levels. Quercetin also improved lipidmetabolism by regulating SREBPs and LDLR protein expres-sion, which are controlled by Akt. This flavonoid not onlyactivated Akt by stimulating phosphorylation but alsoincreased Akt activity by inducing Akt deacetylation possiblybecause of the effect of quercetin on SIRT1. However, furtherin vitro experiments should be conducted to explore themechanisms of quercetin in diabetes.

5. Conclusion

Quercetin beneficially affects glucose and lipid metabolismdisorder and is probably associated with the upregulatedactivity and protein level of SIRT1 and its influence onAkt signaling pathway. Hence, quercetin shows potentialfor the treatment of glucose and lipid metabolism disorderin diabetes.

Conflicts of Interest

The authors declare that there is no conflict of interestregarding the publication of this article.

Authors’ Contributions

Xi Xie and Jing Peng designed and performed the experi-ments, acquisition, and analysis of data and drafted themanuscript. Li Zhu, Keye Li, Xiaohong Lin, and QianhuiShen helped to perform animal experiments and preparethe manuscript. Qingde Li, Xiaoding Lin, and Guoping Lihave been involved in drafting the manuscript and revisingit critically for important intellectual content.

Acknowledgments

This study was supported by research grants from theNational Natural Science Foundation of China (Grant no.81402984), the Medical Scientific Research Foundation ofGuangdong Province, China (no. A2016591), the Hospital

Pharmacy Research Foundation of Guangdong Province,China (no. 2016A02), and the Education and TeachingResearch Project of Hainan University, China (no. hdjy1709).

References

[1] T. T. Phan, I. J. Lim, S. Y. Chan, E. K. Tan, S. T. Lee, andM. T. Longaker, “Suppression of transforming growth factorbeta/smad signaling in keloid-derived fibroblasts by quercetin:implications for the treatment of excessive scars,” Journal ofTrauma-Injury Infection & Critical Care, vol. 57, no. 5,pp. 1032–1037, 2004.

[2] H. Iskender, E. Dokumacioglu, T. M. Sen, I. Ince, Y. Kanbay,and S. Saral, “The effect of hesperidin and quercetin on oxida-tive stress, NF-κB and SIRT1 levels in a STZ-induced experi-mental diabetes model,” Biomedicine & Pharmacotherapy,vol. 90, pp. 500–508, 2017.

[3] C. Thipkaew, J. Wattanathorn, and S. Muchimapura, “Electro-spun nanofibers loaded with quercetin promote the recoveryof focal entrapment neuropathy in a rat model ofstreptozotocin-induced diabetes,” BioMed Research Interna-tional, vol. 2017, Article ID 2017493, 12 pages, 2017.

[4] J. Roslan, N. Giribabu, K. Karim, and N. Salleh, “Quercetinameliorates oxidative stress, inflammation and apoptosis inthe heart of streptozotocin-nicotinamide-induced adult malediabetic rats,” Biomedicine & Pharmacotherapy, vol. 86,pp. 570–582, 2017.

[5] R. F. de Souza and W. F. De Giovani, “Antioxidant propertiesof complexes of flavonoids with metal ions,” Redox Report,vol. 9, no. 2, pp. 97–104, 2004.

[6] S. Y. Lin, Y. Y. Wang, W. Y. Chen, Y. H. Chuang, P. H. Pan,and C. J. Chen, “Beneficial effect of quercetin on cholestaticliver injury,” The Journal of Nutritional Biochemistry, vol. 25,no. 11, pp. 1183–1195, 2014.

[7] Y. Tang, Y. Li, H. Yu et al., “Quercetin attenuates chronic eth-anol hepatotoxicity: implication of “free” iron uptake andrelease,” Food and Chemical Toxicology, vol. 67, pp. 131–138,2014.

[8] F. R. Seiva, L. G. Chuffa, C. P. Braga, J. P. Amorim, andA. A. Fernandes, “Quercetin ameliorates glucose and lipidmetabolism and improves antioxidant status in postnatallymonosodium glutamate-induced metabolic alterations,” Foodand Chemical Toxicology, vol. 50, no. 10, pp. 3556–3561,2012.

[9] M. M. Alam, D. Meerza, and I. Naseem, “Protective effect ofquercetin on hyperglycemia, oxidative stress and DNA dam-age in alloxan induced type 2 diabetic mice,” Life Sciences,vol. 109, no. 1, pp. 8–14, 2014.

[10] Y. Wang, N. Dang, P. Sun, J. Xia, C. Zhang, and S. Pang,“The effects of metformin on fibroblast growth factor 19,21 and fibroblast growth factor receptor 1 in high-fat dietand streptozotocin induced diabetic rats,” Endocrine Journal,vol. 64, 2017.

[11] J. R. Kuang, Z. H. Zhang, W. L. Leng, X. T. Lei, and Z. W.Liang, “Dapper1 attenuates hepatic gluconeogenesis andlipogenesis by activating PI3K/Akt signaling,” Molecular andCellular Endocrinology, vol. 447, pp. 106–115, 2017.

[12] W. Yang, J. Wang, Z. Chen et al., “NFE2 induces miR-423-5pto promote gluconeogenesis and hyperglycemia by repressingthe hepatic FAM3A-ATP-Akt pathway,” Diabetes, vol. 66,2017.


[13] J. Hao, C. Chen, K. Huang et al., “Polydatin improves glucoseand lipid metabolism in experimental diabetes throughactivating the Akt signaling pathway,” European Journal ofPharmacology, vol. 745, pp. 152–165, 2014.

[14] R. H. Houtkooper, E. Pirinen, and J. Auwerx, “Sirtuins asregulators of metabolism and healthspan,” Nature ReviewsMolecular Cell Biology, vol. 13, no. 4, pp. 225–238, 2012.

[15] X. Ye, M. Li, T. Hou, T. Gao,W. G. Zhu, and Y. Yang, “Sirtuinsin glucose and lipid metabolism,” Oncotarget, vol. 8, no. 1,pp. 1845–1859, 2017.

[16] Z. Mei, X. Zhang, J. Yi, J. Huang, J. He, and Y. Tao, “Sirtuins inmetabolism, DNA repair and cancer,” Journal of Experimental& Clinical Cancer Research, vol. 35, no. 1, p. 182, 2016.

[17] A. Bedalov, S. Chowdhury, and J. A. Simon, “Biology, chemis-try, and pharmacology of Sirtuins,” Methods in Enzymology,vol. 574, pp. 183–211, 2016.

[18] A. Biason-Lauber, M. Böni-Schnetzler, B. P. Hubbard et al.,“Identification of a SIRT1 mutation in a family with type 1 dia-betes,” Cell Metabolism, vol. 17, no. 3, pp. 448–455, 2013.

[19] A. S. Banks, N. Kon, C. Knight et al., “SirT1 gain of functionincreases energy efficiency and prevents diabetes in mice,” CellMetabolism, vol. 8, no. 4, pp. 333–341, 2008.

[20] J. H. Lee, M. Y. Song, E. K. Song et al., “Overexpression ofSIRT1 protects pancreatic β-cells against cytokine toxicity bysuppressing the nuclear factor-κB signaling pathway,” Diabe-tes, vol. 58, no. 2, pp. 344–351, 2009.

[21] K. Huang, J. Huang, X. Xie et al., “Sirt1 resists advancedglycation end products-induced expressions of fibronectinand TGF-β1 by activating the Nrf2/ARE pathway in glomeru-lar mesangial cells,” Free Radical Biology & Medicine, vol. 65,pp. 528–540, 2013.

[22] V. B. Pillai, N. R. Sundaresan, and M. P. Gupta, “Regulationof Akt signaling by sirtuins: its implication in cardiachypertrophy and aging,” Circulation Research, vol. 114, no. 2,pp. 368–378, 2014.

[23] N. R. Sundaresan, V. B. Pillai, D. Wolfgeher et al., “The deace-tylase SIRT1 promotes membrane localization and activationof Akt and PDK1 during tumorigenesis and cardiac hypertro-phy,” Science Signaling, vol. 4, no. 182, article ra46, 2011.

[24] J. Zhang, “The direct involvement of SirT1 in insulin-inducedinsulin receptor substrate-2 tyrosine phosphorylation,” Jour-nal of Biological Chemistry, vol. 282, no. 47, pp. 34356–34364, 2007.

[25] W. Wang, C. Wang, X. Q. Ding et al., “Quercetin andallopurinol reduce liver thioredoxin-interacting protein toalleviate inflammation and lipid accumulation in diabeticrats,” British Journal of Pharmacology, vol. 169, no. 6,pp. 1352–1371, 2013.

[26] C. Wang, Y. Pan, Q. Y. Zhang, F. M. Wang, and L. D. Kong,“Quercetin and allopurinol ameliorate kidney injury in STZ-treated rats with regulation of renal NLRP3 inflammasomeactivation and lipid accumulation,” PLoS One, vol. 7, no. 6,article e38285, 2012.

[27] I. B. Gomes, M. L. Porto, M. C. Santos et al., “Renoprotective,anti-oxidative and anti-apoptotic effects of oral low-dosequercetin in the C57BL/6J model of diabetic nephropathy,”Lipids in Health and Disease, vol. 13, p. 184, 2014.

[28] X. Xie, T. Lan, X. Chang et al., “Connexin43 mediates NF-κBsignalling activation induced by high glucose in GMCs:involvement of c-Src,” Cell Communication and Signaling,vol. 11, no. 1, p. 38, 2013.

[29] P. S. Gillies and C. J. Dunn, “Pioglitazone,” Drugs, vol. 60,no. 2, pp. 333–343, 2000.

[30] U. Smith, “Pioglitazone: mechanism of action,” InternationalJournal of Clinical Practice, vol. 121, pp. 13–18, 2001.

[31] J. Waugh, G. M. Keating, G. L. Plosker, S. Easthope, and D. M.Robinson, “Pioglitazone: a review of its use in type 2 diabetesmellitus,” Drugs, vol. 66, no. 1, pp. 85–109, 2006.

[32] R. Belfort, S. A. Harrison, K. Brown et al., “A placebo-controlled trial of pioglitazone in subjects with nonalcoholicsteatohepatitis,” The New England Journal of Medicine,vol. 355, no. 22, pp. 2297–2307, 2006.

[33] X. Wang, Y. Liu, Z. Yang et al., “Glucose metabolism-relatedprotein 1 (GMRP1) regulates pancreatic beta cell proliferationand apoptosis via activation of Akt signalling pathway in ratsand mice,” Diabetologia, vol. 54, no. 4, pp. 852–863, 2011.

[34] P. Cohen andM. Goedert, “GSK3 inhibitors: development andtherapeutic potential,” Nature Reviews Drug Discovery, vol. 3,no. 6, pp. 479–487, 2004.

[35] P. B. Iynedjian, “Molecular physiology of mammalian glucoki-nase,” Cellular and Molecular Life Sciences, vol. 66, no. 1,pp. 27–42, 2009.

[36] P. B. Iynedjian, R. A. Roth, M. Fleischmann, and A. Gjinovci,“Activation of protein kinase B/cAkt in hepatocytes issufficient for the induction of expression of the gene encodingglucokinase,” Biochemical Journal, vol. 351, no. 3, pp. 621–627,2000.

[37] J. R. Krycer, L. J. Sharpe, W. Luu, and A. J. Brown, “The Akt–SREBP nexus: cell signaling meets lipid metabolism,” Trendsin Endocrinology & Metabolism, vol. 21, no. 5, pp. 268–276,2010.

[38] X. H. Li, C. Chen, Y. Tu et al., “Sirt1 promotes axonogenesis bydeacetylation of Akt and inactivation of GSK3,” MolecularNeurobiology, vol. 48, no. 3, pp. 490–499, 2013.

[39] J. Yoshino, K. F. Mills, M. J. Yoon, and S. Imai, “Nicotinamidemononucleotide, a key NAD+ intermediate, treats the patho-physiology of diet- and age-induced diabetes in mice,” CellMetabolism, vol. 14, no. 4, pp. 528–536, 2011.

[40] P. Aksoy, C. Escande, T. A. White et al., “Regulation of SIRT 1mediated NAD dependent deacetylation: a novel role for themultifunctional enzyme CD38,” Biochemical and BiophysicalResearch Communications, vol. 349, no. 1, pp. 353–359, 2006.

[41] C. Escande, V. Nin, N. L. Price et al., “Flavonoid apigenin is aninhibitor of the NAD+ase CD38: implications for cellularNAD+ metabolism, protein acetylation, and treatment ofmetabolic syndrome,” Diabetes, vol. 62, no. 4, pp. 1084–1093,2013.


Submit your manuscripts athttps://www.hindawi.com

Stem CellsInternational

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinson’s Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com

Documents

Full-Text PDF - Hindawi Publishing Corporationdownloads.hindawi.com/journals/jdr/2017/3417306.pdf · Research Article Quercetin Improves Glucose and Lipid Metabolism of Diabetic Rats: