Hepatic overexpression of ATP synthase β subunit activates...

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Hepatic overexpression of ATP synthase β subunit activates PI3K/Akt pathway to ameliorate hyperglycemia of diabetic mice Running title: ATP synthase β subunint and hyperglycemia Chunjiong Wang 1,2# , Zhenzhen Chen 1# , Sha Li 1# , Yuan Zhang 3 , Shi Jia 1 , Jing Li 1 , Yujing Chi 1 , Yifei Miao 1 ,Youfei Guan 1,4 and Jichun Yang 1* 1 Department of Physiology and Pathophysiology, Key Laboratory of Molecular Cardiovascular Science Peking (Beijing) University Health Science Center Beijing 100191, China 2 Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin 300070, China 3 Department of Laboratory Medicine, Peking University Third Hospital, Beijing 100191, China 4 Shenzhen University Diabetes Center, Shenzhen University Health Science Center, Shenzhen 518060, China # These authors contributed equally to this work *Correspondence to: Jichun Yang, Ph.D. Department of Physiology and Pathophysiology, Peking (Beijing) University Health Science Center, 38 Xueyuan Road, Beijing, China 100191 Tel: (+86) 10-82805613 E-mail: [email protected] Page 1 of 57 Diabetes Diabetes Publish Ahead of Print, published online December 2, 2013

Transcript of Hepatic overexpression of ATP synthase β subunit activates...

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Hepatic overexpression of ATP synthase β subunit activates PI3K/Akt

pathway to ameliorate hyperglycemia of diabetic mice

Running title: ATP synthase β subunint and hyperglycemia

Chunjiong Wang1,2#, Zhenzhen Chen1#, Sha Li1#, Yuan Zhang3, Shi Jia1, Jing Li1,

Yujing Chi1, Yifei Miao1,Youfei Guan1,4 and Jichun Yang1*

1 Department of Physiology and Pathophysiology, Key Laboratory of Molecular Cardiovascular Science

Peking (Beijing) University Health Science Center Beijing 100191, China

2 Department of Physiology and Pathophysiology, Tianjin Medical University,

Tianjin 300070, China 3 Department of Laboratory Medicine,

Peking University Third Hospital, Beijing 100191, China 4 Shenzhen University Diabetes Center,

Shenzhen University Health Science Center, Shenzhen 518060, China

# These authors contributed equally to this work

*Correspondence to:

Jichun Yang, Ph.D.

Department of Physiology and Pathophysiology,

Peking (Beijing) University Health Science Center,

38 Xueyuan Road, Beijing, China 100191

Tel: (+86) 10-82805613

E-mail: [email protected]

Page 1 of 57 Diabetes

Diabetes Publish Ahead of Print, published online December 2, 2013

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Abstract

ATP synthase β subunit (ATPSβ) had been previously shown to play an important role

in controlling ATP synthesis in pancreatic β cells. This study aimed to investigate the

role of ATPSβ in regulation of hepatic ATP content and glucose metabolism in

diabetic mice. Both ATPSβ expression and ATP content were reduced in the livers of

type 1 and type 2 diabetic mice. Hepatic overexpression of ATPSβ elevated cellular

ATP content, and ameliorated hyperglycemia of STZ-induced diabetic mice and db/db

mice. ATPSβ overexpression increased phosphorylated Akt (pAkt) levels and reduced

PEPCK and G6pase expression levels in the livers. Consistently, ATPSβ

overexpression repressed hepatic glucose production in db/db mice. In cultured

hepatocytes, ATPSβ overexpression increased intracellular and extracellular ATP

content, elevated cytosolic free calcium level and activated Akt independent of insulin.

ATPSβ-induced increase in cytosolic free calcium and pAkt levels was attenuated by

inhibition of P2 receptors. Notably, inhibition of Calmodulin (CaM) completely

abolished ATPSβ-induced Akt activation in liver cells. Inhibition of P2 receptors or

CaM blocked ATPSβ-induced nuclear exclusion of forkhead box O1 (FOXO1) in liver

cells. In conclusion, a decrease in hepatic ATPSβ expression in the liver, leading to the

attenuation of ATP-P2 receptor-CaM-Akt pathway, may play an important role in the

progression of diabetes.

Keywords: diabetes; ATPSβ; ATP; PI3K-Akt; P2 receptor

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Introduction

In the past decades, diabetes had become one of the major diseases threatening

our health with an estimated global prevalence of 6.4% in 2010 (1). Liver is the key

tissue that releases glucose into the circulation in fasting status, and an increase in

hepatic glucose production due to insulin deficiency or insulin resistance is the central

event in the development and progression of type 1 or type 2 diabetes (2). Moreover,

liver is also one of the key tissues for lipid metabolism.

Adenosine triphosphate (ATP) serves as an energy molecule as well as a signal

molecule in many cells (3). We had previously shown that leucine upregulates ATPSβ

to enhance ATP synthesis and insulin secretion in rat islets, type 2 diabetic human

islets and rattus INS-1 cells. Overexpression or knockdown of ATPSβ increases or

reduces cellular ATP levels in INS-1 cells (4; 5). Overall, these findings suggest that

ATPSβ plays a crucial role in controlling ATP synthesis.

In the livers of STZ-induced type 1 diabetic mice, HFD-induced type 2 diabetic

mice and a methionine and choline deficient (MCD) diet-induced non-alcoholic

steatohepatitis (NASH) rats, ATP content was significantly reduced (6-8). Hepatic

ATP content is also decreased in insulin resistant and type 2 diabetic patients (9;

10). A decrease in hepatic ATP/AMP ratio stimulates food intake by signals via the

vagus nerve to the brain, which may lead to obesity and insulin resistance in humans

(11). In obese healthy subjects, hepatic ATP content was inversely related to body

mass index (BMI), decreasing steadily with increasing BMI (12). Hepatic

knockdown of PEPCK, one of the key gluconeogenic enzymes, ameliorated

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hyperglycemia and insulin resistance with increased hepatic ATP content in db/db

mice (13). Overall, all these studies revealed that a decrease in ATP content is

associated with an increase in glucose production in the livers of diabetic animals and

humans. Given the crucial role of ATPSβ in controlling mitochondrial ATP synthesis

(4; 5), we hypothesized that its expression in the livers may be deregulated and

associated with increased hepatic glucose production under diabetic conditions.

In this study, we reported that ATPSβ expression is reduced and correlated with

ATP content in the livers of type 1 and type 2 diabetic mice. Hepatic overexpression

of ATPSβ increased cellular ATP content, and suppressed gluconeogenesis, leading to

the amelioration hyperglycemia of type1 and type2 diabetic mice.

Experimental procedures

Construction of Adenovirus expressing rattus ATPSβ - Adenovirus expressing

rattus ATPSβ was constructed using the ATPSβ cDNA coding sequence cloned from

INS-1 cells in our previous study (4) with a 6xHis tag inserted in the N-terminus.

Overexpression of ATPSβ in the livers of diabetic mice - To assess the impact of

high-fat diet on hepatic ATPSβ expression, 8-week old C57BL/6 mice were fed on

45% high-fat diet (HFD) or a control diet (Diet #MD45%fat & #MD10%fat,

Medicience Ltd, China) for 12 weeks (14). The mRNA and protein expression of

ATPSβ or other genes in the livers was analyzed by real time PCR and western

blotting assays. For overexpression of ATPSβ in the liver, 8-12 week female db/db

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mouse on BKS background were chosen for experiment. 1.0x109 pfu Ad-ATPSβ or

Ad-GFP were injected into the db/db mice via tail vein as previously described

(16)(15). At the 3rd and 7th day post virus injection, oral glucose tolerance tests were

performed. On the 8th day, the fed animals were sacrificed for experimental analysis.

8 week old male C57BL/6 mice were injected intraperitoneally for 7 consecutive days

with STZ (50mg/kg, sigma) freshly dissolved in citrate buffer (pH=4.2). Control mice

were injected with citrate buffer. Mice with the fasting blood glucose (FBG) level

greater than 16.4mmol/L at 30 days post final STZ injection were chosen for

experiments. The mice were treated with Ad-ATPSβ or Ad-GFP as db/db mice.

Fasting blood glucose was monitored at 3 days post viral injection. On the 4th day, the

fed animals were sacrificed for experimental analysis. Serum was collected for insulin,

TG and total CHO levels determination, and liver was taken for biochemical analysis.

All procedures were approved by the Institutional Animal Care and Use Committee of

Peking University Health Science Center.

Oral Glucose Tolerance Tests - Mice were fasted for 6 hours (8 am to 2 pm) in a

cage with fresh bedding prior to oral glucose tolerance test (OGTT). Mice were orally

administered with glucose at the dose of 3g/kg and blood glucose levels were

monitored at 0, 15, 30, 60, 90 and 120 minutes post glucose load using a Freestyle

brand glucometer (Roche) via blood collected from the tail (15). The blood glucose

concentrations at 0 minute were determined as fasting blood glucose. At the same

time, blood samples were taken at time points (0, 15, 30 and 60 min) from the tail

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vein for detecting insulin levels. Plasma insulin levels were measured using the

Rat/Mouse Insulin ELASA kit (MILIPORE) (16).

Pyruvate tolerance tests - The protocol for pyruvate tolerance tests (PTT) was

detailed elsewhere (17). In brief, db/db mice infected with Ad-ATPSΒ or Ad-GFP for

7 days were fasted for 16 hours and were injected intraperitoneally with pyruvate

(0.75mg/Kg body weight in saline). Blood glucose levels were measured from the tail

vein at indicated times using a Freestyle brand glucometer.

Insulin tolerance tests - Insulin tolerance tests (ITT) were done with an

intraperitoneal injection of insulin (3 U/kg body weight into mice after 6 hours of

fasting (18). Plasma glucose was measured using blood drawn from the tail vein at

designated time points (0, 15, 30, 60, 90, 120 minutes post insulin injection) (19).

Immunoprecipitation and immunoblot assays - The immunoprecipitation was

performed using Protein G Argrose bead as described previously (20). 200µg of liver

protein was precipitated with anti-ubiquitin or IgG, and separated by 10-12% SDS gel.

Immunoblot was performed, and the membrane was developed with ECL.

Cell culture - Human hepatocellular carcinoma (HepG2) cells was purchased from

American Type Culture Collection (Rockville, MD), and cultured at 37°C in 5%

CO2–95% air in DMEM medium. The cells were infected with 20 MOI of Ad-GFP or

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Ad-ATPSβ for 48 hours. For insulin stimulation, the cells were serum-starved for 12

hours followed by treatment with 100 nM insulin (NOVO) for 5 minutes and then

lysed in fresh Roth Lysis buffer. For inhibition of PI3K, the infected cells were treated

with indicated concentrations of wortmannin (inhibitor of PI3K), PIK75 (inhibitor of

p110α) or TGX221 (inhibitor of p110β) for 30 minutes before being lysed. The

infected cells were treated with indicated concentrations of PPADS (antagonist of P2

receptor), U73122 (inhibitor of IP3R) and CPZ (inhibitor of CaM) for 30 minutes to 1

hour before experimental assay. The lysate was centrifuged at 13,000 rpm at 4°C for

10 minutes. 20-100 µg total proteins were subjected to western blotting assay

immediately.

Primary hepatocyte culture - Primary mouse hepatocyte was cultured as previously

described (4). In brief, 5-6-week-old male C57BL/6 mice were anaesthetized with

10% chloral hydrate, and then a catheter was placed in inferior vena cava. The liver

was perfused with 1 ml of heparin (320 µ/ml), 40 ml of solution I (Krebs’s solution +

0.1 mM EGTA), and 30 ml of solution II (Krebs’s solution + 2.74 mM CaCl2 + 0.05%

Collagenase I), respectively. The perfused liver was passed though a 400 screening

size filter by flushing with RPMI 1640 medium. The hepatocytes were collected by

centrifuge at 50 g for 2 minutes. Hepatocytes were re-suspended with 1640 medium

and planted in 6-well plates for experiments after three washes with RPMI 1640.

Immunoblotting - Cells or liver tissues were lysed in fresh Roth lysis buffer (50 mM

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HEPES, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 5 mM EGTA, 20 mM Na

pyrophosphate, 20 mM NaF, 0.2 mg/mL PMSF, 0.01mg/mL leupeptin, and 0.01

mg/mL aprotinin, pH 7.4). The lysate was centrifuged at 4 °C at 13 000 rpm for 10

min. The protein concentration in the supernatant was determined by bicinchoninic

acid assay. Twenty-200 µg of total protein were separated by 12-15% SDS-PAGE.

Proteins in the gel were transferred to the Hybond-C Extra membrane (Amersham

Biosciences) at 120V for 2 h at 4°C. The membrane was washed once with TBST (1.2

g/L Tris Base, 5.84 g/L NaCl, 0.1% Tween-20, pH 7.5) before blocking in TBST

containing 1% BSA at room temperature for 1 h. The membrane was incubated in

1:200 ~ 1:1000 primary antibodies at 4°C overnight. The membrane was washed five

times with TBST, and incubated in 1:5000 peroxidase-conjugated secondary

antibodies at room temperature for 1 h before washing as above, and then developed

with ECL. After immunoblotting assays of target proteins, the membrane was stripped

with 0.2 N NaOH and reprobed for EIF5 or other house-keeping protein as loading

control. Anti-ATPSβ, pAkt, Akt, FOXO1, Fas, mSREBP1 and SREBP1 antibodies

were from Abcam. Anti-ATPSα, 6His-tag, G6Pase, Ubiquitin, EIF5 and β-actin

antibodies were from Santa. Ant- ATPSγ and ATPSd antibodies were form Bioworld.

Anti-CoxIV antibodies were from Invitrogen. Peroxidase-conjugated secondary

antibodies were from Santa. Activation of Akt was evaluated by analyzing

phosphorylation at Ser473 site.

Real-time quantitative PCR assay - Total RNA of tissues and cells were extracted

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with RNApure High-purity Total RNA Rapid Extraction Kit (BioTeke Corporation).

Quantification of target gene expression was performed via quantitative real-time

PCR. The complementary DNA was synthesized with the use of RevertAidTM First

Strand cDNA Synthesis Kit (Fermentas K1622). Target gene mRNA level was

normalized to that of β-actin in the same sample as detailed previously (21). Each

sample was measured in duplicate or triplicate in each experiment. Moreover, melting

curve for each PCR product was analyzed to ensure the specificity of the

amplification product. All the primer sequences for real time PCR assays were listed

in supplementary table 1.

ATP content determination - ATP content was assessed by bioluminescence method

using ATP-Lite Assay Kit (Vigorous Biotechnology Beijing Co., Ltd). For hepatic

ATP content determination, 1ml lysis buffer was added into per 100mg frozen tissue.

For cellular ATP content determination, 300 ul lysis buffer was added into the 35mm

dish. For extracellular ATP content determination, HepG2 cell culture medium was

collected. The lysate ATP content or the ATP content in medium was measured by

luminometric determination of the luciferin-luciferase reaction. For determination of

relative ATP level in the cells, the ATP content values (nmol) were first normalized to

the protein mount (nmol/mg protein) in the same sample, and then normalized to the

control values. For determination of relative ATP level in the medium, the absolute

concentration was determined and normalized to the control value.

Cellular calcium measurement - HepG2 cells seeded on coverslips were loaded with

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1µM Fura-2 AM (Molecular Probes) for 10min, and then imaged under Olympus ix71

fluorescence microscope. The emission intensities under 340nm and 380nm

illumination were recorded every 1 second, and the ratio of the emission densities

(F340/F380) reflects the intracellular free calcium concentration (5). For basal

calcium concentration measurement, infected HepG2 cells were treated with indicated

concentrations of PPADS and Suramin for 1 hour.

Mitochondria isolation - Mitochondria were isolated from liver tissue or HepG2

cells using the Mitochondria/Cytosol Fractionation Kit (Pierce) according to

the manufacture's protocol. In brief, liver tissue or HepG2 cells were homogenized in

Mito-Cyto extraction buffer provided by the Kit, and then the lysate was centrifuged

at 800g for 5 min twice to pellet the nucleus and cell debris. The supernatant was

collected and centrifuged at 10,000g for 10 min to pellet mitochondria, which was

washed three times with lysis buffer to clean contaminated cytoplasmic proteins (22).

Immunofluorescence - To detect the translocation of FOXO1, HepG2 cells were

infected with Ad-GFP or Ad-ATPSβ for 48 hours and treated with of PPADS (50µM)

and CPZ (100µM) for 1 hour before been fixed with 4% paraformaldehyde for 15

minutes and permeabilized with 0.5% Triton X-100 for 10 minutes. Nonspecific

binding was blocked in 1% BSA at room temperature for 10 minutes, followed by

incubation with anti-FOXO1 antibody overnight at 4°C. The slides were then

incubated with Alexa fluo 594 antibodies (1:200). Images were obtained

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using Confocal microscopy.

Statistical analysis - Data are presented as mean ± S.E.M. Statistical significance of

differences between groups was analyzed by unpaired Student’s t test or by one-way

analysis of variance (ANOVA) when more than two groups were compared.

Results

ATPSβ expression was reduced in the livers of db/db and HFD-fed diabetic mice.

To determine the potential role of ATPSβ in the progression of hepatic insulin

resistance and type 2 diabetes, its expression in the livers of db/db and HFD-fed

diabetic mice was analyzed. The mRNA (Figure 1A) and protein (Figure 1B) levels of

ATPSβ were significantly reduced in the livers of db/db mice. In contrast, the mRNA

levels of α, γ, δ and ε subunits of F1 domain, and b, c and d subunits of F0 domain

were not significantly different between db/db and db/m mouse livers (Suppl Figure

1A). The protein levels of α and γ subunits of F1 domain, and d subunits of F0

domain were confirmed to remain unchanged between db/db and db/m mouse livers

(Suppl Figure 1B). Hepatic ATP content was also reduced in db/db mice when

compared with db/m mice (Figure 1C). The mRNA and protein levels of ATPSβ, and

ATP content were also reduced in the livers of HFD-fed diabetic mice (Figure D-F).

Similarly, the mRNA levels of α, γ, δ, ε, b, c and d subunits of ATP synthase were not

significantly different between HFD and NC mouse livers (Suppl Figure 1C). The

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protein levels of α, γ and d subunits were also confirmed to remain unchanged in

HFD-fed diabetic mouse livers (Suppl Figure 1D).

Hepatic overexpression of ATPSβ ameliorated hyperglycemia in db/db mice. The

efficacy of Ad-ATPSβ treatment on cellular ATPSβ protein level was firstly

determined in HepG2 cells (Suppl Figure 2). Then, ATPSβ was overexpressed in the

livers of db/db mice via tail vein injection of Ad-ATPSβ to evaluate its impact on ATP

content and glucose metabolism. At 3 days post virus injection, Ad-ATPSβ-treated

mice exhibited significant improvement of fasting hyperglycemia and glucose

intolerance when compared with Ad-GFP-treated mice (Figure 2A-B). At 7 days post

virus injection, the fasting hyperglycemia and glucose intolerance was markedly

attenuated in Ad-ATPSβ-treated mice when compared with Ad-GFP-treated mice

(Figure 2C). The fasting blood glucose of mice at 0, 3 and 7 days post viral injection

was shown in Figure 2D. At the 8th day post virus injection, the serum insulin levels in

Ad-ATPSβ-treated mice were significantly lower than that in control mice (Figure 2E).

ATPSβ overexpression also reduced hepatic lipid deposition in db/db mice (Suppl

Figure 3).

ATPSβ overexpression activated Akt and repressed gluconeogenic genes in the

livers of db/db mice. Ad-ATPSβ treatment increased ATPSβ protein level in the livers

of db/db mice (Figure 3A). Moreover, overexpressed ATPSβ was shown to be

predominantly located in mitochondria (Figure 3B). In contrast, injection of

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Ad-ATPSβ had little effect on the mRNA and protein levels of ATPSβ in skeletal

muscle and pancreas, indicating the specificity of ATPSβ overexpression in the liver

(Suppl Figure 4). ATPSβ overexpression elevated cellular ATP content, increased

phosphorylated Akt (pAkt) levels and reduced the protein level of gluconeogenic gene

G6Pase in the livers of db/db mice (Figure 3C-E). The mRNA levels of PEPCK and

G6Pase in the livers were significantly repressed after ATPSβ overexpression (Figure

3F). In vivo, OGTT, insulin secretion, insulin tolerance test (ITT) and pyruvate

tolerance test (PTT) assays indicated there is no significant difference between saline-

and Ad-GFP-treated mice (Suppl Figures 5 and 6). Moreover, ATPSβ overexpression

failed to affect insulin secretion in db/db mice (Suppl Figure 5D-E). In vitro, Ad-GFP

infection also failed to significantly affect Akt activation in HepG2 cells (Suppl

Figure 5F). These findings suggested that the virus itsself at the dose used in the

current study has little effect on glucose metabolism and Akt activation in mouse liver

cells. ITT assays indicated that the global insulin sensitivity was improved by hepatic

overexpression of ATPSβ (Suppl Figure 6A-B). PTT assays revealed that hepatic

glucose production was repressed after ATPSβ overexpression (Suppl Figure 6C-D).

Overall, these findings were consistent with the decrease in gluconeogenic genes and

the attenuation of hyperglycemia in db/db mice after hepatic ATPSβ overexpression.

ATPSβ activated PI3K-Akt signaling pathway through P2 receptor. Because Akt

plays a crucial role in controlling glucose metabolism, the effect of ATPSβ on Akt

activation was further analyzed in HepG2 cells and primary cultured mouse

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hepatocytes. In HepG2 cells, immunofluoresence staining and immunoblotting assays

indicated that Ad-ATPSβ treatment increased ATPSβ protein level in mitochondria

(Suppl Figures 7-8). ATPSβ overexpression significantly elevated intracellular and

extracellular ATP levels in both HepG2 cells (Figure 4A) and primary cultured mouse

hepatocytes (Figure 4B). In HepG2 cells infected by Ad-GFP, insulin potently

stimulated Akt activation (Figure 4C). Ad-ATPSβ treatment markedly elevated pAkt

levels (Figure 4C) when compared with Ad-GFP treated cells without insulin

stimulation. ATPSβ overexpression also augmented insulin-stimulated Akt activation

in HepG2 cells (Figure 4C). Similarly, ATPSβ also induced Akt activation in primary

cultured mouse hepatocytes in the absence of insulin stimulation (Figure 4D).

ATPSβ-induced Akt activation was completely blocked by PI3K inhibitor wortmannin

in HepG2 cells in the absence of insulin stimulation (Figure 4E). Inhibition of PI3K

p110α catalytic subunit blocked ATPSβ-induced Akt activation by about 70-80%,

whereas inhibition of PI3K p110β catalytic subunit had little effect on ATPSβ-induced

Akt activation in the absence of insulin (Figure 4E). Overall, these results suggested

that ATPSβ-induced Akt activation requires the activity of PI3K p110α catalytic

subunit but is independent of insulin.

ATP can be released to function as a signal molecule in various cell types (23; 24).

In HepG2 cells, ATPSβ-induced Akt activation was significantly attenuated by ATP

receptor P2 receptor antagonist PPADS (Figure 5A). Pretreatment with another P2

receptor antagonist suramin also attenuated ATPSβ-induced Akt activation (Suppl

Figure 9A). Furthermore, inhibition of P2 receptor downstream molecule PLC using

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U73122 also attenuated ATPSβ-induced Akt activation (Figure 5B). ATPSβ

overexpression elevated cytosolic free calcium level, which was inhibited by PPADS

and suramin in HepG2 cells (Figure 5C). Because an increase in cytosolic free

calcium level has been shown to activate PI3K/Akt signaling axis through CaM in

several cell types (25; 26), we further analyzed whether ATPSβ induced Akt activation

via calcium-CaM signaling pathway. The results indicated that depletion of

extracellular calcium partially attenuated (Suppl Figure 9B), whereas inhibition of

CaM using CPZ completely abolished ATPSβ-induced Akt activation in HepG2 cells

(Figure 5D).

Inhibition of P2 receptor or CaM blocked ATPSβ-induced translocation of

FOXO1. Activation of Akt plays a crucial role in suppressing gluconeogenesis by

phosphorylating and inactivating FOXO1, the key transcriptor controlling the

transcription of gluconeogenic genes PEPCK and G6Pase (27). As expected, ATPSβ

overexpression significantly promoted nuclear exclusion of FOXO1 with the

activation of Akt in HepG2 cells (Figure 6). In support, inhibition of P2 receptors or

CaM blocked ATPSβ-promoted nuclear exclusion of FOXO1 with the attenuation of

Akt activation (Figure 6).

Hepatic overexpression of ATPSβ ameliorated hyperglycemia of streptozocin

(STZ)-induced type 1 diabetes mice. In the livers of STZ-induced type 1 diabetic

mice, the mRNA and protein levels of ATPSβ expression was also significantly

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reduced with a decrease in ATP content (Figure 7A-C). Hepatic overexpression of

ATPSβ for 3 days elevated cellular ATP content and ameliorated fasting

hyperglycemia in STZ-induced type 1 diabetic mice (Figure 7C-E). ATPSβ

overexpression increased pAkt levels and reduced mRNA and protein levels of

PEKCK and G6Pase in the livers of STZ-treated mice, suggesting that hepatic

gluconeogenesis was inhibited (Figure 7F-H).

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Discussion

In the livers of type 1 and type 2 diabetic mice, ATPSβ expression was reduced

and positively correlated with ATP content. Hepatic overexpression of ATPSβ

elevated cellular ATP content, suppressed hepatic glucose production, and improved

hyperglycemia, hyperinsulinemia, insulin resistance and fatty liver in db/db mice.

Hepatic overexpression of ATPSβ also elevated cellular ATP and ameliorated

hyperglycemia in STZ-induced diabetic mice. ATPSβ overexpression increased pAkt

levels and repressed the expression of gluconeogenic genes in the livers of these

diabetic mice. These findings suggested that inhibition of hepatic glucose production

will attenuate hyperglycemia and global insulin resistance in type 2 diabetic mice.

Similarly, it had been previously reported that silencing of hepatic PEPCK suppressed

hepatic glucose production and improved global insulin resistance in db/db mice (13).

In vitro, ATPSβ overexpression increased intracellular and extracellular ATP levels in

HepG2 cells and primary cultured mouse hepatocytes. In support of our previous (4; 5)

and current findings that ATPSβ plays a crucial role in controlling ATP synthesis,

hydrogen peroxide induces ATPSβ expression and increases cellular ATP level in

melanocytes (28). Proteomic analysis revealed that ATPSβ expression was reduced

with a decrease in ATP content in mouse livers after ischemia-reperfusion treatment

(29). No change of other subunits of ATP synthase was found in ischemia-reperfusion

injured mouse livers in the same study (29).

Extracellular ATP can activate PI3K-Akt pathway through P2 receptors in several

cell types. Ishikawa et al found that Pannexin 3 releases ATP into extracellular space,

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which activates PI3K/Akt pathway via P2 receptors to promote the proliferation of

osteoblasts (30). Electrical pulses can stimulate skeletal muscle cells to release ATP,

which enhances glucose uptake via activation of PI3K-Akt pathway through P2

receptors (31). Extracellular ATP also activates PI3K-Akt pathway to attenuate

ischemia-induced apoptosis of human endothelial cells (32). P2 receptors were

divided into P2X receptors and P2Y receptors. P2X receptors are ligand-gated ion

channels which are permeable for calcium (33), while P2Y receptors are G

protein-coupled receptors which stimulate PLC to increase IP3, resulting in calcium

release from internal storage (23). Liver cells and HepG2 cells have been shown to

release ATP. P2 receptors are also constitutively expressed in liver cells and HepG2

cells (24; 34). Exposure to exogenous ATP has been reported to elevate cytosolic free

calcium level in HepG2 cells and primary cultured mouse hepatocytes (35). PPADS is

a specific inhibitor for P2X subtype, whereas suramin is considered to be a

broad-spectrum P2 receptor inhibitor (36). Both PPADS and suramin significantly

attenuated ATPSβ-induced increase in cytosolic free calcium level and Akt activation

in HepG2 cells. Depletion of extracellular calcium also attenuated ATPSβ-induced

Akt activation. Furthermore, U73122, an inhibitor of P2Y receptor downstream

molecule PLC also attenuated ATPSβ-induced Akt activation in HepG2 cells. These

results revealed the involvement of both P2X and P2Y receptors in ATPSβ-induced

increase in cytosolic free calcium level and Akt activation in liver cells.

Cytosolic free calcium level is crucial second messenger in ATP/P2 receptor

signaling pathway. Increased cytosolic free calcium level activates CaM, which can

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activate PI3K via direct association with its p85 regulatory subunit, leading to

activation of Akt (37). Importantly, inhibition of CaM using CPZ completely

abolished ATPSβ-induced Akt activation in HepG2 cells. ATPSβ–induced Akt

activation requires the activity of PI3K p110α catalytic subunit but is independent of

insulin signaling. These findings were consistent with recent discoveries that p110α

predominantly mediated Akt activation in liver cells. Deletion of p110α blunted Akt

activation in response to insulin, whereas deletion of p110β had little effect on

insulin-stimulated Akt activation in the liver (38). Furthermore, ATPSβ

overexpression reduced hepatic and serum TG content with little effect on liver and

serum CHO levels in db/db mice. The protein levels of key lipogenic genes FAS and

mature SREBP-1 were reduced in the livers of db/db mice after ATPSβ

overexpression (Suppl Figure 3D). FOXO1 is a crucial transcriptor controlling the

transcription of glucogeogenic enzymes PEPCK and G6Pase (27). Insulin-stimulated

activation of Akt can phosphorylate FOXO1 and promote its translocation from

nucleus to cytoplasma, suppressing the transcription of gluconeogenic genes and

hepatic gluconeogenesis (27). ATPSβ overexpression promotes nuclear exclusion of

FOXO1, which can be inhibited by antagonisms of P2 receptor or CaM. Overall, these

findings revealed that ATPSβ overexpression elevated ATP synthesis and secretion in

liver cells. Released ATP activates both P2X and P2Y receptors to increase cytosolic

free calcium level, which activates CaM-PI3K-Akt signaling axis to suppress

gluconeogenesis independent of insulin. However, it should be noted that elevated

intracellular ATP levels will inhibit the activity of ATP sensitive K+ (K(ATP)) channel

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and increase the influx of extracellular calcium through L-type voltage-gated calcium

channel (39; 40), which may also contribute to ATPSβ-induced increase in free

cytosolic calcium and Akt activation. Interestingly, although we found that ATPSβ

overexpression for 7 days significantly reduced the mRNA level PEPCK in db/db

mouse livers, its protein level remained unchanged (Figure 3D-F, Suppl Figure

10A-C). The decrease in PEPCK mRNA level was confirmed by two different set of

primers according to the mRNA sequence of PEPCK (Suppl Figure 10C). At 3 days

post ATPSβ overexpression, both the mRNA and protein levels of PEPCK were

significantly reduced in the livers of db/db mice (data not shown) and STZ-induced

diabetic mice (Figure 7). Co-immunoprecipitation assay revealed that the

ubiquitination of PEPCK protein was reduced at 7 days after ATPSβ overexpression,

suggesting that the ubiquitin-mediated degradation of PEPCK protein might decrease

(Suppl Figure 10D). A decrease in PEPCK protein degradation might be a protective

mechanism against persistent suppression of hepatic glucose production induced by

long-term of ATPSβ overexpression.

It had been intensively studied that excessive accumulation of free fatty acids

(FFAs), in particular saturated FFAs such as palmitate, plays an important role in the

progression of insulin resistance in various tissues (41). Hojlund et al found that

ATPSβ protein level is reduced in skeletal muscle of insulin-resistant and type 2

diabetic patients (42; 43). In vivo, FFAs induced insulin resistance in skeletal muscle

cells with a decrease in ATP generation (44). Rooibos ameliorates

palmitate-induced insulin resistance in C2C12 muscle cells with increased cellular

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ATP levels (45). In support, inhibition of ATP synthase using oligomycin reduces

cellular ATP levels and induces insulin resistance in cultured human myotubes (46). In

the current study, we found that palmitate downregulated ATPSβ and induced insulin

resistance in HepG2 cells, which was reversed by overexpression of ATPSβ (Suppl

Figure 11). Palmitate has been shown to induce insulin resistance by stimulating

reactive oxygen species (ROS) production in hepatocytes (47). We further found that

ATPSβ overexpression reduced palmitate-induced ROS production in HepG2 cells

(Suppl Figure 12). ATPSβ also tended to reduce ROS production in basal condition in

HepG2 cells (Suppl Figure 12). In lipid stressed condition, an increase in electron

transfer on respiratory chain as well as a decrease in ATPSβ expression may

contribute to ROS overproduction and oxidative stress, which is one of the main

causes of hepatic insulin resistance (47; 48). These findings suggest that repression of

ATPSβ expression, leading to a decrease in ATP synthesis and a possible increase in

ROS production, might be a novel mechanism for lipid-induced insulin resistance in

liver and muscle cells.

Activation of adenosine 5'-monophosphate (AMP)-activated protein kinase

(AMPK) exerts beneficial effects on amelioration of hyperglycemia (49). The activity

of AMPK is modulated by various factors such as an increase in AMP/ATP ratio,

adiponectin and metformin (49). Anti-diabetic drug metformin can target

mitochondrial complex I to inhibit ATP production (50; 51), leading to the activation

of AMPK (52), which at least partially mediates metformin’s hypoglycemic effects.

However, some deleterious effects of AMPK activation including inhibition of insulin

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secretion (53), induction of pancreatic β cell and renal medullary interstitial cell

apoptosis (54; 55) had also been reported. Given the role of ATP as the key energy

storage molecule and an important signaling molecule (30-32), activation of

ATPSβ-ATP-P2 receptor signaling may have some unique advantages than activation

of metformin-AMPK signaling in the treatment of type 2 diabetes.

In summary, the present study demonstrated that ATPSβ expression is

positively correlated with ATP content in the livers of diabetic mice. A decrease in

ATPSβ expression in the liver, leading to the reduction of ATP content and attenuation

P2 receptor-PI3K-Akt signaling pathway, may play an important role in the

progression of diabetes. Upregulating hepatic ATPSβ might be an attractive method

for treatment of both type 1 and type 2 diabetes (Figure 8).

Acknowledgments

This study was supported by grants from the Ministry of Science and Technology

(2012CB517504/2011ZX09102) and the Natural Science Foundation of China

(81170791/30870905/81200625/81322011). This study was also supported by a grant

from Beijing Natural Science Foundation (7122107).

No potential conflicts of interest relevant to this article were reported.

C.W., Z.C., S.L., S.J., Y.Z. and Y.M. researched data, contributed to discussion.

C.W. wrote Manuscript. J.L. and Y.C. reviewed/edited manuscript. C.W., Y.G. and J.Y.

designed the study, and revised/edited manuscript. Dr. Jichun Yang is the guarantor of

this work and, as such, had full access to all the data in the study and takes

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responsibility for the integrity of the data and the accuracy of the data analysis

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Figure legends

Figure 1. ATPSβ expression was reduced in the livers of db/db and HFD-fed

diabetic mice. (A) The mRNA level of ATPSβ was reduced in the livers of db/db

mice. (B) The protein level of ATPSβ was reduced in the livers of db/db mice. (C)

Hepatic ATP content was reduced in db/db mice. N=5, *p<0.05 versus db/m mice. (D)

The mRNA level of ATPSβ was reduced in the livers of mice fed on a high fat diet

(HFD) for 12 weeks. (E) The protein level of ATPSβ was reduced in the livers of

HFD-fed mice. (F) Hepatic ATP content was reduced in HFD-fed mice. N=5, *p<0.05

versus control mice. NC, normal chow; HFD, high fat diet.

Figure 2. Hepatic overexpression of ATPSβ markedly ameliorated fasting

hyperglycemia and glucose intolerance in db/db mice. (A-C) Oral glucose

tolerance test before (A), or at 3 (B) and 7 (C) days post tail vein injection of Ad-GFP

or Ad-ATPSβ. At 3 and 7 days post virus injection, Ad-ATPSβ-transduced mice

exhibited significant improvement of hyperglycemia and glucose intolerance. (D) The

fasting blood glucose before, or at 3 and 7 days post viral injection. (E) Ad-ATPSβ

treatment reduced serum insulin levels. N=8-10, *p<0.05 versus Ad-GFP group.

Figure 3. ATPSβ overexpression repressed gluconeogenic genes in the livers of

db/db mice. (A) Ad-ATPSβ treatment increased ATPSβ protein level in the livers. (B)

Ad-ATPSβ treatment increased ATPSβ protein level in the mitochondria.

Mitochondria were isolated from db/db mouse livers, and immuno blotting assays

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were performed using antibodies against ATPSβ or 6xHis tag. Mi, mitochondrial

fraction. (C) ATPSβ overexpression elevated ATP content in the livers. (D-E) ATPSβ

overexpression on the protein levels of gluconeogenic genes in the livers.

Representative images were shown in (D) and quantitative data shown in (E). (F)

ATPSβ overexpression reduced the mRNA levels of G6Pase and PEPCK in the livers.

N=5-8, *p<0.05 versus Ad-GFP group.

Figure 4. ATPSβ overexpression activated Akt via PI3K in liver cells. (A-B)

ATPSβ overexpression increased intracellular and extracellular ATP levels in HepG2

cells (A) and primary cultured mouse hepatocytes (B). (C) ATPSβ activated Akt

independent of insulin in HepG2 cells. The infected cells were serum starved for 12

hours and treated with or without 100nM insulin for 5 minutes before pAkt levels

were analyzed. (D) ATPSβ activated Akt independent of insulin in primary cultured

mouse hepatocytes. (E) Effect of inhibition of PI3K on ATPSβ-induced Akt activation

in HepG2 cells. Infected HepG2 cells were treated with Wotmannin (1µM), p110α

inhibitor PIK75 (100nM) or p110β inhibitor TGX221 (100nM) for 30 minutes before

pAkt levels were analyzed. D, DMSO; W, wortmannin; α, p110α inhibitor PIK75; β,

p110β inhibitor TGX221. N=4-5, *p<0.05 versus control cells; #p<0.05 versus

Ad-GFP-infected cells in the presence of insulin.

Figure 5. ATPSβ induced Akt activation via P2 receptor/calcium/CaM signaling

pathway in HepG2 cells. (A) Inhibition of P2 receptors attenuated ATPSβ-induced

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Akt activation in HepG2 cells. Infected cells were treated with PPADS (50µM) for 1

hour before pAkt levels were analyzed. (B) Inhibition of PLC attenuated

ATPSβ-induced Akt activation in HepG2 cells. Infected cells were treated with PLC

inhibitor U73122 (5µM) for 1 hour before pAkt levels were analyzed. (C) Inhibition

of P2 receptors attenuated ATPSβ-induced increase in cytosolic free calcium level.

Infected cells were treated with PPADS or suramin (50µM) for 1 hour before cellular

calcium levels were analyzed. The calcium levels were obtained from more than 300

single cells in at least 3 independent experiments. (D) Inhibition of CaM completely

abolished ATPSβ-induced Akt activation. Infected cells were treated with CaM

antagonist CPZ (100µM) for 1 hour before pAkt levels were analyzed. N=5, *p<0.05

versus control cells; #p<0.05 versus Ad-ATPSβ-treated cells.

Figure 6. ATPSβ promoted nuclear exclusion of FOXO1 in HepG2 cells. In the

absence of insulin, ATPSβ overexpression promoted translocation of FOXO1 from

nucleus to cytoplasm, which was blocked by P2 receptor antagonist PPADS or CaM

inhibitor CPZ. The images shown here were obtained using confocal microscopy, and

the representatives of at least 3 independent experiments.

Figure 7. Hepatic overexpression of ATPSβ ameliorated fasting hyperglycemia in

STZ-induced type 1 diabetic mice. (A-B) The mRNA (A) and protein (B) levels of

ATPSβ were reduced in the livers STZ-treated mice.. (N=5, *p<0.05 versus control

mice). (C) Ad-ATPSβ treatment elevated ATP content in the livers. (D) Ad-ATPSβ

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treatment increased ATPSβ protein level in the livers of STZ-induced diabetic mice.

(E) Hepatic overexpression of ATPSβ ameliorated fasting hyperglycemia of

STZ-treated mice. 3 days post viral injection, fasting blood glucose was monitored.

(F-G) ATPSβ overexpression increased pAkt levels and reduced the protein levels of

G6Pase and PEPCK in the livers. (H) ATPSβ overexpression reduced the mRNA

levels of G6Pase and PEPCK in the livers. N=9, *p<0.05, **p<0.01 versus control

mice, #p<0.05 versus Ad-GFP treated mice.

Figure 8. Proposed mechanism of ATPSβ in regulation of glucose metabolism in

the livers. ATPSβ enhances ATP synthesis and elevates extracellular ATP level, which

activates P2 receptors to increase cytosolic free calcium levels. Finally, elevated

cytosolic calcium levels activate the PI3K-Akt signaling pathway in a CaM-dependent

manner, leading to the suppression of hepatic gluconeogenesis. ATPSβ, ATP synthase

β subunit; P2X, P2X receptor; P2Y, P2Y receptor; CaM, Calmodulin;. “→” represents

activation or enhancement, “─┤” represents repression.

Supplemental Figure Legends

Figure 1. The expression levels of ATP synthase subunits in diabetic mouse livers.

A) The mRNA levels of ATP synthase subunits in livers of db/db mice. B) The protein

levels of ATP synthase subunits in the livers of db/db mice. The representative gel

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images were shown in upper panel, and quantitative data shown in lower panel. C)

The mRNA levels of ATP synthase subunits in livers of HFD-mice. C57BL/6 mice

were fed on a high-fat diet (HFD) for 12 weeks. D) The protein levels of ATP

synthase subunits in the livers of HFD mice. The representative gel images were

shown in upper panel, and quantitative data were shown in lower panel. NC, normal

chow; HFD, high fat diet. N=5, *p<0.05 versus db/m or NC mice.

Figure 2. Characterization of adenovirus expressing ATPSβ. HepG2 cells were

infected with different dose (MOI) of Ad-ATPSβ or Ad-GFP for 48 hours. ATPSβ

protein level was analyzed by western blotting assays. (A) Immunoblotting assay

using antibodies against ATPSβ. (B) Immunoblotting assay using antibodies against

6xHis tag. The images shown here were the representatives of at least 3 independent

experiments.

Figure 3. ATPSβ overexpression reduced lipid deposition in the livers of db/db

mice. (A) Morphological and Oil Red O staining assays of lipid deposition in the

livers. (B-C) Quantitative assay of TG content in the livers (B) and in serum (C). (D)

ATPSβ overexpression reduced the protein levels of FAS and mature SREBP-1 in the

livers. N=5, *p<0.05 versus control mice.

Figure 4. Tail-vein injection of Ad-ATPSβ on ATPSβ protein levels in skeletal

muscle and pancreas of db/db mice. A-B) Ad-ATPSβ injection had little effect on

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ATPSβ protein (A) and mRNA (B) levels in skeletal muscle of db/db mice. C-D)

Ad-ATPSβ injection had little effect on ATPSβ protein (C) and mRNA (D) levels in

pancreas of db/db mice. N=5-8, there is no statistically significance between two

groups of mice.

Figure 5. Hepatic overexpression of ATPSβ improved glucose tolerance in db/db

mice. A-C) OGTT data of mice at 0, 3 and 7 days post viral injection. Three groups of

db/db mice were treated with saline, Ad-GFP or Ad-ATPSβ via tail-vein injection,

respectively. D) Insulin secretion of mice at 7 days post viral injection. N=8, *p<0.05

versus Ad-GFP-treated mice, and there is no statistically significance between saline-

and Ad-GFP-treated mice. E) AUC analyses of data shown in (D). F) Ad-GFP

infection failed to affect Akt activation in HepG2 cells. The cells were treated with

saline, Ad-GFP or Ad-ATPSβ for 48 hours before pAkt levels were assayed. N=8,

*p<0.05 versus Ad-GFP-treated cells, and there is no statistically significance

between saline- and Ad-GFP-treated cells.

Figure 6. Overexpression of ATPSβ repressed hepatic glucose production in

db/db mice. A) Insulin tolerance tests (ITT) of mice at 7 days post viral injection. B)

Area under curve (AUC) analyses of data shown in (A). C) Pyruvate tolerance tests

(PTT) of mice at 7 days post viral injection. D) Area under curve (AUC) analyses of

data shown in (C). N=6, *p<0.05 versus Ad-GFP-treated mice, and there is no

statistically significance between saline- and Ad-GFP-treated mice.

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Figure 7. Immunoflurescence staining revealed that overexpressed ATPSβ was

located in mitochondria of HepG2 cells. Overexpressed ATPSβ was mainly located

in mitochondria. COX IV, Cytochrome c oxidase subunit 4, mitochondrial marker.

The images were captured by confocal microscopy and the representatives of at least

3 independent experiments.

Figure 8. Overexpressed ATPSβ protein was located in mitochondria of HepG2

cells. Mitochondrial and cytosolic fractions were isolated, and immunoblotting was

performed to analyze the distribution of ATPSβ protein. The images shown here were

the representatives of at least 3 independent experiments.

Figure 9. Suramin attenuated ATPSβ-induced Akt activation in HepG2 cells. A)

Infected HepG2 cells were treated with indicated concentration of suramin for 1 hour

before pAkt levels were analyzed. B) ATPSβ-induced Akt activation was partially

dependent on the presence of extracellular calcium. Ad-ATPSβ-infected cells were

treated with calcium free medium plus 0.5mM EGTA for 2 hours before pAkt levels

were analyzed. N=5, *p<0.05 versus control cells infected with Ad-GFP, #p<0.05

versus Ad-ATPSβ-infected cells.

Figure 10. Hepatic overexpression of ATPSβ on gluconeogenic genes expression.

A-B) ATPSβ overexpression on the protein levels of pAkt, G6Pase and PEPCK in

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db/db mouse livers. The representative gel images were shown in (A), and

quantitative data shown in (B). C) ATPSβ overexpression on the mRNA levels of

PEPCK in db/db mouse livers. Two sets of primers according to PEPCK mRNA were

used. D) ATPSβ overexpression on ubiquitination of PEPCK protein in db/db mouse

livers. IP, immunoprecipation; IB, immunoblotting; Ub, ubiquitin. E) ATPSβ

overexpression on ATP levels in mouse livers. N=6, *p<0.05 versus Ad-GFP-treated

mice, and there is no statistically significance between saline- and Ad-GFP-treated

mice.

Figure 11. ATPSβ overexpression protected against palmitate-induced insulin

resistance in HepG2 cells. (A-B) Chronic exposure to palmitate repressed ATPSβ

expression in HepG2 cells. HepG2 cells treated with palmitate (0.2mM) for 24 hours

before experimental assays (N=3, *p<0.05). (C) HepG2 cells were infected with

Ad-GFP or Ad-ATPSβ for 24 hours, and then treated with 0.2mM palmitate for 24

hours. The cells were stimulated with insulin (100nM) for 5 minutes before pAkt

levels were analyzed. (N=3, *p<0.05). FFA, palmitate; INS, insulin.

Figure 12. ATPSβ overexpression reduced palmitate-induced ROS production in

HepG2 cells. For ROS determination, HepG2 cells were treated with Ad-LacZ or

Ad-ATPSβ for 42 hours and then treated with 0.2mM palmitate for 6 hours (0.1M

NaOH+50g/L BSA as control). Then the cells were loaded by 10µM DCFH-DA for 20

minutes and washed twice with FBS free medium. Then, HepG2 cells were collected

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and the fluorescence intensity was measured by flow cytometry. ROS level was

determined using DCFH-DA Method. N=3,*P<0.05 versus 0 hour; #<0.05 versus 6

hour palmitate treatment.

Page 36 of 57Diabetes

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db/m db/db

0.0

0.5

1.0

*

Re

lati

ve

AT

P c

on

ten

t

(fo

ld o

f C

on

)

Figure 1

NC HFD0.0

0.5

1.0

*

Re

lati

ve

AT

P c

on

ten

t

(fo

ld o

f C

on

)

A

B

C

db/m db/db0.0

0.5

1.0

1.5

*R

ela

tiv

e A

TP

mR

NA

(fo

ld o

f C

on

)

HFD

ATPSβ

eIF5

NC HFD0.0

0.5

1.0

1.5

*

Re

lati

ve

AT

PS

β p

rote

in

(fo

ld o

f C

on

)

NC

D

E

F

NC HFD0.0

0.5

1.0

1.5

*

Rela

tiv

e A

TP

mR

NA

(fo

ld o

f C

on

)

0.0

0.5

1.0*

db/m db/db

Rela

tiv

e A

TP

pro

tein

(fo

ld o

f C

on

)

ATPSβ

db/m db/db

eIF5

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Figure 2A

B

Ad-GFP Ad-ATPSβ0

10

20

30

40

50

*

Se

rum

in

su

lin

(ng

/mL

)

0 30 60 90 1200

10

20

30

40

50

60Ad-GFP

Ad-ATPSβ

Day 0

Time (minute)

Blo

od

glu

co

se

(mm

ol/L

)

Day 3

0 30 60 90 1200

10

20

30

40

50Ad-GFP

Ad-ATPSβ

*

** *

**

Time (minute)

Blo

od

glu

co

se

(mm

ol/L

)

0 30 60 90 1200

10

20

30

40Ad-GFP

Ad-ATPSβ

Day 7

Time (minute)

**

* * *

*Blo

od

glu

co

se

(mm

ol/L

)

0 70

5

10

15

20

Ad-GFP

Ad-ATPSβ

3

* *

Days post viral injection

Fa

sti

ng

blo

od

glu

co

se

(m

mo

l/L

)

C

D

E

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G6Pase PEPCK0.0

0.5

1.0

1.5 Ad-GFP

Ad-ATPSβ

* *

Re

lati

ve

mR

NA

le

ve

l

(fo

ld o

f C

on

)

pAkt/Akt G6Pase PEPCK0

1

2

3 Ad-GFP

Ad-ATPSβ

*

*

Re

lati

ve

pro

tein

le

ve

l

(fo

ld o

f c

on

)

Figure 3A

B

Ad-GFP Ad-ATPSβ0.0

0.5

1.0

1.5*

Re

lati

ve

AT

P c

on

ten

t

(fo

ld o

f C

on

)

C

Ad-GFP Ad-ATPSβ

Ad-GFP Ad-ATPSβ0

1

2

3 *R

ela

tiv

e A

TP

pro

tein

(fo

ld o

f C

on

)

EIF5

ATPSβ

6xHis tag pAkt

Akt

G6Pase

PEPCK

eIF5

Ad-GFP Ad-ATPSβ

D

E

Fβactin

ATPSβ

Mi

Ad-GFP Ad-ATPSβ

Cytoplasm

6His-tag

COXIV

Ad-GFP Ad-ATPSβ

ATPSβ

Mitochondria

Page 39 of 57 Diabetes

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Figure 4

Ad-G

FP

Ad-A

TPSβ

Ad-G

FP

Ad-A

TPSβ

0

1

2

3

4

**

*Cell Medium

Re

lati

ve

AT

P c

on

ten

t

(fo

ld o

f C

on

)

A

C

D

E

Ad-GFP Ad-ATPSβ0

5

10

15

20

25 0 nM INS

100 nM INS*

*

*

#

Re

lati

ve

pA

kt/

Ak

t le

ve

l

(fo

ld o

f C

on

)

pAkt

Akt

ATPSβ

eIF5

Ad-GFP Ad-ATPSβ

Insulin - + - +

Ad-G

FP

Ad-A

TPSβ

Ad-G

FP

Ad-A

TPSβ

0

1

2

*

*Cell Medium

Re

lati

ve

AT

P c

on

ten

t

(fo

ld o

f C

on

)

Ad-GFP Ad-ATPSβ0

1

2

3

4 *

Re

lati

ve

pA

kt/

Ak

t le

ve

l

(fo

ld o

f C

on

)

pAkt

Akt

eIF5

Ad-GFP Ad-ATPSβ

DW

ort

p110α

p110β D

Wort

p110α

p110β

0

5

10

15

20 Ad-GFP

Ad-ATPSβ

**

Re

lati

ve

pA

kt/

Ak

t le

ve

l

(fo

ld o

f C

on

)

pAkt

Akt

Ad-GFP Ad-ATPSβ

D W α β D W α β

eIF5

B

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Figure 5

0

1

2

3

4*

#

Ad-GFP Ad-ATPSβ

U73122 - - +

Re

lati

ve

pA

kt/

Ak

t le

ve

l

(fo

ld o

f c

on

)

pAkt

Akt

eIF5

Ad-GFP Ad-ATPSβ

- - +U73122

PPADS - + - +

Ad-GFP Ad-ATPSβ

pAkt

Akt

EIF5

0

5

10

15

*

Ad-GFP Ad-ATPSβ

PPADS - + - +

Re

lati

ve

pA

kt/

Ak

t le

ve

l

(fo

ld o

f c

on

)

#

pAkt

Akt

eIF5

0

1

2

3

4

5

#

Ad-GFP Ad-ATPSβ

CPZ - - +

*R

ela

tiv

e p

Ak

t/A

kt

le

ve

l

(fo

ld o

f c

on

)

Ad-GFP Ad-ATPSβ

- - +CPZ

A

B

C

D

Ad-G

FP

Ad-A

TPSβ

PPA

DS

sura

min

0.0

0.50.540.550.560.570.580.590.60

* # #

34

0/3

80

Page 41 of 57 Diabetes

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FOXO1 DAPI Merge

Ad-GFP

Ad-ATPSβ

Ad-ATPSβ

+ PPADS

Ad-ATPSβ

+ CPZ

Figure 6 Page 42 of 57Diabetes

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Figure 7

Con STZ

0.0

0.5

1.0

*

Con STZATPSβ

eIF5

Rela

tiv

e A

TP

pro

tein

(fo

ld o

f C

on

)

Con STZ0.0

0.5

1.0

*

Rela

tiv

e A

TP

mR

NA

(fo

ld o

f C

on

)

Ad-GFP Ad-ATPSβ0

1

2

#

ATPSβ

6xHis tag

eIF5

Ad-GFP Ad-ATPSβ

Rela

tiv

e A

TP

pro

tein

(fo

ld o

f C

on

)

Con Ad-GFP Ad-ATPSβ

0.0

0.5

1.0

**

#

Re

lati

ve

AT

P c

on

ten

t

(fo

ld o

f C

on

)

0

5

10

15

20

25

Con

STZ+Ad-GFP

STZ+Ad-ATPSβ

0 day 3 day

#

**** **

Fa

sti

ng

blo

od

glu

co

se

(mm

ol/L

)

pAkt

/Akt

G6P

ase

PEPCK

0

1

2

3

4

5Ad-GFP

Ad-ATPSβ

#

Re

lati

ve

pro

tein

le

ve

l

(fo

ld o

f C

on

)

##

A

B

C

GD

E

F

pAkt

Akt

PEPCK

G6Pase

eIF5

Ad-GFP Ad-ATPSβG6P

ase

PEPCK

0.0

0.5

1.0

1.5 Ad-GFP

Ad-ATPSβ

#

Re

lati

ve

mR

NA

le

ve

l

(fo

ld o

f C

on

)

#

H

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Figure 8

ATPSβ

ATP release

P2X P2Y

Ca2+/CaM/PI3K

Akt

Gluconeogenic

genes

Page 44 of 57Diabetes

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ATPSα

ATPSβ

ATPSγ

ATPSδ

ATPSε

ATPSb

ATPSc

ATPSd

0.0

0.5

1.0

1.5 db/m

db/db

*

Re

lati

ve

mR

NA

le

ve

l

(fo

ld o

f C

on

)

ATPSα ATPSγ ATPSd0.0

0.5

1.0

1.5 db/m

db/db

Re

lati

ve

pro

tein

le

ve

l

(fo

ld o

f C

on

)

ATPSα

EIF5

ATPSd

db/m db/db

ATPSγ

β-actin

ATPSα

ATPSβ

ATPSγ

ATPSδ

ATPSε

ATPSb

ATPSc

ATPSd

0.0

0.5

1.0

1.5 NC

HFD

*

Re

lati

ve

mR

NA

lev

el

(fo

ld o

f C

on

)

ATPSα

EIF5

ATPSd

NC HFD

ATPSγ

β-actin

ATPSα ATPSγ ATPSd0.0

0.5

1.0

1.5 NCHFD

Re

lati

ve

pro

tein

le

ve

l

(fo

ld o

f C

on

)

A C

B D

Supplemental Figure 1Page 45 of 57 Diabetes

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20 10 20 50 100 MOI

Ad-ATPSβ

ATPSβ

β actin

A

Ad-GFP

6His-tag

βactin

20 25 50 5 10 20 MOI

B

Ad-ATPSβAd-GFP

Supplemental Figure 2 Page 46 of 57Diabetes

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Ad-GFP Ad-ATPSβ0

100

200

300

400

*

Se

rum

TG

le

ve

l

(m

g/d

l)

Ad-GFP Ad-ATPSβ0

5

10

15

20

*

He

pa

tic

TG

co

nte

nt

(m

g/g

.tis

su

e)

A

B

Ad-GFP Ad-ATPSβ C

D

FAS mSREBP-10.0

0.5

1.0

1.5 Ad-GFP

Ad-ATPSβ

**

Re

lati

ve

pro

tein

le

ve

l

(fo

ld o

f C

on

)

Ad-GFP Ad-ATPSβ

FAS

mSREBP-1

SREBP-1

eIF5

Supplemental Figure 3Page 47 of 57 Diabetes

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Ad-GFP Ad-ATPSβ0.0

0.5

1.0

1.5

Rela

tiv

e A

TP

pro

tein

(fo

ld o

f C

on

)

Ad-GFP Ad-ATPSβ0.0

0.5

1.0

1.5

Re

lativ

e A

TP

mR

NA

(fo

ld o

f C

on

)

Ad-GFP Ad-ATPSβ0.0

0.5

1.0

1.5

2.0R

ela

tiv

e A

TP

mR

NA

(fo

ld o

f C

on

)

Ad-GFP Ad-ATPSβ0.0

0.5

1.0

1.5

Rela

tiv

e A

TP

pro

tein

(fo

ld o

f C

on

)

Muscle Pancreas

ATPSβ

EIF5

Ad-GFP Ad-ATPSβ

β-actin

ATPSβ

EIF5

Ad-GFP Ad-ATPSβ

β-actin

A C

B D

Supplemental Figure 4 Page 48 of 57Diabetes

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0 30 60 90 1200

20

40

60Saline

Ad-GFP

Ad-ATPSβ

Time (minute)

** *

* **

Day 3

Blo

od

glu

co

se

(m

mo

l/L

)

0 30 60 90 1200

10

20

30

40

50 Saline

Ad-GFP

Ad-ATPSβ

Day 0

Time (minute)

Blo

od

glu

co

se

(m

mo

l/L

)

0 30 60 90 1200

20

40

60 Saline

Ad-GFP

Ad-ATPSβ

Day 7

*

* * **

*

Time (minute)

Blo

od

glu

co

se

(m

mo

l/L

)

pAKT

AKT

EIF5

ATPSβGFPSaline

β-actin

Saline Ad-GFP Ad-ATPSβ0

2

4

6

ns

*

Re

lati

ve

pA

kt/

Ak

t le

ve

l

(fo

ld o

f C

on

)

A D

B

0 15 30 45 600

10

20

30

40 Saline

Ad-GFP

Ad-ATPSβ

*

Time (minute)

Ins

ulin

le

ve

l (n

g/m

L)

C

Supplemental Figure 5

Saline Ad-GFP Ad-ATPβ0

500

1000

1500

AU

C (

ng

/mL

.min

ute

)E

F

Page 49 of 57 Diabetes

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Sal

ine

Ad-G

FP

Ad-A

TPSβ

0

500

1000

1500

**

ITT

AU

C (

mm

ol/L

*min

ute

)ITT at Day 7

0 30 60 90 1200

5

10

15

20

25

Ad-GFP

Ad-ATPSβ

Saline

****

** ** ****

Time (minute)

Blo

od

glu

co

se

(m

mo

l/L

)

PTT at Day 7

0 30 60 90 120 150 1800

10

20

30

Saline

Ad-GFP

Ad-ATPSβ

****

**** ********

Time (minute)

Blo

od

glu

co

se

(m

mo

l/L

)

Salin

e

Ad-G

FP

Ad-A

TPβ0

1000

2000

3000

4000

5000

**

PTT

AU

C (

mm

ol/L

*min

ute

)

A C

B D

Supplemental Figure 6 Page 50 of 57Diabetes

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COX IV DAPI MergeATPSβ

Ad-LacZ

Ad-ATPSβ

Supplemental Figure 7Page 51 of 57 Diabetes

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Ads- GFP ATPSβ GFP ATPSβ

Mitochondria cytoplasma

ATPSβ

COXIV

βactin

Supplemental Figure 8 Page 52 of 57Diabetes

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Ad-G

FP

Ad-A

TPSβ

Ad-A

TPSβ

Ad-A

TPSβ

0

2

4

6

8

10 *

suramin(μM) 0 0 10 50

#

#

Re

lati

ve

pA

kt/

Ak

t le

ve

l

(fo

ld o

f C

on

)

suramin 0 0 10 50 (μM)

pAkt

Akt

eIF5

Ad-GFP Ad-ATPSβ

0

1

2

3

4

5

Ad-GFP Ad-ATPSβ

Ca2+ + + -

*#

Re

lati

ve

pA

kt/

Ak

t le

ve

l

(fo

ld o

f C

on

)

Ad-GFP Ad-ATPSβ

pAkt

Akt

eIF5

Ca2+ + + ─

A B

Supplemental Figure 9Page 53 of 57 Diabetes

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Saline Ad-GFP Ad-ATPSβ0.0

0.5

1.0

1.5

2.0

*Saline

Ad-GFP

Ad-ATPSβ

Re

lati

ve

AT

P c

on

ten

t

(fo

ld o

f C

on

)

pAKT

AKT

EIF5

G6Pase

PEPCK

β-actin

pAkt

/Akt

G6P

ase

PEPCK

0.0

0.5

1.0

1.5

2.0

2.5 Saline

Ad-GFP

Ad-ATPSβ*

*

Re

lati

ve

pro

tein

le

ve

l

(fo

ld o

f C

on

)

G6Pase PEPCK PEPCK0.0

0.5

1.0

1.5 SalineAd-GFPAd-ATPSβ

* * *

Primer set 1 Primer set 2

Re

lati

ve

mR

NA

le

ve

l

(fo

ld o

f C

on

)

A C

B

EIB: anti-PEPCK

Ubiquitin-input:

IB: anti-Ubiquitin

Ad-GFP Ad-ATPSβ

IP: Ub Ub Ub Ub IgG

D

Supplemental Figure 10 Page 54 of 57Diabetes

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Con FFA

0.0

0.5

1.0

*

Rela

tiv

e A

TP

pro

tein

(fo

ld o

f C

on

)Con FFA

ATPSβ

eIF5

C

C+IN

SFFA

FFA+IN

S C

C+IN

SFFA

FFA+IN

S

0

2

4

6

8 Ad-GFP

Ad-ATPSβ

*

*

Re

lati

ve

pA

kt/

Ak

t le

ve

l

(fo

ld o

f C

on

)

pAkt

Akt

INS - + - + - + - +

FFA - - + + - - + +

Ad-GFP Ad-ATPSβ

eIF5

A

B

C

Supplemental Figure 11Page 55 of 57 Diabetes

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0h 6h

0.0

0.5

1.0

1.5

2.0 Ad-LacZ

Ad-ATPSβ *#

Palmitate

p=0.07

Re

lati

ve

RO

S le

ve

l

(fo

ld o

f C

on

)

Supplemental Figure 12 Page 56 of 57Diabetes

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Table 1. List of oligonucleotide primer pairs used in real time RT-PCR and RT-PCR

analysis.

Gene sense antisense

ATPSβ(H,M) 5'-AACATTGTTGGCAATGAGCA-3' 5'-ATGTAGCCCGTGAAGACCTC-3'

ATPSα 5'-TGTCCGCTTACATTCCAACA-3' 5'-CTTCATGGTACCTGCCACCT-3'

ATPSε 5'-GGCTGGACTCAGCTACATCCGGT-3' 5'-TGCCAGAACAGGGGCCCACA-3'

ATPSγ 5'-CCGTGCAGCCGCAATGGAT-3' 5'-TACAGAGCCAAAGAACCTGTCCCA-3'

ATPSδ 5'-CTTTGCCTCCCCGACGCAGG-3' 5'-ACGGAGCCGCTGCTCACAAA-3'

ATPSb 5'-TCAGAAGCGCCATTACCTCT-3' 5'-TTGGCAATGGTCTCCTTTTC-3'

ATPSc 5'-CCATCTAAGCAGCCTTCCTG-3' 5'-CCCAGAATGGCATAGGAGAA-3'

ATPSd 5'- CTGCGATTGACTGGGCTTAC -3' 5'- AGACACAAACTCAGCACAGC -3'

G6Pase 5'-AGGAAGGATGGAGGAAGGAA-3' 5'-TGGAACCAGATGGGAAAGAG-3'

PEPCK

(set 1)

5'-ATCTTTGGTGGCCGTAGACCT-3' 5'-CCGAAGTTGTAGCCGAAGAA-3'

PEPCK

(set 2)

5'-ACCCAAGAGCAGAGAGACAC-3' 5'- ATGCCCATCCGAGTCATGAT -3'

GK 5'-TATGAAGACCGCCAATGTGA-3' 5'-CACTGAGCTCTCATCCACCA-3'

β-actin 5'-AGCCATGTACGTAGCCATCC -3' 5'-GCTGTGGTGGTGAAGCTGTA -3'

M: mouse; H: human. If not indicated, all the primer sequences are referred to mouse orgin.

Page 57 of 57 Diabetes