Hepatic overexpression of ATP synthase β subunit activates...
Transcript of Hepatic overexpression of ATP synthase β subunit activates...
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]
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Diabetes Publish Ahead of Print, published online December 2, 2013
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
Page 18 of 57Diabetes
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
Page 20 of 57Diabetes
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
Page 21 of 57 Diabetes
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
Page 22 of 57Diabetes
responsibility for the integrity of the data and the accuracy of the data analysis
Page 23 of 57 Diabetes
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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
Page 28 of 57Diabetes
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
Page 29 of 57 Diabetes
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
Page 30 of 57Diabetes
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
Page 31 of 57 Diabetes
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
Page 32 of 57Diabetes
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.
Page 33 of 57 Diabetes
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
Page 34 of 57Diabetes
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
Page 35 of 57 Diabetes
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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(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
Sβ
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
Sβ
mR
NA
(fo
ld o
f C
on
)
0.0
0.5
1.0*
db/m db/db
Rela
tiv
e A
TP
Sβ
pro
tein
(fo
ld o
f C
on
)
ATPSβ
db/m db/db
eIF5
Page 37 of 57 Diabetes
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
Page 38 of 57Diabetes
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
Sβ
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
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
Page 40 of 57Diabetes
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
FOXO1 DAPI Merge
Ad-GFP
Ad-ATPSβ
Ad-ATPSβ
+ PPADS
Ad-ATPSβ
+ CPZ
Figure 6 Page 42 of 57Diabetes
Figure 7
Con STZ
0.0
0.5
1.0
*
Con STZATPSβ
eIF5
Rela
tiv
e A
TP
Sβ
pro
tein
(fo
ld o
f C
on
)
Con STZ0.0
0.5
1.0
*
Rela
tiv
e A
TP
Sβ
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
Sβ
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
Page 43 of 57 Diabetes
Figure 8
ATPSβ
ATP release
P2X P2Y
Ca2+/CaM/PI3K
Akt
Gluconeogenic
genes
Page 44 of 57Diabetes
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
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
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
Ad-GFP Ad-ATPSβ0.0
0.5
1.0
1.5
Rela
tiv
e A
TP
Sβ
pro
tein
(fo
ld o
f C
on
)
Ad-GFP Ad-ATPSβ0.0
0.5
1.0
1.5
Re
lativ
e A
TP
Sβ
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
Sβ
mR
NA
(fo
ld o
f C
on
)
Ad-GFP Ad-ATPSβ0.0
0.5
1.0
1.5
Rela
tiv
e A
TP
Sβ
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
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
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
COX IV DAPI MergeATPSβ
Ad-LacZ
Ad-ATPSβ
Supplemental Figure 7Page 51 of 57 Diabetes
Ads- GFP ATPSβ GFP ATPSβ
Mitochondria cytoplasma
ATPSβ
COXIV
βactin
Supplemental Figure 8 Page 52 of 57Diabetes
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
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
Con FFA
0.0
0.5
1.0
*
Rela
tiv
e A
TP
Sβ
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
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
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