Inhibition of pyruvate dehydrogenase kinase 2 protects ... · TCA cycle to generate ATP to meet hepatic energy demands (4). Whether acetyl-CoA produced by β-oxidation forms ketone

1

Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis

through modulation of TCA cycle anaplerosis and ketogenesis

Authors Younghoon Go1811

Ji Yun Jeong11011

Nam Ho Jeoung211

Jae-Han Jeon18

Bo-

Yoon Park19

Hyeon-Ji Kang19

Chae-Myeong Ha19

Young-Keun Choi1 Sun Joo Lee

1 Hye

Jin Ham8 Byung-Gyu Kim

8 Keun-Gyu Park

18 So Young Park

3 Chul-Ho Lee

4 Cheol Soo

Choi5 Tae-Sik Park

5 W N Paul Lee

6 Robert A Harris

712 and In-Kyu Lee

189 12

1Department of Internal Medicine Kyungpook National University School of Medicine

Daegu South Korea

2Department of Pharmaceutical Science and Technology Catholic University of Daegu

Gyeongsan South Korea

3Department of Physiology College of Medicine Yeungnam University Daegu South Korea

4Disease Model Research Center Korea Research Institute of Bioscience amp Biotechnology

Daejeon South Korea

5Lee Gil Ya Cancer and Diabetes Institute Gachon University of Medicine and Science

Inchon South Korea

6Department of Pediatrics Harbor-UCLA Medical Center 1000 West Carson Street

Torrance CA 90502 USA

7Roudebush VA Medical Center and the Department of Biochemistry and Molecular Biology

Indiana University School of Medicine Indianapolis IN 46202 USA

8Leading-edge Research Center for Drug Discovery and Development for Diabetes and

Metabolic Disease Kyungpook National University Daegu South Korea

9BK21 plus KNU Biomedical Convergence Programs at Kyungpook National University

Page 1 of 45 Diabetes

Diabetes Publish Ahead of Print published online July 6 2016

2

Daegu South Korea

Contact

In-Kyu Lee MD PhD

Division of Endocrinology and Metabolism

Kyungpook National University School of Medicine

50 Samduck-2Ga Jung-Gu Daegu South Korea 700-721

Tel +82-53-420-5564 Fax +82-53-426-2046 E-mail leeiknuackr

Robert A Harris PhD

Roudebush VA Medical Center and the Department of Biochemistry and Molecular Biology

Indiana University School of Medicine Indianapolis IN 46202

Tel + 327-440-8941 Fax + 317-988-3180 E-mail raharrisiuedu

Additional Footnotes

10Present address Department of Internal Medicine Soonchunhyang University Gumi

Hospital Gumi South Korea

11Co-first author

12Co-corresponding author

Page 2 of 45Diabetes

3

ABSTRACT

Hepatic steatosis is associated with increased insulin resistance and tricarboxylic acid

(TCA) cycle flux but decreased ketogenesis and pyruvate dehydrogenase complex (PDC)

flux This study examined whether hepatic PDC activation by inhibition of pyruvate

dehydrogenase kinase 2 (PDK2) ameliorates these metabolic abnormalities

Wild-type (WT) mice fed a HFD exhibited hepatic steatosis insulin resistance and

increased levels of pyruvate TCA cycle intermediates and malonyl-CoA but reduced

ketogenesis and PDC activity due to PDK2 induction Hepatic PDC activation by PDK2

inhibition attenuated hepatic steatosis improved hepatic insulin sensitivity reduced hepatic

glucose production increased capacity for β-oxidation and ketogenesis and decreased the

capacity for lipogenesis These results were attributed to altered enzymatic capacities and a

reduction in TCA anaplerosis that limited the availability of oxaloacetate (OAA) for the TCA

cycle which promoted ketogenesis

The present study reports that increasing hepatic PDC activity by inhibition of PDK2

ameliorates hepatic steatosis and insulin sensitivity by regulating TCA cycle anaplerosis and

ketogenesis The findings suggest PDK2 is a potential therapeutic target for non-alcoholic

fatty liver disease (NAFLD)

INTRODUCTION

Hepatic steatosis is induced by a number of lipid metabolic abnormalities including

increased de novo lipogenesis inhibited triacylglyceol (TG) release enhanced FA influx from

adipose tissue and reduced hepatic FA oxidation and ketogenesis (1) Recently it was

proposed that dysregulation of ketone body metabolism could potentially contribute to the

pathogenesis of non-alcoholic fatty liver disease (NAFLD) Reduced ketogenesis exacerbates


4

diet-induced hepatic steatosis and hyperglycemia (2) and a ketotic diet reduces the body

weight of mice as much as a calorie-restricted diet (3) However the precise mechanism by

which impaired ketogenesis contributes to hepatic steatosis is still unclear

In the well-fed state acetyl-CoA produced from glucose is converted to FAs for storage

of excess energy or oxidized to CO2 in the TCA cycle to generate ATP by oxidative

phosphorylation In the fasting state two-thirds of the free FAs entering the liver are

converted to acetyl-CoA by β-oxidation and then further processed to ketone bodies which

act as an energy source for peripheral tissues The other one-third of FAs is utilized by the

TCA cycle to generate ATP to meet hepatic energy demands (4) Whether acetyl-CoA

produced by β-oxidation forms ketone bodies or enters the TCA cycle is determined by

anaplerotic influx of TCA cycle intermediates The conversion of pyruvate to OAA by

pyruvate carboxylase (PC) is one of the most important sources of anaplerosis in the liver

Pyruvate can also be converted to acetyl-CoA by oxidative decarboxylation mediated by

PDC PDC activity is inhibited via phosphorylation of the pyruvate dehydrogenase E1α

subunit (PDHE1α) which is mediated by increased expression of PDKs during fasting or in

an insulin-resistant state (5-7) Four isoforms of PDK (PDK1-4) are expressed in a tissue-

specific manner with unique expression profiles in response to different physiological

conditions (8) Among these isoforms PDK2 is the major PDK responsible for regulation of

PDC activity in the liver (8-10)

Recently it was reported that decreased PDC activity and enhanced pyruvate

carboxylation due to hepatic insulin resistance contribute to increased hepatic

gluconeogenesis in obese subjects with hepatic steatosis (1112) Because of competition for

pyruvate the balance between PDC and PC activity may play a critical role in metabolic

dysfunction caused by obesity and insulin resistance In this study we examined the


5

possibility that activation of PDC by PDK2 inhibition reduces the anaplerotic flux of

pyruvate into the TCA cycle which may increase ketogenesis and prevent hepatic steatosis

induced by a HFD Indeed we found that PDK2 inhibition ameliorates hepatic steatosis and

insulin resistance suggesting that PDK2 is a potential therapeutic target for NAFLD

RESERCH DESIGN AND METHODS

Animal Experiments

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

Kyungpook National University For the NAFLD mice model 8-week-old male WT

(C57BL6J) and PDK2 KO mice (13) were fed a HFD in which 20 of the calories were

derived from carbohydrates and 60 from fat (Research Diets D12492 pellets) Control WT

and PDK2 KO mice were fed an isocaloric LFD in which 70 of the calories were derived

from carbohydrates and 10 from fat (Research Diets D12450B pellets) The mice were

housed and maintained on a 12 h lightdark cycle at 22 plusmn 2degC After the mice were sacrificed

tissues were rapidly collected and freeze-clamped with Wollenberger tongs at the temperature

of liquid nitrogen and stored at -80degC for analysis

Construction of PDK2 DN Recombinant Adenovirus and Its Infection In Vivo

pCMV6-KanNeo mouse PDK2 plasmid was purchased from OriGene The PDK2

dominant-negative (DN) mutant R157A (1415) was generated by site-directed mutagenesis

with the QuickChange II site-directed mutagenesis kit (Stratagene) using the following

primers forward 5rsquo-CCAGTACTTCCTGGACGCCTTCTACCTCAGC-3rsquo and reverse 5rsquo-

GCTGAGGTAGAAGGCGTCCAGGAAGTACTGG-3rsquo Recombinant adenovirus for PDK2

DN mutant was generated using pAd-Track-CMV shuttle vector as described previously (16)


6

Recombinant PDK2 DN and GFP adenovirus were amplified in AD-293 cells and purified by

CsCl gradient centrifugation Adenovirus titers were determined using Adeno-X rapid titer

(BD Bioscience) according to the manufacturerrsquos instructions After 12 weeks of HFD

feeding in WT mice recombinant adenoviruses (10 times 109 plaque-forming units) expressing

PDK2 DN and GFP constructs were delivered by tail vein injection On day 3 after

adenovirus infection glucose tolerance tests were performed in overnight-fasted mice by

intraperitoneal (ip) injection of glucose (1 gkg body weight) On day 7 after adenovirus

infection mice were sacrificed after overnight fasting After the mice were sacrificed tissues

were rapidly collected and freeze-clamped with Wollenberger tongs at the temperature of

liquid nitrogen

Measurement of PDC Activity

PDC activity was spectrophotometrically measured in a 96-well plate reader by coupling

to the reaction catalyzed by arylamineacetyltransferase as described previously (7) One unit

of PDC activity corresponds to the acetylation of 1 micromol of p-(p-aminophenyl azo)

benzenesulfonate per min at 30degC

Statistical Analysis

Statistical significance was determined by the unpaired Studentrsquos t-test when two groups

were compared Values are presented as the means plusmn SEM of the indicated number of

independent samples P values less than 005 were considered statistically significant

See the Supplementary materials and methods section for additional details


7

RESULTS

Up-Regulation of PDK2 Reduces Hepatic PDC Activity in HFD-Fed Mice

PDK2 expression is increased in the livers of obese animals (917) its role in the

development of hepatic metabolic diseases remains unclear We examined PDC activity and

the expression of PDK isoenzymes in the livers of WT mice fed a HFD As shown in Figure

1A PDC activity was significantly lower in the livers of mice fed a HFD than in those of

mice fed a LFD The reduction in hepatic PDC activity was associated with significant

increases in the phosphorylation of PDHE1α (Fig 1B) PDK2 mRNA levels were

significantly higher in the livers of HFD-fed mice compared to LFD-fed mice (Fig 1C)

Conversely the level of the PDK4 which is induced by a HFD in skeletal muscle and

heart(5) was markedly decreased in the livers of HFD-fed mice (Fig 1C) The protein levels

of PDK2 and PDK4 showed the same pattern as their respective mRNAs (Fig 1D) To

confirm that PDK2 regulates hepatic PDC activity in HFD-fed mice PDC activity and

phosphorylation of PDH E1α were measured in the livers of WT and PDK2 KO mice As

expected from the data shown in Figure 1A the HFD reduced hepatic PDC activity in WT

mice but not in PDK2 KO mice (Fig 1E) Consistent with this finding PDHE1α

phosphorylation was decreased in PDK2 KO mice compared to WT mice (Fig 1F) To

examine PDC activity change by PDK2 deficiency in other tissue we measured the

phosphorylation state of PDHE1α in muscle heart and kidney Phosphorylation of PDHE1α

was similar between HFD-fed PDK2 KO mice and HFD-fed WT mice in these tissues

(supplementary Fig 1) The serum levels of pyruvate lactate and FFA were not different

between WT and PDK2 KO mice after HFD feeding (Supplementary Fig 2) These findings

indicate that PDK2 is a major regulator of PDC activity in the liver of HFD-fed mice


8

PDK2 Deficiency Prevents HFD-Induced Hepatic Steatosis

To elucidate the role of PDK2 in the development of hepatic steatosis WT and PDK2 KO

mice were fed a HFD to induce NAFLD After 16 weeks of HFD feeding body weight gain

in HFD-fed PDK2 KO mice was significantly lower than that of HFD-fed WT mice (Fig 2A)

Even though the body weights of HFD-fed PDK2 KO mice were lower than those of WT

mice there were no differences in food consumption or physical activity (Supplementary Fig

3A and 3B) Relative to HFD-fed WT mice HFD-fed PDK2 KO mice exhibited greater

energy expenditure (Supplementary Fig 3C)

The liverbody weight ratio of PDK2 KO mice fed a HFD was significantly lower than

that of WT mice (Fig 2B) The number and the size of the lipid droplets were greatly

decreased in livers of PDK2 KO mice fed a HFD (Fig 2C) However no difference in

phenotype in epididymal adipose tissue (EAT) was observed between WT and PDK2 KO

mice on either diet (Fig 2C) Consistent with the histological analysis the hepatic TG

content was significantly lower in PDK2 KO mice than in WT mice after HFD feeding (Fig

2D) The level of hepatic glycogen was also significantly reduced in PDK2 KO mice fed a

HFD compare to WT mice fed a HFD (Fig 2E) Serum activities of AST and ALT markers of

liver injury were also significantly elevated in WT mice fed a HFD but not in PDK2 KO

mice (Fig 2F and 2G) The expression of genes involved in inflammation including tumor

necrosis factor alpha (TNFα) monocyte chemoattractant protein-1 (MCP-1) and

plasminogen activator inhibitor-1 (PAI-1) were significantly lower in PDK2 KO mice than in

WT mice fed a HFD (Supplementary Fig 4A) These data indicate that PDK2 deficiency

could ameliorate HFD-induced hepatic steatosis injury and inflammation


9

PDK2 Deficiency Ameliorates Hepatic Insulin Resistance and Reduces Hepatic Glucose

Production

In HFD-fed mice PDK2 deficiency lowered fasting blood glucose and serum insulin

levels by 15 and 63 respectively relative to WT mice (Fig 3A and 3B) HFD-fed PDK2

KO mice processed glucose more efficiently than HFD-fed WT mice as evidenced by a 30

decrease in the area under the curve (AUC) during GTT (Fig 3C) However with LFD-fed

mice the elimination of glucose was similar between PDK2 KO mice and WT mice (Fig 3C)

which is well agreed with previous data (18) These observations suggest that PDK2

deficiency prevents impaired glucose homeostasis induced by a HFD

To better delineate the mechanism responsible for improved hepatic glucose homeostasis

in HFD-fed PDK2 KO mice we performed a hyperinsulinemic-euglycemic clamp study In

HFD-fed WT mice the glucose infusion rate (GIR) was lower hepatic glucose production

(HGP) was higher during the clamp and whole body glucose turnover was lower (Fig 3D)

than in LFD-fed WT mice However in HFD-fed PDK2 KO mice the GIR was increased

two-fold compared to HFD-fed WT mice (Fig 3D) Furthermore both basal and clamped

HGP were significantly lower in HFD-fed PDK2 KO mice than in HFD-fed WT mice (Fig

3D) Nevertheless whole body glucose turnover was not different between HFD-fed PDK2

KO mice and WT mice (Fig 3D) HGP measured by the pyruvate tolerance test (PTT) was

lower in HFD-fed PDK2 KO mice than in HFD-fed WT (Fig 3E) To evaluate insulin

signaling we examined the phosphorylation levels of AKT and FoxO1 in the liver and

muscle of HFD-fed WT and PDK2 KO mice after insulin injection Insulin-stimulated

phosphorylation of AKT and FoxO1 were significantly increased in the livers of PDK2 KO

mice compared to those of WT mice (Fig 3F) Interestingly in muscle the levels of AKT

and FoxO1 phosphorylation did not differ between HFD-fed PDK2 KO mice and HFD-fed


10

WT mice (Supplementary Fig 5) suggesting that the liver is the major organ affected by

PDK2 deficiency in HFD-fed mice Hepatic insulin resistance caused by obesity may be

caused by increased intracellular DAG and long chain acyl-CoA esters leading to activation

of protein kinase Cε (PKCε) which negatively affects insulin signaling (19) To examine

whether PDK2 deficiency ameliorates hepatic insulin resistance induced by a HFD we

measured DAG levels in the livers of HFD-fed PDK2 KO mice and HFD-fed WT mice The

amounts of 160 181 160-181 and total DAG were markedly lower in the livers of HFD-

fed PDK2 KO mice than in those of HFD-fed WT mice (Fig 3G) Furthermore the

phosphorylation of PKCε at Ser729 was significantly reduced in the livers of HFD-fed PDK2

KO mice compared to HFD-fed WT mice (Fig 3H) These results suggest that activation of

hepatic PDC by PDK2 deficiency ameliorates HGP and hepatic insulin resistance induced by

a HFD

PDK2 Deficiency Reduces TCA cycle Intermediates and Malonyl-CoA in HFD-Fed Mice

Altered hepatic TCA cycle anaplerosis and ketogenesis are associated with hepatic

steatosis and insulin resistance (20) We hypothesized that hepatic PDC activation by PDK2

deficiency shifts the fate of pyruvate from TCA cycle anaplerosis by PC to oxidative

decarboxylation by PDC This shift would reduce the levels of TCA cycle intermediates

thereby reducing TCA cycle flux Thus we measured hepatic pyruvate lactate and TCA

cycle intermediates in WT and PDK2 KO mice Levels of lactate and pyruvate in HFD-fed

PDK2 KO mice were significantly lower than in HFD-fed WT mice (Supplementary Fig 6A

and 6B) However the [lactate][pyruvate] ratio an index of the cytosolic free

[NADH][NAD] ratio was not different between two groups (Supplementary Fig 6C)

Hepatic TCA cycle intermediates (OAA citrate and succinate) were significantly higher in


11

HFD-fed WT mice than in LFD-fed WT mice (Fig 4A) By contrast the TCA cycle

intermediates in HFD-fed PDK2 KO mice were dramatically lower than in HFD-fed WT

mice (Fig 4A) Despite the increase in hepatic PDC activity caused by PDK2 deficiency

there were no differences in the levels of hepatic acetyl-CoA and ATP in PDK2 KO mice

compared to WT mice fed either diet (Fig 4B) Most importantly the increase in malonyl-

CoA levels in the livers of WT mice fed a HFD was reduced significantly in the livers of

HFD-fed PDK2 KO mice (Fig 4B) Since malonyl-CoA is a major negative regulator of β-

oxidation via inhibition of CPT1 (21) we measured hepatic β-HB levels as an index of

ketogenesis The levels of hepatic β-HB were significantly decreased in HFD-fed WT mice

compared to LFD-fed WT mice (Fig 4C) confirming that the reduction in ketogenesis is

associated with hepatic steatosis induced by a HFD However β-HB levels were completely

restored in HFD-fed PDK2 KO mice compared to HFD-fed WT mice (Fig 4C) We then

determined the rate of β-HB production in WT and PDK2 KO mice using a stable isotope

([13

U]β-HB) dilution method and found that the decreased rate of β-HB production in HFD-

fed WT mice was restored in HFD-fed PDK2 KO mice (Fig 4D)

Next we examined the expression of genes involved in β-oxidation ketogenesis and

lipogenesis in the livers of WT and PDK2 KO mice As expected the expression of genes

involved in β-oxidation and ketogenesis including peroxisome proliferator-activated receptor

gamma coactivator 1-alpha (PGC1α) peroxisome proliferator-activated receptor gamma

coactivator 1-beta (PGC1β) carnitine palmitoyltransferase 1-liver (CPT1-L) peroxisome

proliferator-activated receptor alpha (PPARα) and 3-hydroxy-3-methylglutaryl-CoA synthase

2 (HMGCS2) were markedly down-regulated in the livers of WT mice fed a HFD compared

to a LFD However the expression levels of these genes were either partially or completely

restored in the livers of PDK2 KO mice (Fig 4E) The expression of genes involved in


12

lipogenesis such as sterol regulatory element-binding protein 1c (SREBP1c) fatty acid

synthase (FAS) acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1)

was reduced in HFD-fed PDK2 KO mice relative to HFD-fed WT mice (Supplementary Fig

7) These findings suggest that activation of hepatic PDC reduced TCA cycle activity by

decreasing the anaplerotic influx of OAA This resulted in reduction of malonyl-CoA in liver

of PDK2 KO mice fed a HFD

Alteration of Anaplerotic Flux into the TCA cycle by PDK2 Deficiency Promotes

Ketogenesis

In the previous results a decrease in TCA cycle intermediates and an increase in ketone

body were observed in the liver of PDK2 KO mice fed a HFD (Fig 4) These results suggest

that increased PDC activity by PDK2 deficiency might diminish hepatic PC flux and TCA

cycle flux To examine this hypothesis we employed stable isotopomer flux study with 13

C6-

glucose supplement with 2 mM octanoate as a FA substrate in primary hepatocytes isolated

from WT and PDK2 KO mice The PDC flux (measured by acetyl-CoA[M+2] enrichment) in

primary hepatocytes of PDK2 KO mice was significantly increased compared to that of WT

hepatocytes (Fig 5A) However PC flux (measured by aspartate[M+3] and citrate[M+3]) and

TCA flux (measured by citrate[M+2] glutamate[M+2] succinate[M+2] and aspartate[M+2])

in primary hepatocyte of PDK2 KO mice were significantly lower than that of WT mice (Fig

5A) These results indicate that TCA flux is decreased due to reduced anaplerotic PC flux

even though the PDC flux is increased which suggests that PDK2 deficiency leads to the

production of ketone bodies from FA rather than complete oxidation to CO2 by TCA cycle

In order to examine this hypothesis we measured the production of β-HB and CO2 with

octanoate in the primary hepatocytes obtained from WT mice and PDK2 KO mice Oxidation


13

of [14

C1]-octanoate to 14

CO2 was significantly reduced and the rate of β-HB production

increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and

5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA

cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to

that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is

responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)

Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose

Homeostasis in HFD-Fed Mice

It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the

PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs

inactivates their enzymatic activity showing these motifs are indispensable for kinase activity

(1415)

To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were

infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-

GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)

indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)

The number and the size of the lipid droplets and TG levels were reduced in the livers of

HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig

6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and

succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN

(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the

two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated

mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)


14

These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis

by decreasing TCA cycle anaplerosis and increasing ketogenesis

To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation

of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were

measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition

improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I

respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate

the impaired glucose homeostasis induced by a HFD

DISCISSION

As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis

and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism

depending upon the tissue and the physiological condition of the organism (22) In the insulin

resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis

rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and

hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been

recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about

the molecular mechanisms that are involved The fate of pyruvate is decided by the relative

activity of PC to PDC In this study we provide evidence that decreased PDC activity by

increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed

mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic

steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-

fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared

to HFD-fed WT mice


15

The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may

promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably

caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate

which would reduce the rate of ATP production in the TCA cycle Complete oxidation of

palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when

palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between

PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle

FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO

mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-

oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The

observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports

the promotion of β-oxidation and ketogenesis in PDK2-deficient mice

Recently it was reported that increased anaplerotic flux into the TCA cycle (from

pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)

Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats

with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is

likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC

activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC

and TCA cycle flux using 13

C6-glucose isotopomer in primary hepatocytes obtained from WT

and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency

significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by

acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different

between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and

PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to


16

pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition

the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the

decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed

that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)

Together with our results showing reduced expression of inflammatory markers in the livers

of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux

as a major source of electrons for oxidative stress in cells Although testing the effect of

PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the

limits of the present study decreased oxidative metabolism by the TCA cycle is a likely

mechanism for the protective effect of PDK2 deficiency

Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the

HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp

As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that

activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has

been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex

vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of

pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge

which may reduce HGP as well as lipogenesis in PDK2 KO mice

DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance

(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced

hepatic DAG accumulation and PKCε activation and therefore had significantly improved

hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased

serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice

In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could


17

prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose

homeostasis most likely by augmentation of FA consumption associated with reduction of

TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of

ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of

treatment for NAFLD and its complications

ARTICLE INFORMATION

Acknowledgments

We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical

Research Center South Korea) for help with the analysis of hepatic metabolites using LC-

MSMS

Funding

This research was supported by the National Research Foundation of Korea (NRF) grant

awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the

Korea Health Technology RampD Project through the Korea Health Industry Development

Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea

(HI16C1501)

Duality of Interest

No potential conflicts of interest relevant to this article were reported

Author contributions


18

YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and

wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the

experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL

analyzed and discussed the data

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13 Dunford EC Herbst EA Jeoung NH et al PDH activation during in vitro muscle

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14 Klyuyeva A Tuganova A Popov KM The carboxy-terminal tail of pyruvate

dehydrogenase kinase 2 is required for the kinase activity Biochemistry 20054413573-

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15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase

kinase-4 structures reveal a metastable open conformation fostering robust core-free basal

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16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system

for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514

17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate

dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high

saturated fat diet FEBS J 20122791883-1893

18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and

hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839

19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-

alcoholic fatty liver disease J Biol Chem 200427932345-32353

20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the

pathology of diet-induced hepatic insulin resistance and fatty liver J Lipid Res

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21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin

Invest 20121221958-1959

22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science

201233741-43

23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure

mechanism and regulation of pyruvate carboxylase Biochem J 2008413369-387


injury and hyperglycemia J Clin Invest 20141245175-5190

25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and

starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-

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26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-


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related diseases Rev Endo Metabol 20141599-110

27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-

13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an

insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524

28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents

hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745

29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in

nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385

Figure Legends

Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2

(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n

= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The

bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic

expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)

Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16

weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver

of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show

quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005

and

p lt 001

Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD

(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and

representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)


22

Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)

obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)

(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and

PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005

p lt 001 and

p

lt 0001

Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic

Glucose Production and Insulin Resistance Induced by a HFD

(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and

PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)

hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (

p lt 001 WT-

HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a

HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G

and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and

PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show

quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005

p lt 001 and

p lt 0001

Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and

Malonyl-CoA and Increases Ketogenesis

(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and

succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =

6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA

(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production


23

rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of

[U-13

C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-

oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean

plusmn SEM p lt 005

p lt 001

p lt 0001

Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis

in Mouse Primary Hepatocytes

(A) The incorporation of 13

C atoms from 13

C6-glucose to acetyl-CoA[M+2] (PDC flux red)

aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]

succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary

hepatocytes were treated with 25 mM 13

C6-glucose and 2 mM sodium octanoate for 2 h (n =

5) The enrichment of 13

C isotopomer were determined by LC-MS (B) Oxidation of [1-14

C]

sodium octanoate to 14

CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and

PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)

with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were

determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in

the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM

p lt

001

p lt 0001

Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents

Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice

(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance

of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels

of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and


24

malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and

H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or

Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =

5) All data are presented as the mean plusmn SEM p lt 005

p lt 001

p lt 0001

Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via

PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice


A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005

WT PDK2 KO WT PDK2KO

LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT

WT PDK2 KOWT PDK2 KO

LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD

LFD HFD LFD HFD LFD HFD LFD HFD

G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2

-oxidationketogenesis

E

PGC1 PGC1 CPT1-L


Figure 5

A

C

Control Octanoate OctanoateGlutamine

-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7

Induced pathwayReduced pathway




Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang

Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-

Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee

Robert A Harris and In-Kyu Lee

SUPPLEMENTARY MATERIALS

Primary Hepatocyte Culture

Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice

by the collagenase perfusion method described previously (1) To measure 14

CO2 production

primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-

Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200

mCimmol [1-14

C]sodium octanoate (American Radiolabeled Chemicals) 14

CO2 was

collected after incubation for 2 h For stimulation of ketone body production cells were

incubated in media containing 2 mM sodium octanoate for 4 h (2)

13C-Isotopomer labeling studies

Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM

medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed

with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate

(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer


25 mM of [U-13

C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium

octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then

collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite

extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After

incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm

for 5 min All the samples were lysophilized and resuspended in 300 l of water containing

01 formic acid prior to LC-MSMS analysis Analytes were separated on a

pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera

coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase

consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected

ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information

and raw spectrum intensity data of which was extracted respectively from within a retention

time range of each multiple reaction monitoring (MRM) scan performed simultaneously

Subsequently 13

C-MIDs (mass isotopomer distributions) were determined and corrected for

natural isotope abundance from the SIM scan data of target metabolite isotopomer

Western Blot Analysis

Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20

mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL

EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and

phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay

Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and

transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary

antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)

phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)


PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α

(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)

Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)

Quantitative Real-Time RT-PCR

Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according

to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a

cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the

Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR

system (Applied Biosystems) 36B4 was used for normalization The primer sequences are

given in the Supplementary Table 1

Measurement of Metabolites in Liver

Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization

with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic

pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods

Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP

acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem

mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard

such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels

were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were

assessed by LC-MSMS as described previously (11) with a bench-top tandem mass

spectrometer


Glucose and Pyruvate Tolerance Tests

Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip

injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood

glucose was measured with an Accu-Chek glucometer (Roche)

Measurement of Serum Parameters

Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical

analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)

Measurement of Energy Expenditure Using an Indirect Calorimetry System

Food intake physical activity CO2 production rates O2 consumption rates and energy

expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE

systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark

cycles at 22 plusmn 2degC

Hyperinsulinemic-Euglycemic Clamp Study

Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA

USA) were inserted into the right internal jugular veins of anesthetized mice After overnight

fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the

jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp

was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1

min-1

and 20 glucose was infused at a variable rate to maintain euglycemia To determine

insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-

3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h

before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected


via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2

(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the

[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of

the clamp Hepatic glucose production during the clamps was determined by subtracting the

glucose infusion rate from the whole body glucose uptake rate

References

1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of

fasting glucose metabolism Nature 20054371109-1111

2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls

fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104

3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous

extraction of proteins and metabolites from cells in culture MethodsX 2014174-80






6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate

In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145

7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-

hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290

8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of


enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608

9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic

analysis in Bergmeyer HU(ed) 1974 p1562-1565

10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic

systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG

mice Anal Bioanal Chem 20114001895-1904

11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel

transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic

insulin signaling J Biol Chem 201128638035-38042

12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle

blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-

E409


Supplementary Table 1 Primer sequences used for real-time RT-PCR

Gene Forward primer (5-3) Reverse primer (5-3)

PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC

GGAGCAATCCCAGCAGTGAA

GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT

TGATCTTGTCCTGTTTAGCCTTGT

PDK4

TNFa

MCP-1

PAI-1

CCATGAGAAGAGCCCAGAAGA

GACGTGGAACTGGCAGAAGAG

ACCTGGATCGGAACCAAATG

CACCCCTTCCAGAGTCCCATA

GAACTTTGACCAGCGTGTCTACAA

CCGCCTGGAGTTCTGGAA

CCTTAGGGCAGATGCAGTTTTAA

GCTGAAACACTTTTACTCCGAAGTT

SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT

FAS

ACC1

ACCTGGTAGACCACTGCATTGAC

CGCTCAGGTCACCAAAAAGAAT

CCTGATGAAACGACACATTCTCA

GGGTCCCGGCCACATAA

SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT

PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA

PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA

CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG

PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT

HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT

36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC


Supplementary Fig 1

Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of

heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD


Supplementary Fig 2

Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2

KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-

fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented

as the mean plusmn SEM p lt 005


Supplementary Fig 3

Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a

HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in

WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt

005 p lt 001


Supplementary Fig 4

Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT

and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-

fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the

mean plusmn SEM p lt 005

p lt 001

p lt 0001


Supplementary Fig 5

Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD

Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and

PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the

protein bands detected by immunoblot analysis


Supplementary Fig 6

Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic

lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO

mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001


Supplementary Fig 7

Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and

PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on

overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented


p lt 001

p lt 0001


2

Daegu South Korea

Contact

In-Kyu Lee MD PhD

Division of Endocrinology and Metabolism

Kyungpook National University School of Medicine

50 Samduck-2Ga Jung-Gu Daegu South Korea 700-721

Tel +82-53-420-5564 Fax +82-53-426-2046 E-mail leeiknuackr

Robert A Harris PhD

Roudebush VA Medical Center and the Department of Biochemistry and Molecular Biology

Indiana University School of Medicine Indianapolis IN 46202

Tel + 327-440-8941 Fax + 317-988-3180 E-mail raharrisiuedu

Additional Footnotes

10Present address Department of Internal Medicine Soonchunhyang University Gumi

Hospital Gumi South Korea

11Co-first author

12Co-corresponding author


3

ABSTRACT

















INTRODUCTION







4


























5






Animal Experiments


















6










liquid nitrogen












7

RESULTS
























8
























9


Production
























10












a HFD













11














([13













12








Ketogenesis





C6-













13

of [14

C1]-octanoate to 14












(1415)













14









DISCISSION














to HFD-fed WT mice


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


3

ABSTRACT

















INTRODUCTION







4


























5






Animal Experiments


















6










liquid nitrogen












7

RESULTS
























8
























9


Production
























10












a HFD













11














([13













12








Ketogenesis





C6-













13

of [14

C1]-octanoate to 14












(1415)













14









DISCISSION














to HFD-fed WT mice


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


4


























5






Animal Experiments


















6










liquid nitrogen












7

RESULTS
























8
























9


Production
























10












a HFD













11














([13













12








Ketogenesis





C6-













13

of [14

C1]-octanoate to 14












(1415)













14









DISCISSION














to HFD-fed WT mice


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


5






Animal Experiments


















6










liquid nitrogen












7

RESULTS
























8
























9


Production
























10












a HFD













11














([13













12








Ketogenesis





C6-













13

of [14

C1]-octanoate to 14












(1415)













14









DISCISSION














to HFD-fed WT mice


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


6










liquid nitrogen












7

RESULTS
























8
























9


Production
























10












a HFD













11














([13













12








Ketogenesis





C6-













13

of [14

C1]-octanoate to 14












(1415)













14









DISCISSION














to HFD-fed WT mice


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


7

RESULTS
























8
























9


Production
























10












a HFD













11














([13













12








Ketogenesis





C6-













13

of [14

C1]-octanoate to 14












(1415)













14









DISCISSION














to HFD-fed WT mice


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


8
























9


Production
























10












a HFD













11














([13













12








Ketogenesis





C6-













13

of [14

C1]-octanoate to 14












(1415)













14









DISCISSION














to HFD-fed WT mice


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


9


Production
























10












a HFD













11














([13













12








Ketogenesis





C6-













13

of [14

C1]-octanoate to 14












(1415)













14









DISCISSION














to HFD-fed WT mice


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


10












a HFD













11














([13













12








Ketogenesis





C6-













13

of [14

C1]-octanoate to 14












(1415)













14









DISCISSION














to HFD-fed WT mice


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


11














([13













12








Ketogenesis





C6-













13

of [14

C1]-octanoate to 14












(1415)













14









DISCISSION














to HFD-fed WT mice


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


12








Ketogenesis





C6-













13

of [14

C1]-octanoate to 14












(1415)













14









DISCISSION














to HFD-fed WT mice


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


13

of [14

C1]-octanoate to 14












(1415)













14









DISCISSION














to HFD-fed WT mice


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


14









DISCISSION














to HFD-fed WT mice


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


15



























16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


16


























17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


17






ARTICLE INFORMATION

Acknowledgments



MSMS

Funding





(HI16C1501)

Duality of Interest




18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


18





REFERENCES


Clin Invest 2004114147-152










Metab 2008295E46-54








19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


19



Biophys 20003811-7









2011531175-1181



201114804-810






13582





20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


20












2012531080-1092


Invest 20121221958-1959


201233741-43







383



21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


21









Figure Legends










and

p lt 001





22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


22





p lt 001 and

p

lt 0001






p lt 001 WT-






p lt 001 and

p lt 0001








23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


23


[U-13



plusmn SEM p lt 005

p lt 001

p lt 0001




C atoms from 13








C]







p lt

001

p lt 0001







24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


24





p lt 001

p lt 0001




A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


A B

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

LFD HFD

C

Rel

ativ

e m

RN

A l

eve

ls

PDK1 PDK2 PDK3 PDK4

mouse liver

D E

FWT-HFD PDK2 KO-HFD

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK1

PDK2

PDK3

PDK4

-tubulin

mouse liver

Hep

atic

PD

C a

ctiv

ity

(Ug

wei

gh

t)

LFD HFD

PDK1

LFD HFD

PDK2

PDK3

PDK4

-tubulin

mouse liver

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S29

3)P

DH

E1

pP

DH

E1

(S30

0)P

DH

E1

Figure 1


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


Figure 2

A B

Bo

dy

wei

gh

t (g

)

Weeks

p lt 005


LFD HFD

Liv

erb

od

y w

eig

ht

()

E

LFD HFD

D

Ser

um

AS

T (

UL

)

LFD HFD

LFD HFD

Liver

EAT


LFD HFD

Hep

atic

TG

(m

g

g t

issu

e)

Ser

um

AL

T (

UL

)

LFD HFD

F

C

G

Hep

atic

gly

cog

en

(mg

g t

issu

e)

LFD HFD


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


Figure 3

D

E F

Time (min)

pA

KT

AK

T

pF

oxO

1F

ox

O1

WT-HFD PDK2 KO-HFD

pFoxO1 (S256)

FoxO1

pAKT (S473)

AKT

Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +

A B C

Blo

od

glu

cose

(m

gd

L)

GIR

(m

gk

gm

in)

Bas

al H

GP

(m

gk

gm

in)

Cla

mp

HG

P (

mg

kg

min

)

Glu

cose

tu

rno

ver

(mg

kg

min

)

Insulinstimulated

Insulinstimulated

DA

G (

nm

olg

tis

sue)

pPKC (S729)

PKC

-tubulin

WT-HFD PDK2 KO-HFD

mouse liver

mouse liver

Blo

od

glu

cose

(m

gd

L)

LFD HFD

Ser

um

in

sulin

(n

gm

L)

LFD HFD

Blo

od

glu

cose

(m

gd

L)

Time (min)

AU

C (

mg

dL

x m

in)

LFD HFD


G H

pP

KC(

S72

9)P

KC


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


Figure 4

B

A

C

Hep

atic

-H

B

(m

olg

tis

sue)

-H

B p

rod

uct

ion

rat

e(m

gm

ink

g)

Hep

atic

OA

A

(m

olg

tis

sue)

LFD HFD

Hep

atic

cit

rate

(

mo

lg t

issu

e)

LFD HFD

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

LFD HFD

LFD HFD

Hep

atic

ace

tyl-

Co

A

(AU

)

LFD HFD

Hep

atic

AT

P (

AU

)

LFD HFD

Hep

atic

mal

on

yl-C

oA

(A

U)

LFD HFD

D

LFD HFD

Rel

ativ

e m

RN

A l

eve

ls

PPAR HMGCS2


E

PGC1 PGC1 CPT1-L


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


Figure 5

A

C


-H

B p

rod

uct

ion

rat

e(

mo

lmin

mg

pro

tein

)

14C

O2

pro

du

ctio

n

(dp

m1

5 x

106

cells

)

B

D

E

nri

chm

ent

PDC flux PC flux

E

nri

chm

ent

E

nri

chm

ent

TCA cycle flux


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


Figure 6

D

Hep

atic

OA

A

(m

olg

tis

sue)

Hep

atic

cit

rate

(

mo

lg t

issu

e)

Hep

atic

su

ccin

ate

(m

olg

tis

sue)

E

Hep

atic

-H

B

(m

olg

tis

sue)

F

Hep

atic

ace

tyl-

Co

A

(AU

)

Hep

atic

A

TP

(A

U)

Hep

atic

m

alo

nyl

-Co

A(A

U)

GB

loo

d g

luco

se (

mg

dL

)

H

Ser

um

in

sulin

(n

gm

l)

I

Blo

od

glu

cose

(m

gd

L)

AU

C (

mg

dL

x m

in)

pPDHE1 (S293)

pPDHE1 (S300)

PDHE1

PDK2

-tubulin

mouse liver

B C

Hep

atic

TG

(m

gg

tis

sue)

A

Ad-GFP Ad-PDK2 DN


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


Figure 7













CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001












CO2 production



mCimmol [1-14


CO2 was









25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


25 mM of [U-13














Subsequently 13

































spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001






















spectrometer




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001




















min-1











References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001







References





























E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001













E409




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001




PDK1

PDK2

PDK3

CACCACGCGGACAAAGG

CCCCGTCCCCGTTGTC


GCCCAGCGTGACGTGAA

TCGCAGGCATTGCTGGAT


PDK4

TNFa

MCP-1

PAI-1






CCGCCTGGAGTTCTGGAA




FAS

ACC1




GGGTCCCGGCCACATAA









Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


Supplementary Fig 1




Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


Supplementary Fig 2






Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


Supplementary Fig 3




005 p lt 001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


Supplementary Fig 4





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


Supplementary Fig 5






Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


Supplementary Fig 6





Supplementary Fig 7





p lt 001

p lt 0001


Supplementary Fig 7





p lt 001

p lt 0001


Documents

Inhibition of pyruvate dehydrogenase kinase 2 protects ... · TCA cycle to generate ATP to meet hepatic energy demands (4). Whether acetyl-CoA produced by β-oxidation forms ketone