Activation of Pyruvate Dehydrogenase by Sodium Dichloroacetate

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Activation of Pyruvate Dehydrogenase by Sodium Dichloroacetate Shifts Metabolic Consumption from Amino Acids to Glucose in IPEC-J2 Cells and Intestinal Bacteria in Pigs Rui An, Zhiru Tang, Yunxia Li, Tiejun Li, Qingqing Xu, Jifu Zhen, Feiru Huang, Jing Yang, Cheng Chen, Zhaoliang Wu, Mao Li, Jiajing Sun, Xiangxin Zhang, Jinchao Chen, Liuting Wu, Shengjun Zhao, Qingyan Jiang, Weiyun Zhu, Yulong Yin, and Zhihong Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05800 • Publication Date (Web): 23 Feb 2018 Downloaded from http://pubs.acs.org on February 24, 2018

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Journal of Agricultural and Food Chemistry

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Running title: Shifting from AA to glucose metabolism using PDH

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Title: Activation of Pyruvate Dehydrogenase by Sodium Dichloroacetate Shifts

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Metabolic Consumption from Amino Acids to Glucose in IPEC-J2 Cells and Intestinal

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Bacteria in Pigs

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Authors:

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Rui An,† Zhiru Tang,†,△ Yunxia Li,‡,△Tiejun Li,§,△Qingqing Xu,† Jifu Zhen,† Feiru Huang,ǁ

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Jing Yang,† Cheng Chen,† Zhaoliang Wu,† Mao Li,† Jiajing Sun,† Xiangxin Zhang,† Jinchao

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Chen,† Liuting Wu,† Shengjun Zhao,⊥Qingyan Jiang,#,* Weiyun Zhu,�,* Yulong Yin,§,*

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Zhihong Sun†,*

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Affiliations:

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†

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Technology, Southwest University, Chongqing 400715, P. R. China.

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‡

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China.

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§

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P. R. China.

Laboratory for Bio-feed and Molecular Nutrition, College of Animal Science and

Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu 611130, P. R.

Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Changsha 410125,

ACS Paragon Plus Environment


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ǁ

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430070, P. R. China.

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College of Animal Science and Technology, Huazhong Agricultural University, Wuhan

⊥

School of Animal Science and Nutritional Engineering, Wuhan Polytechnic University,

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Wuhan 430023, P. R. China.

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#

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510642, P. R. China.

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�

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210095, P. R. China.

College of Animal Science and Technology, Huanan Agricultural University, Guangzhou

College of Animal Science and Technology, Nanjing Agricultural University, Nanjing

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△

Equally to first author.

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*

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J.); [email protected] (Y. L. Y.); [email protected] (Z.H.S.).

Corresponding author. [email protected] (W. Y. Z.); [email protected] (Q. Y.

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ABSTRACT

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The extensive metabolism of amino acids (AA) as fuel is an important reason for the low

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use efficiency of protein in pigs. In this study, we investigated whether regulation of the

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pyruvate dehydrogenase kinase (PDK)/pyruvate dehydrogenase alpha 1 (PDHA1)

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pathway affected AA consumption by porcine intestinal epithelial (IPEC-J2) cells and

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intestinal bacteria in pigs. The effects of knockdown of PDHA1 and PDK1 with small

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interfering RNA (siRNA) on nutrient consumption by IPEC-J2 cells were evaluated.

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IPEC-J2 cells were then cultured with sodium dichloroacetate (DCA) to quantify AA and

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glucose consumption and nutrient oxidative metabolism. The results showed that

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knockdown of PDHA1 using siRNA decreased glucose consumption but increased total

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AA (TAA) and glutamate (Glu) consumption by IPEC-J2 cells (P < 0.05). Opposite

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effects were observed using siRNA targeting PDK1 (P < 0.05). Additionally, culturing

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IPEC-J2 cells in the presence of 5 mM DCA markedly increased the phosphorylation of

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PDHA1 and PDH phosphatase 1, but inhibited PDK1 phosphorylation (P < 0.05). DCA

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treatment also reduced TAA and Glu consumption and increased glucose depletion (P
0.05; Fig. 3F).

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Culturing intestinal (cecal) bacteria with DCA increased glucose consumption but reduced

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TAA and Glu consumption (P < 0.05; Fig. 3G). The GDH2 activity in intestinal bacteria

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was decreased by DCA addition (P < 0.05), whereas GDH1 activity in intestinal bacteria

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was increased (P < 0.05; Fig. 3H).



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Discussion

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The activity of PDH is closely related to glucose metabolism.39,40 PDK is an inhibitor of

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PDH and a key regulatory enzyme in glucose metabolism.41–43 PDK activation inhibits

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PDH, decreasing the contribution of glucose oxidation to the tricarboxylic acid (TCA)

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cycle.44 The regulatory role of PDH/PDK in glucose metabolic homeostasis has been

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extensively studied in cancer cells.41 The results in this study showed that siRNA targeting

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PDHA1 decreased glucose consumption by IPEC-J2 cells, but increased TAA and Glu

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consumption. Moreover, siRNA targeting PDK1 had opposite effects on glucose, TAA, and

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Glu consumption by cells. Glucose and AAs, which are important sources of cellular

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energy, compete and interact with each other.45,46 The capacity for tissues or organs to

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preferentially utilize glucose and AA as fuels and to be able to rapidly switch between them

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is termed metabolic flexibility. The results in this study indicated that regulating PDH was

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indeed an appropriate measure for controlling cellular glucose and AA catabolism in the

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intestinal epithelial cells of pigs.

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Having established that PDH was a suitable target, we then successfully reprogrammed

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AA and glucose metabolism in IPEC-J2 cells using DCA. Specifically, we found that

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culturing IPEC-J2 with 5 mM DCA facilitated PDH activity in the cells by decreasing the

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phosphorylation of PDK1 and enhancing the phosphorylation of PDP1, thereby leading to a

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metabolic shift from AA to glucose/pyruvate oxidation and a decrease in AA consumption


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by cells. DCA, which is an inhibitor of PDK,47 binds to the N-terminal domain of PDK and

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promotes conformational changes, leading to the inactivation of kinase activity and

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reactivating glucose oxidation.48 DCA addition also caused increases in mRNA and protein

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expression of GLUT1 and GLUT4 in cells, indicating that DCA increased the uptake of

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glucose from culture medium. These changes were consistent with the results of glucose

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oxidation and consumption. Culturing cells with DCA had no effect on the intracellular

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concentrations of citric acid, oxalacetic acid, fumaric acid, and malic acid, suggesting that

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DCA did not change the supply of metabolites in the TCA cycle, despite causing a

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metabolic shift in cells from AA to glucose/pyruvate oxidation. We also observed that

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culturing intestinal bacteria with DCA increased glucose consumption but reduced TAA

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and Glu consumption. Among AAs, Glu is the most important contributor to intestinal

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oxidative energy metabolism;49 GDH1 is responsible for catalyzing α-ketoglutarate to Glu,

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whereas GDH2 has the opposite role. In this study, changes in GDH2 and GDH1 activities

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in intestinal bacteria in response to DCA addition were consistent with the results of AA

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consumption by intestinal bacteria. In contrast to IPEC-J2 cells, we only measured the

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effects of DCA on the consumption of glucose and AAs by intestinal bacteria and the

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GDH2 and GDH1 activities in intestinal bacteria, and did not detect the expression (or

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phosphorylation) of PDK and PDH following treatment with DCA. The influences of PDK

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and PDHA1 knockdown on glucose and AA catabolism in bacteria were also not measured

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because the components of fecal bacteria are very complex, making such samples



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unsuitable for these analyses. Our findings are consistent with previous studies showing

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that DCA treatment enhanced pyruvate flux into the mitochondria and promoted glucose

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oxidation.48,50

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DCA, which is a mitochondria-targeting small molecule that can penetrate most tissues

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after oral administration,51 has been used to treat congenital lactic acidosis in mitochondrial

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diseases for over 30 years.52–56 In addition, DCA was found to reverse the Warburg effect

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and induce apoptosis in tumor cells by increasing the flux of pyruvate into the

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mitochondria and promoting glucose oxidation.47,51 DCA is a generic agent that is both

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inexpensive and safe for human use;52,54–56 indeed, DCA does not induce renal, pulmonary,

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hepatic, bone marrow, or cardiac toxicity.55 Most of the side effects of DCA are mild, with

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the most serious side effect being reversible peripheral neuropathy.57 Because of its safety

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and low price, DCA should be suitable for use in a low-protein diet to improve the

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efficiency of AA use and reduce porcine nitrogen excretion.

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In conclusion, our data suggested that the PDK/PDH axis was a regulatory target for

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shifting AA oxidation to pyruvate/glucose metabolism. Culturing cells with DCA increased

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PDHA1/PDP1 phosphorylation while inhibiting PDK1 phosphorylation. Addition of DCA

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also reduced AA consumption by test cells and intestinal bacteria while increasing glucose

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depletion. The results indicate that DCA may be suitable as a dietary additive to decrease

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nitrogen excretion by increasing glucose depletion.

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ACKNOWLEDGEMENTS

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We gratefully acknowledge the National Science and Technology Major Project of China

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(2017YFD0500504), the National “948” Project from the Ministry of Agriculture of China

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(2015Z74), and the National Natural Science Foundation of China (31772610).

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Notes

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The authors declare no competing financial interest.

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(53) Kuroda, Y.; Ito, M.; Toshima, K.; Takeda, E.; Naito, E.; Hwang, T. J.; Hashimoto, T.;

512

Miyao, M.; Masuda, M.; Yamashita, K.; Adachi, T.; Suzuki, Y.; Nishiyama, K. Treatment of

513

chronic congenital lactic acidosis by oral administration of dichloroacetate. J. Inherit.

514

Metab. Dis. 1986, 9, 244–252

515

(54) Stacpoole, P. W.; Barnes, C. L.; Hurbanis, M. D.; Cannon, S. L.; Kerr, D. S. Treatment

516

of congenital lactic acidosis with dichloroacetate. Arch. Dis. Child. 1997, 77, 535–541.

517

(55) Stacpoole, P. W.; Kerr, D. S.; Barnes, C.; Bunch, S. T.; Carney, P. R.; Fennell, E. M.;

518

Felitsyn, N. M.; Gilmore, R. L.; Greer, M.; Henderson, G. N.; Hutson, A. D.; Neiberger, R.

519

E.; O'Brien, R. G.; Perkins, L. A.; Quisling, R. G.; Shroads, A. L.; Shuster, J. J.; Silverstein,

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J. H.; Theriaque, D. W.; Valenstein, E. Controlled clinical trial of dichloroacetate for


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treatment of congenital lactic acidosis in children. Pediatrics 2006, 117, 1519–1531.

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(56) Stacpoole, P. W.; Gilbert, L. R.; Neiberger, R. E.; Carney, P. R.; Valenstein, E.;

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Theriaque, D. W.; Shuster, J. J. Evaluation of long-term treatment of children with

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congenital lactic acidosis with dichloroacetate. Pediatrics 2008, 121, e1223–e1228.

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(57) Kaufmann, P.; Engelstad, K.; Wei, Y.; Jhung, S.; Sano, M. C.; Shungu, D. C.; Millar,

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DiMauro, S.; De Vivo, D. C. Dichloroacetate causes toxic neuropathy in MELAS: a

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randomized, controlled clinical trial. Neurology 2006, 66, 324–330.

529 530 531 532 533 534 535 536 537 538 539 540



541

Page 28 of 36

Table 1. Primer sequences for amplification of genes in the mucosa of the jejunum Primer sequence

Length of PCR product (bp)

GLUT1

Sense: 5’-CTGTCGTGTCGCTGTTCGT-3’

141

Antisense: 5’-TCAGGTAGGACATCCAGGGTA-3’ GLUT4

Sense: 5′-TCCTGATGACTGTGGCTCTG-3’

235

Antisense: 5′-TCCGCAACATACTGGAAACT-3’ PDHA1

Sense: 5’-GTCAGGAAGCTTGTTGCGTG-3’

208

Antisense: 5’-CCATTGCCCCCGTAGAAGTTPDK1

Sense: 5’-AGAGTTGCCCGTTAGGTTGG -3’

240

Antisense: 5’-ACCACACCTTGAGCCATTGT3’ GAPDH

Sense: 5’-GGTCGGAGTGAACGGATTT-3’ Antisense: 5’-ATTTGATGTTGGCGGGAT-3’

542 543


245

Page 29 of 36


A

B 120

120

a

100 Relative level (%)

Relative level (%)

100

a

80 60

b

40 20 0

80 60 b 40 20 0

siCtrol

siPDHA1

siCtrol

mRNA expression of PDHA1

mRNA expression of PDK1

C

D

30

***

20 10 Glucose 0 TAA -10 -20

30

***

**

Glutamate

% change of nutrient consuption by siRNA against PDK1

% change of nutrient consuption by siRNA against PDHA1

40

544

siPDK1

**

20 10

TAA

Glutamate

0 -10

Glucose

-20 -30 -40 -50

545 546 547 548 549 550 551


*** ***


Page 30 of 36

552

Figure 1. Effects of siRNA targeting PDHA1 and PDK1 on cellular nutrient consumption.

553

(A and B) Intracellular expression of PDHA1 (A) and PDK1 mRNAs (B) in IPEC-J2 cells

554

transfected with either nontargeted siRNA (siCtrl) or siRNA against PDHA1 or PDK1. (C

555

and D) siRNA against PDHA1 (C) and PDK1 (D) altered the nutrient consumption profile

556

by cells. n = 6 for A and B. All data are means ± SEM.

557

superscripted letters within the same index indicate significant difference (P < 0.05). ***

558

and ** indicate P < 0.001 and P < 0.01, respectively.

559 560


a,b

Values with different

Page 31 of 36


A

B 5 mM DCA

0 mM DCA PDK1

75 KD

p-PDK1

p-PDK1

50KD 55 KD

PDK1

43-KD 55-KD

% change by 5 mM DCA

0

β-Actin

40-KD

NS

-20 -40 -60 -80 -100

C

***

D 5 mM DCA

80-KD

400.0

p-PDP1

60-KD 72-KD PDP1 55-KD 55-KD


0 mM DCA

300.0 200.0 100.0 NS 0.0

β-Actin

40-KD

***

PDP1

p-PDP1

F

E 5 mM DCA

0 mM DCA

500.0

p-PDHA1

38-KD 55-KD 43-KD

PDHA1

55-KD 40-KD

β-Actin


***

50-KD

400.0 300.0 200.0 100.0

NS

0.0

561 562


PDHA1

p-PDHA1


563

Figure 2. Effects of culturing cells with 5 mM DCA on PDK1, PDHA1, and PDP1 protein

564

expression and phosphorylation in cells. (A–F) Cells were cultured with DCA (5 mM), and

565

the phosphorylation and expression of PDK1 (A and B), PDP1 (C and D), and PDHA1 (E

566

and F) were evaluated. n = 6 for A–F. All data are means ± SEM. NS and ** indicate P >

567

0.05 and P < 0.01, respectively.

568 569 570 571 572 573 574 575 576 577 578 579 580 581


Page 32 of 36

Page 33 of 36


A

B 5 mM DCA 0 mM DCA

% changes of mRNA concentration by 5 mM DCA

100 *** 80

50-KD

GLUT4

*** 60

37-KD 75-KD 50-KD

40 20

55-KD 0 GLUT1

GLUT4

β-acti n

40-KD

D

C

60

450 ***

400

% change of nutrient oxidaation by 5 mM DCA

% changes of protein expression by 5 mM DCA

GLUT1

350 300 250 200 ***

150 100 50

*** ***

40 Glutamate oxidation

20 0 Pyruvate oxidation

-20

Glucose oxidation

-40

0 GLUT4

F

% change of nutrient consumption by 5 mM DCA

50 40

***

30 20 TAA

10

Glutamate

0 -10

Glucose

-20 -30 -40 -50

*** ***

% change of the concentrations of cellular metabolites by 5 mM DCA

E

***

-60

GLUT1

10 8

NS NS

6 4

NS

2

Oxalacetic acid

0 -2

Citric acid

Fumaric acid

-4 -6

582


NS

Malic acid


G

H 60

150

***

*** 40 TAA

20

Glutamate

0 -20

Glucose

-40 -60 -80 -100

***

% change of GDH1 and GDH2 activity by 5 mM DCA

% change of nutrient consumption by 5 mM DCA

Page 34 of 36

100 50

GDH2

0 GDH1 -50 -100 -150

***

*** -200

583 584 585 586 587 588 589 590 591 592 593 594 595 596


Page 35 of 36


597

Figure 3. Shifting AA consumption to glucose metabolism by culturing IPEC-J2 cells and

598

cecal bacteria with DCA. (A–C) IPEC-J2 cells were cultured with 5 mM DCA, and

599

changes in mRNA (A) and protein expression (B and C) of GLUT1 and GLUT2; pyruvate,

600

glucose, and Glu oxidation (D); and glucose, TAA, and glucose consumption (E) were

601

evaluated. (F) Citric acid cycle metabolite contents were determined in IPEC-J2 cells after

602

DCA addition. (G) Effects of DCA addition on glucose, TAA, and glucose consumption by

603

intestinal bacteria. (H) Percent changes of GDH1 and GDH2 activities in intestinal bacteria

604

following the addition of 5 mM DCA. n = 6 for A–F. All data are means ± SEM. NS and

605

*** indicate P > 0.05 and P < 0.001, respectively.

606 607 608 609 610 611 612 613 614 615 616



617

Page 36 of 36

Shifting AA oxidation to glucose metabolism DCA

Pyruvate/Glucose

618 PDK

PDH

DCA

PDP

NH3

619

GDH1

Glu

GDH2 DCA AA except Glu/Gln

Precursors for urea


Activation of Pyruvate Dehydrogenase by Sodium Dichloroacetate

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