Cell Metabolism, Volume 9

Supplemental Data

Fasting-Induced Hypothermia and Reduced Energy Production in Mice Lacking Acetyl-CoA Synthetase 2

Iori Sakakibara, Takahiro Fujino, Makoto Ishii, Toshiya Tanaka, Tatsuo Shimosawa, Shinji Miura, Wei Zhang, Yuka Tokutake, Joji Yamamoto, Mutsumi Awano, Satoshi Iwasaki, Toshiyuki Motoike, Masashi Okamura, Takeshi Inagaki, Kiyoshi Kita, Osamu Ezaki, Makoto Naito, Tomoyuki Kuwaki, Shigeru Chohnan, Tokuo Yamamoto, Robert E. Hammer, Tatsuhiko Kodama, Masashi Yanagisawa, and Juro Sakai

Supplemental Experimental Procedures

Assay Procedure for Malonyl-CoA : Acetyl-CoA Cycling

Mouse tissues were quickly (<1 min) removed and frozen in liquid nitrogen. Frozen

tissue was weighed, pulverized, transferred to 1 ml of ice-cold 0.3 M sulfuric acid,

sonicated for 10 min and centrifuged at 10,000 x g for 5 min at 4°C. Supernates were

carefully adjusted to pH 6.0 on ice. The reaction mixture for malonyl-CoA, which is

based on the methods of Takamura et al. (Cha et al., 2005; Hu et al., 2003; Takamura et

al., 1985), contained the appropriate amount of neutralized tissue extract, 50 mM

Tris_HCl (pH 7.2), 10 mM magnesium sulfate, 1 mM 2-mercaptoethanol, 2 mM

oxaloacetate, and 1 unit of citrate synthase (Sigma) in a total volume of 200 μl. After 20

min at 25°C, the reaction was terminated by placing the tube in an ice slush. The

‘‘cycling’’ reaction is then initiated by addition of 50 mM malonate, 10 mM ATP, and

1.0 unit of malonate decarboxylase (from Pseudomonas putida; Takamura et al.) and

incubated at 30°C for 20 min followed by the addition of 1.0 unit of acetate kinase

(EC2.7.2.1). After 20 min of incubation, 100 μl of 2.5 M neutralized hydroxylamine

were added and the incubation was continued for an additional 20 min at 25°C. The

reaction was terminated by addition of 0.3 ml of 10 mM ferric chloride dissolved in 25

mM trichloroacetic acid and 1 M HCl. The quantity of acetylhydroxamate formed was

measured at A540. For measurement of acetyl-CoA, acetyl-CoA, which is based on

the methods of Takamura et al. (Cha et al., 2005; Hu et al., 2003; Takamura et al., 1985),

contained the appropriate amount of neutralized tissue extract, 50 mM Tris_HCl (pH

7.2), 10 mM magnesium sulfate, and 1 mM 2-mercaptoethanol in a total volume of 200

μl. After 20 min at 25°C, the reaction was terminated by placing the tube in an ice slush

and incubated at 30°C for 20 min followed by the addition of 1.0 unit of acetate kinase

(EC2.7.2.1). After 20 min of incubation, 100 μl of 2.5 M neutralized hydroxylamine

were added and the incubation was continued for an additional 20 min at 25°C. The

reaction was terminated by addition of 0.3 ml of 10 mM ferric chloride dissolved in 25

mM trichloroacetic acid and 1 M HCl. The quantity of acetylhydroxamate formed was

measured at O.D. 540.

Assay Procedure for Acetyl-CoA

Mouse tissues were quickly (<1 min) removed and frozen in liquid nitrogen. Frozen

tissue was weighed, pulverized, transferred to 1 ml of ice-cold 0.3 M sulfuric acid,

sonicated for 10 min and centrifuged at 10,000 x g for 5 min at 4°C. Supernates were

carefully adjusted to pH 6.0 on ice. The reaction mixture for measurement of

acetyl-CoA, which is based on the methods of Takamura et al. (Cha et al., 2005; Hu et

al., 2003; Takamura et al., 1985), contained the appropriate amount of neutralized tissue

extract, 50 mM Tris-HCl (pH 7.2), 10 mM magnesium sulfate, and 1 mM

2-mercaptoethanol in a total volume of 200 μl. After 20 min at 25°C, the reaction was

terminated by placing the tube in an ice slush and incubated at 30°C for 20 min

followed by the addition of 1.0 unit of acetate kinase (EC2.7.2.1). After 20 min of

incubation, 100 μl of 2.5 M neutralized hydroxylamine were added and the incubation

was continued for an additional 20 min at 25°C. The reaction was terminated by

addition of 0.3 ml of 10 mM ferric chloride dissolved in 25 mM trichloroacetic acid and

1 M HCl. The quantity of acetylhydroxamate formed was measured at O.D. 540.

Acetyl-CoA Carboxylase Activity

Acetyl-CoA carboxylase activity was determined by the H14CO3- fixation assay

(Nakanishi and Numa, 1970). The H14CO3- fixation assay follows the rate of

acetyl-CoA-dependent H14CO3- incorporation into acid-stable material, i. e.

malonyl-CoA. Tissue extract was first preincubated at 37°C for 30min in a mixture

(final volume, 1.0 ml) containing 50 mM Tris-C1 pH 7.5, 10 mM potassium citrate, 10

mM MgCl, 3.75 mM GSH and 0.75 mg/ml bovine serum albumin. The reaction was

then initiated by adding an aliquot of the preincubated enzyme to an assay mixture (final

volume, 0.8 ml) containing 50 mM Tris-HC1 pH 7.5, 10 mM potassium citrate, 10 mM

MgCl,, 3.75 mM ATP, 0.125 mM acetyl-CoA, 3.75 mM GSH, 0.75 mg/ ml bovine

serum albumin and 12.5 mM KHI4CO3 (specific activity, 3.0 x 105 counts x min-1 x

pmole-l). Following incubation at 37°C for 10 min, the reaction was terminated with

0.2 ml of 5 N HC1. After centrifugation, an aliquot of the supernatant fluid was taken to

dryness in a scintillation counting vial at 80°C for 45-60min. 0.5ml of water and 10ml

of scintillator solution were added, and 14C activity was determined with the use of a

liquid scintillation spectrometer.

NAD+ and NADH Measurements

NAD+ and NADH nucleotides concentration in skeletal muscle and BAT of 12 week old

male mice were directly measured by NAD+ /NADH Assay kit (Biochain Insitute, Inc.)

according to the manufacturer’s instructions. In brief, approximately 10 mg of frozen

tissue was homogenized in 100 μl of acid extraction buffer for NAD+ and 100 μl of

alkali extraction for NADH concentration measurement, respectively. After

neutralization, the concentration of nucleotides was measured at 565 nm by an alcohol

dehydrogenase cycling reaction. Values for both nucleotides were detected within the

linear range.

Measurement of Adenine Nucleotides Contents

The gastrocnemius muscle was homogenized from 12 week old male mice with 1.0 N

perchloric acid and centrifuged at 10,000 x g for 15 min. After neutralization of the

supernatant with calcium carbonate, adenine nucleotide concentrations were determined

by high performance liquid chromatography (Phenomenex Luna 5 μ NH2; mobile phase,

sodium phosphatase buffer; detector, 260 nm; Torrance, CA) as described previously

(Miura et al., 2006; Scott et al., 1992).

Body Composition Analysis

Mice were anesthesised with pentobarbital sodium, Nembutal (0.08 mg/g body weight;

Abbot Laboratories, Chicago, IL), and scanned with a Lunar PIXI mus2 densitometer

(Lunar Corp., Madison, WI), equipped for dual-energy X-ray absorptiometry (DEXA)

(Miura et al., 2006; Nagy and Clair, 2000).

Supplemental Reference

Cha, S.H., Hu, Z., Chohnan, S., and Lane, M.D. (2005). Inhibition of hypothalamic fatty acid synthase triggers rapid activation of fatty acid oxidation in skeletal muscle. Proc Natl Acad Sci U S A 102, 14557-14562. Hu, Z., Cha, S.H., Chohnan, S., and Lane, M.D. (2003). Hypothalamic malonyl-CoA as a mediator of feeding behavior. Proc Natl Acad Sci U S A 100, 12624-12629. Miura, S., Tomitsuka, E., Kamei, Y., Yamazaki, T., Kai, Y., Tamura, M., Kita, K., Nishino, I., and Ezaki, O. (2006). Overexpression of peroxisome proliferator-activated receptor gamma co-activator-1alpha leads to muscle atrophy with depletion of ATP. Am J Pathol 169, 1129-1139. Nagy, T.R., and Clair, A.L. (2000). Precision and accuracy of dual-energy X-ray absorptiometry for determining in vivo body composition of mice. Obes Res 8, 392-398. Nakanishi, S., and Numa, S. (1970). Purification of rat liver acetyl coenzyme A carboxylase and immunochemical studies on its synthesis and degradation. Eur J Biochem 16, 161-173. Scott, M.D., Baudendistel, L.J., and Dahms, T.E. (1992). Rapid separation of creatine, phosphocreatine and adenosine metabolites by ion-pair reversed-phase high-performance liquid chromatography in plasma and cardiac tissue. J Chromatogr 576, 149-154. Takamura, Y., Kitayama, Y., Arakawa, A., Yamanaka, S., Tosaki, M., and Ogawa, Y. (1985). Malonyl-CoA: acetyl-CoA cycling. A new micromethod for determination of acyl-CoAs with malonate decarboxylase. Biochim Biophys Acta 834, 1-7.

Figure S1. AceCS2-Deficient Mice Exhibited Growth Retardation during Milk Suckling (A) Body weight change and (B) body length of male and female mice fed a normal chow (male AceCS2+/+; n=9, AceCS2+/-; n=17, AceCS2-/-; n=7, female AceCS2+/+; n=9, AceCS2 +/-; n=17, AceCS2-/-; n=7). (C) Representative picture of 4 week old male AceCS2+/+ and AceCS2-/- mice. (D) Food intake/ body weight of male mice (4 or 20 weeks old) were measured (7-9 per genotype). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to AceCS2+/+.

Figure S2. Mitochondrial Morphogenesis and mRNA Expression Levels of Fed and 48 Hr Fasted Mice (A) Male mice (12 weeks old) were singly housed and given water ad libitum. Food was removed from cages in the morning. BAT and skeletal muscle analysis by transmission electron microscopy. Scale bar, 1 μm. (B) Relative mRNA expression levels of UCP1, SIRT3, PGC-1α and PPARδ in BAT of male mice (12 weeks old, 6-7 per genotype). **p < 0.01 compared to AceCS2+/+.

Figure S3. Relative mRNA Expression Levels (A) and Restoration of Hypoglycemia by Pyruvate (B) in LC/HF Diet Fed Male Mice (A) Relative mRNA expression levels of glucose 6 phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), and alanine aminotransferase 1 (ALT1) (liver), and branched chain aminotransferase 2 (BCAT2) (skeletal muscle) in LC/HF diet fed male mice (4 weeks old) and in 12 week old normal chow diet fed male mice, (6-7 per genotype). *p < 0.05 compared to AceCS2+/+. LC/HF, low carbohydrate high fat diet, NC, normal chow diet. (B) LC/HF diet fed male mice (4 weeks old) were received an i.p. injection of 500 mg/kg sodium pyruvate (WAKO) dissolved in water. Blood glucose levels were determined ***p < 0.05 compared to AceCS2+/+. (AceCS2-/-; n=5, AceCS2-/-; n=4).

Figure S4. Comparison of Plasma Parameter, Body Temperature, and Body Weight of AceCS2-/- Mice that Survived or Died while Fed an LC/HF Diet (A-E) Male mice (4 weeks old) were fed a ketogenic diet. (A) Core rectal temperature. (B) Blood glucose level. (C) Plasma NEFA level. (D) Plasma ketone body level. (E) Body weight. (AceCS2-/- dead; n=4, AceCS2-/- survived; n=3).

Figure S5. Malonyl-CoA Levels and ACC Activity of AceCS2-/- Mice Fed an LC/HF Diet AceCS2-/- and AceCS2+/+ mice (male, 26 week old) were fed a LC/HF diet. Malonyl-CoA levels and ACC activity of liver, BAT and SM were determined (AceCS2+/+; n=8, AceCS2-/-; n=8). ***p < 0.001 compared to AceCS2+/+. SM, skeletal muscle.

Figure S6. Oxygen Consumption of AceCS2-/- Mice Fed an LC/HF Diet Not Corrected for Body Weight Oxygen consumption (VO2, left panel), and average of VO2 (right panel) were determined in male mice (26 weeks old) fed a LC/HF diet by indirect calorimetry (AceCS2+/+; n=8, AceCS2-/-; n=7). The data was not corrected for body weight.

Table S1. Metabolic Parameters of AceCS2+/+ and AceCS2-/- Mice at 2 Weeks Old

and 26 Weeks Old

2-4 weeks old 26 weeks old

+/+ -/- +/+ -/-

Glucose (mg/dL) 125 ± 14 110 ± 12 261 ± 14 249 ± 10

Cholesterol (mg/dL) 70 ± 4 71 ± 2 45 ± 3 32 ± 1 *

Triglycerides (mg/dL) 75 ± 7 81 ± 11 75 ± 6 77 ± 4

NEFA (μEql/L) 387 ± 37 403 ± 41 354 ± 40 319 ± 58

Ketone body (mΜ) 0.489 ± 0.058 0.521 ± 0.052 0.205 ± 0.038 0.281 ± 0.062

Insulin (ng/mL) 0.36 ± 0.15 0.46 ± 0.14 2.30 ± 0.26 2.57 ± 0.96

Leptin (pg/mL)

Growth hormone (ng/ml)

IGF-1 (ng/ml)

Acetate (mM)

187 ± 25

1.40 ± 0.32

392 ± 60

0.09 ± 0.04

100 ± 12 *

1.37 ± 0.35

352 ± 41

2.20 ± 0.31

161 ± 29

NE

NE

NE

172 ± 56

NE

NE

NE

Male mice (26 weeks old, 7-9 per genotype) were fed a normal diet. Male mice (2-4

weeks old, n =7-9 per genotype) were fed milk from their mother. Assays of blood

samples were performed on isolated plasma. * p<0.05 compared to AceCS2+/+ mice.

NE, not examined.

Table S2. Body Composition, Food Intake, Weights, and Metabolic Parameters of

AceCS2-/- Mice at 12 Weeks Age

Fed Fasted Parameter

+/+ -/- +/+ -/-

Body Weight (g) 27.2 ± 1.2 25.3 ± 1.8* 20.8 ± 0.9 18.6 ± 1.5**

Lean Body Mass (g) 20.8 ± 2.0 17.1 ± 1.4* NE NE

Fat content (%) 14.3 ± 1.3 15.5 ± 2.2 NE NE

Epididymal fat (g) NE NE 0.052 ± 0.033 0.033 ± 0.02

BMD (mg/cm2) 45.6 ± 2.6 42.1 ± 2.2** NE NE

Food intake (g/day) 3.4 ± 0.8 2.8 ± 0.3 0 0

Glucose (mg/dL) 174 ± 15 185 ± 26 120 ± 22.9 113 ± 18

Cholesterol (mg/dL) 48.9 ± 7.71 47.2 ± 9.42 31.6 ± 7.57 33.3 ± 11.9

Triglycerides (mg/dL) 0.717 ± 0.20 0.662 ± 0.19 1.08 ± 0.29 0.979 ± 0.27

NEFA (mEql/L) 0.717 ± 0.20 0.662 ± 0.19 1.08 ± 0.29 0.979 ± 0.27

Ketone body (mM) 0.210 ± 0.064 0.255 ± 0.14* 2.54 ± 0.831 2.08 ± 1.22

Insulin (ng/mL) 1.19 ± 0.97 1.08 ± 0.57 0.29 ± 0.14 0.132 ± 0.03*

Leptin (pg/mL) 248 ± 94 234 ± 23.2 42.9 ± 27.7 20.6 ± 30.0

Tissue acetate (mmol/mg protein)

Liver

SM

BAT

4.73 ± 0.52

3.29 ± 0.60

3.53 ± 0.37

9.01 ± 1.12**

6.84 ± 0.67***

9.18 ± 0.63***

5.24 ± 0.38

3.48 ± 0.40

3.71 ± 0.12

10.42 ± 3.05*

8.91 ± 2.24*

11.38 ± 3.16*

Male mice (12 weeks old, AceCS2+/+; n = 18, AceCS2-/-; n= 14) were fed on normal

chow diet. To analyze the metabolic parameters of fed and fasted mice, mice were

divided into two groups: Fed and Fasted. The fed group was fed ad libitum and the

fasted group was fasted 48 hr. AceCS2-/- mice weighed less (body weight and lean

body mass) than AceCS2+/+ mice when measured at 12 weeks of age. Fat content per

body weight of AceCS2-/- and AceCS2+/+ mice did not differ significantly. Food

intake and blood analysis of these mice fed or fasted are shown. Asterisks denote the

level of statistical significance between the AceCS2+/+ and AceCS2-/- groups (Student’s

t test). * p<0.05, ** p<0.01 compared to AceCS2+/+ mice. NE, not examined.

BMD, bone mineral density. SM, skeletal muscle.

Table S3. Constituents of the LC/HF Diet (TD96355, Harlan Teklad)

Component g/kg

Casein

DL-methionine

Shortning (Crisco)

Corn Oil

Cellulose

Vitamin mix (Teklad 40060)

Choline bitartrate

TBHQ (antioxidant)

Mineral mix, Ca-P Deficient (TD79055)

Calcium phosphate, dibasic

Calcium carbonate

Magnesium oxide

173.3

2.6

586.4

86.2

87.97

13.0

2.5

0.13

20.0

19.3

8.2

0.4