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Pivotal role of O-GlcNAc modification in cold-induced thermogenesis by brown

adipose tissue through mitochondrial biogenesis

Natsuko Ohashi1, Katsutaro Morino

1, Shogo Ida

1, Osamu Sekine

1, Mengistu Lemecha

1,

Shinji Kume1, Shi-Young Park

2, Cheol Soo Choi

2,3, Satoshi Ugi

1 and Hiroshi Maegawa

1

1Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan

2Korea Mouse Metabolic Phenotyping Center, Lee Gil Ya Cancer and Diabetes Institute,

Gachon University, Incheon, Korea.

3Department of Internal Medicine, Gachon University Gil Medical Center, Incheon,

Korea

Running title: Role of O-GlcNAc modification in BAT

Word count: 4188 words

Number of tables and figures: 6

Corresponding author:

Katsutaro Morino M.D., Ph.D.

Department of Medicine, Shiga University of Medical Science,

Tsukinowa-cho, Seta, Otsu, Shiga 520-2192, Japan

TEL: +81-775-48-2222

FAX: +81-775-43-3858

E-mail: [email protected]

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Diabetes Publish Ahead of Print, published online June 21, 2017

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Abstract

Adipose tissues considerably influence metabolic homeostasis, and both white 1

(WAT) and brown adipose tissue (BAT) play significant roles in lipid and glucose 2

metabolism. O-GlcNAc modification is characterized by the addition of 3

N-acetylglucosamine to various proteins by O-GlcNAc transferase (Ogt), subsequently 4

modulating various cellular processes. However, little is known about the role of 5

O-GlcNAc modification in adipose tissues. Here, we report the critical role of 6

O-GlcNAc modification in cold-induced thermogenesis. Deletion of Ogt in WAT and 7

BAT using adiponectin promoter-driven Cre recombinase resulted in severe cold 8

intolerance with decreased uncoupling protein-1 (Ucp1) expression. Furthermore, Ogt 9

deletion led to decreased mitochondrial protein expression in conjunction with 10

decreased peroxisome proliferator-activated receptor gamma coactivator 1-α protein 11

expression. This phenotype was further confirmed by deletion of Ogt in BAT using 12

Ucp1 promoter-driven Cre recombinase, suggesting that O-GlcNAc modification in 13

BAT is responsible for cold-induced thermogenesis. Hypothermia was significant under 14

fasting conditions. This effect was mitigated following normal diet consumption but not 15

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following consumption of a fatty acid-rich ketogenic diet lacking carbohydrates, 16

suggesting impaired diet-induced thermogenesis, particularly by fat. In conclusion, 17

O-GlcNAc modification is essential for cold-induced thermogenesis and mitochondrial 18

biogenesis in BAT. Glucose flux into BAT may be a signal to maintain BAT 19

physiological responses. 20

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Obesity develops when energy intake exceeds energy expenditure, leading to 22

excess calorie storage in the adipose tissues. Obesity is highly correlated with the 23

development of type 2 diabetes, the metabolic syndrome and cardiovascular disease. 24

Adipose tissues have considerable influence on metabolic homeostasis. Two 25

functionally different types of adipose tissues are present in mammals: white adipose 26

tissue (WAT), which is the primary site of energy storage, and brown adipose tissue 27

(BAT), which is specific to thermogenic energy expenditure. BAT uniquely expresses 28

uncoupling protein 1 (Ucp1) and is rich in mitochondria. Furthermore, it is responsible 29

for active metabolism during cold- or diet-induced thermogenesis and uses glucose and 30

fatty acids as fuel (1-4). 31

Cellular homeostasis between nutrient sensing and energy metabolism is 32

coordinately regulated by complex molecular mechanisms (5). Growing evidence 33

indicates that the hexosamine biosynthetic pathway and its end product, 34

UDP-N-acetylglucosamine (UDP-GlcNAc), act as important nutrient sensors (6-8). In a 35

branch of the glycolytic pathway, fructose-6-phosphate is converted into UDP-GlcNAc 36

through multiple enzymes in the hexosamine biosynthetic pathway. UDP-GlcNAc also 37

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serves as the donor for O-linked N-acetylglucosamine (O-GlcNAc) modification, which 38

is associated with glucose, amino acid and fatty acid metabolism (9). O-GlcNAc 39

modification of proteins on serine/threonine residues occurs in the nucleus, cytoplasm 40

and mitochondria, and its addition is catalyzed by O-GlcNAc transferase (Ogt) (10). 41

Cold-induced thermogenesis is a fundamental function required in mammals 42

to survive in severe environmental changes. BAT is important for maintaining body 43

temperature under cold conditions (11). Recent reports have revealed that, even in 44

humans, significantly increased glucose and fatty acid flux are observed via positron 45

emission tomography with 18F-fluorodeoxyglucose and 18F-fluoro-thiahepadecaoic 46

acid (12). In addition, dramatic changes in both glucose and fatty acid flux occur in the 47

fasting and postprandial states in WAT. Since O-GlcNAc modification is considered a 48

“nutrient sensor” for glucose and fatty acids, O-GlcNAc modification may influence 49

BAT and WAT function. However, little is known about the role of O-GlcNAc 50

modification in adipose tissues. 51

Therefore, in this study, to reveal the physiological role of O-GlcNAc 52

modification in adipose tissues, we analyzed the phenotypes of adipose tissue-specific 53

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Ogt knockout (Ogt-FKO) and BAT-specific Ogt knockout (Ogt-BKO) mice. Here, we 54

demonstrated that O-GlcNAc modification in BAT is essential for thermogenesis and 55

mitochondrial biogenesis. 56

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Research Design and Methods 58

Animal experiments 59

Ogt-KO mice were generated using the Cre-LoxP system. We crossbred Ogt-flox 60

(Ogtf/f

) female mice (Jackson Laboratory, Bar Harbor, ME) with Adipoq-Cre (kindly 61

provided by Dr. Evan Rosen) and Ucp1-Cre (Jackson Laboratory) mice to generate 62

Ogt-FKO and Ogt-BKO mice, respectively. All experiments and analyses were 63

performed with male mice. Because Ogt is present on the X chromosome, the first 64

generation of female mice are heterozygous with X-linked inheritance. Thus, we 65

examined male mice as complete knockout mice (Figures 1A and 4A). All animal 66

handling and experimentation were conducted according to the guidelines of the 67

Research Center for Animal Life Science at Shiga University of Medical Science or the 68

Korea Mouse Metabolic Phenotyping Center at Gachon University. All experimental 69

protocols were approved by the Gene Recombination Experiment Safety Committee 70

and Research Center for Animal Life Science at Shiga University of Medical Science or 71

Gachon University Institutional Animal Care and Use Committee. 72

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Tissue collection 74

Mice were euthanized at various time points by intraperitoneal administration of 10% 75

pentobarbital with sevoflurane inhalation before immediate tissue collection. Inguinal 76

WAT (iWAT), epididymal WAT (eWAT), inter-scapular BAT, gastrocnemius muscle, 77

liver, and pancreas were dissected immediately, snap-frozen in liquid-nitrogen, and 78

stored at −80°C until analysis. 79

80

Blood analysis 81

Blood glucose concentrations were measured with GDH-PQQ glucose test strips 82

(Glutest Sensor®, Sanwa Kagaku, Nagoya, Japan). Plasma insulin levels were 83

measured by an enzyme-linked immunosorbent assay (Morinaga, Tokyo, Japan). Blood 84

ketone levels were measured using a Precision Xceed (Abbott, Chiba, Japan). 85

86

Histological analyses 87

Fixed specimens embedded in paraffin were sectioned (3 µm thicknesses). Antibodies 88

against UCP1 (U6382, Sigma-Aldrich, St. Louis, MO), cytochrome oxidase subunit 4 89

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(COX4; Novus Biologicals, Cambridge, UK), and perilipin (#9349, Cell Signaling 90

Technology, Tokyo, Japan) were used. Transmission electron microscopic analysis was 91

performed with a Hitachi H-7500 (Hitachi, Tokyo, Japan). The adipocyte number and 92

area were calculated using a BZ-H3C (Keyence, Osaka, Japan). 93

94

Acute cold exposure 95

Ogt-FKO, Ogt-BKO, and control mice were deprived of food for 12 h before 96

experiments. The mice were placed into individual cages with bedding and water in a 97

room maintained at 4°C for 3 h. Mouse rectal body temperature was measured using a 98

type T thermocouple rectal probe (RET-3, Physitemp Instruments, Inc., Clifton, NJ). 99

Following cold exposure, mice were euthanized by sevoflurane before immediate 100

inter-scapular BAT and iWAT collection as described above. 101

102

Normal and ketogenic diet consumption during acute cold exposure 103


experiments. Mice were placed into individual cages with bedding, food, and water in a 105

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room maintained at 4°C for 3 h. The mice were fed a normal (protein : fat : 106

carbohydrate = 27.5 : 12.5 : 60 kcal%, CE-2, CREA Japan, Inc., Tokyo, Japan) or 107

ketogenic diet (protein : fat : carbohydrate = 10.4 : 89.5 : 0.1 kcal%, Very Low 108

Carbohydrate Ketogenic Rodent Diet, Research Diets, Inc., New Brunswick) during 109

cold exposure. Before and at 1, 2 and 3 h after cold exposure, mouse rectal body 110

temperatures were assessed. Before and at 3 h after cold exposure, blood glucose 111

concentrations and blood ketone levels were measured. 112

113

Intraperitoneal glucose tolerance test (IPGTT) 114

An IPGTT in Ogt-FKO, Ogt-BKO, and control mice was performed in overnight-fasted 115

mice following an intraperitoneal injection of glucose (1 g/kg body weight) at room 116

temperature or 4°C. Blood glucose levels were determined at 15, 30, 60, 90 and 120 117

min after injection. 118

119

Intraperitoneal insulin tolerance tests (IPITT) 120

An IPITT in Ogt-FKO and control mice was performed an intraperitoneal injection of 121

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insulin (0.5 U/kg body weight) at 6 h after fasting at room temperature. Blood glucose 122

levels were determined at 15, 30, 60, 90 and 120 min after injection. 123

124

Oral glucose and olive oil tolerance tests 125


experiments. The mice were placed into individual cages with bedding and water in a 127

room maintained at 4°C for 3 h. Glucose (20%) or olive oil was administered orally 128

(0.01 ml/g body weight) before and at 1 and 2 h after cold exposure. Before and at 1, 2, 129

and 3 h after cold exposure, mouse rectal body temperatures were measured. Before and 130

at 3 h after cold exposure, blood glucose concentrations and blood ketone levels were 131

measured. 132

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Body fat composition and basal energy balance measurement 134

Fat and lean body masses were measured in mice using the 1H minispec system (LF90II, 135

BrukerOptics, Germany). Basal energy balance, including oxygen consumption, carbon 136

dioxide production rate, respiratory quotient, energy expenditure, and food intake, were 137

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measured (2 days of acclimation followed by 2 days of measurement) using 138

Comprehensive Laboratory Animal Monitoring Systems (CLAMS; Columbus 139

Instruments, Columbus, OH). 140

141

Total RNA preparation and RT-qPCR analysis 142

Total RNA was extracted from tissues using an RNeasy Kit (Qiagen Inc., Valencia, CA). 143

cDNA was synthesized using reverse transcription reagents (Takara Bio, Otsu, Japan). 144

Transcript abundance was assessed by real-time PCR on an Applied Biosystems 7500 145

Real-Time PCR System (Thermo Fisher Scientific K.K., Yokohama, Japan) with SYBR 146

Green (Bio-Rad Laboratories, Hercules, CA). Analytical data were normalized to 147

GAPDH mRNA expression as an internal control. Primer sequences can be found in the 148

Supplemental Data section. 149

150

RT-qPCR analysis of mitochondrial DNA content 151

DNA primers were designed to detect cytochrome oxidase 2 (Cox2) and uncoupling 152

protein 2 (Ucp2) for mitochondrial DNA (mtDNA) and nuclear DNA, respectively. The 153

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ratio of Cox2 to Ucp2 within the samples was used to calculate the mtDNA copy 154

number. Primer sequences can be found in the Supplemental Data section. 155

156

Western blot analysis 157

For western blot analysis, proteins were resolved by SDS-polyacrylamide gel 158

electrophoresis and transferred to nitrocellulose membranes. The membranes were 159

incubated with antibodies against the following molecules: OGT (O6264, 160

Sigma-Aldrich), RL2 (MA1-072, Thermo Scientific, MA), β-tubulin (H-235, Santa 161

Cruz Biotechnology, Santa Cruz, CA), UCP1 (U6382, Sigma-Aldrich), peroxisome 162

proliferator-activated receptor gamma coactivator-1α (PGC-1α; ab54481, Abcam plc, 163

Cambridge, UK), cAMP response element-binding protein (CREB; sc-186, Santa Cruz 164

Biotechnology), pCREB (sc-7978, Santa Cruz Biotechnology), phosho-(Ser/Thr) 165

protein kinase A substrate (pPKA; #9621, Cell Signaling Technology, Tokyo, Japan), 166

phospho-hormone sensitive lipase Ser563 (pHSL; #4139, Cell Signaling Technology), 167

pHSL Ser660 (#4126, Cell Signaling Technology), HSL (#4107, Cell Signaling 168

Technology), cytochrome c oxidase subunit 1 (MTCO1; ab14705, Abcam plc), COX4 169

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(ab14744, Abcam plc), mitochondrial transcription factor A (TFAM; LS-C30495, 170

LifeSpan BioSciences, Seattle, WA), succinate dehydrogenase complex, subunit A 171

(SDHA; ab14715, Abcam plc), pyruvate dehydrogenase (PDH; Novus Biologicals, 172

Cambridge, UK), ubiquitin (Cell Signaling Technology), Autophagy-related gene (ATG) 173

5 (Cell Signaling Technology), ATG7 (Cell Signaling Technology), 174

microtubule-associated protein 1 light chain 3 (LC3; Novus Biologicals), β-actin 175

(A5316, Sigma-Aldrich), pan-actin (sc-1616, Santa Cruz Biotechnology), long-chain 176

acyl coenzyme A dehydrogenase (LCAD) (ACADL; Proteintech Group, Rosemont), 177

and medium-chain acyl coenzyme A dehydrogenase (MCAD) (ACADM; GeneTex, Inc., 178

CA). After additional washing, the membranes were incubated with horseradish 179

peroxidase-linked secondary antibodies followed by chemiluminescence detection. 180

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Stromal vascular (SV) culture and primary brown adipocyte differentiation 182

The interscapular brown fat pad (eight fat pads for each group) was dissected from 183

8-week-old Ogt-BKO and Ogt-flox mice according to a previous report (13). Briefly, 184

tissues were minced and digested with 1.5 U/ml collagenase D (1108874103, Roche) in 185

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10 mM CaCl2 and 2.4 U/ml dispase II (04942078001, Roche) for 40–50 min while 186

shaking at 37°C. Digestion was stopped by adding complete DMEM/F12 containing 187

10% FBS and penicillin/streptomycin (SV culture medium). Cells were collected by 188

centrifugation at 700 × g for 10 min, resuspended, and strained through a 70 µM cell 189

strainer (BD Biosciences). Cells were further filtered through a 40 mm cell strainer to 190

remove clumps and large adipocytes. After further centrifugation as mentioned above, 191

SV cells were re-suspended in SV culture medium and plated onto 6-well 192

collagen-coated dishes. At confluency (day 0), cells were exposed to a differentiation 193

cocktail including 0.25 µM dexamethasone, 10 µg/mL insulin, 1 nM T3, 0.5 µM 194

rosiglitazone, 0.5 mM isobutylmethylxanthine, and 125 µM indomethacin in SV culture 195

medium. At 48 h after induction, the cells were maintained in SV culture medium 196

containing 5 mg/mL insulin and 1 µM rosiglitazone for 6 days. Cells were fully 197

differentiated on day 8. To examine the role of the proteasomal pathway, proteasomal 198

inhibitor MG132 (25 µM) was applied at 3 h before sample collection. All chemicals 199

for cell culture were obtained from Sigma unless indicated otherwise. 200

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Statistical analysis 202

Results are expressed as means ± SEM. Student’s t tests were used to assess differences 203

between two groups. A P-value of less than 0.05 was considered as statistically 204

significant. 205

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Results 206

Ogt-FKO mice display a significantly reduced WAT mass, but an increased BAT mass 207

We first clarified the role of O-GlcNAc modification in WAT and BAT using Ogt-FKO 208

mice (Figure 1A). OGT protein expression was decreased in both WAT and BAT of 209

Ogt-FKO mice compared with those of control mice (Figure 1B). Similarly, O-GlcNAc 210

modifications analyzed by the RL2 antibody were also decreased in these tissues 211

(Figure 1B). Ogt-FKO mice developed normally (Figure 1C), and there were significant 212

reductions in eWAT and iWAT masses (Figure 1D, E). In contrast, BAT in Ogt-FKO 213

mice displayed an increased fat mass. Upon dissection, it was visibly lipid laden and 214

displayed a milky appearance compared with the dark brown color of BAT in control 215

mice (Figure 1F). Histologically, BAT cells in Ogt-FKO mice were substantially 216

enlarged with a WAT-like appearance resulting from accumulation of lipid droplets 217

(Figure 1G, H, Supplementary Figure 1A). Quantitative analysis of cell size revealed 218

that Ogt-FKO mice had significantly smaller adipocytes in eWAT and iWAT, but larger 219

adipocytes in BAT (Figure 1I–K). 220

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Ogt-FKO mice display significant intolerance to cold exposure compared with control 222

mice 223

We next examined the effects of adipose tissue-specific O-GlcNAc modification 224

deficiency in glucose metabolism and thermogenesis. IPGTTs and IPITTs revealed no 225

significant differences between the two genotypes (Figure 2A, B). To assess 226

thermogenesis, a cold exposure test was performed at 4°C for 3 h. Surprisingly, 227

although all mice visibly shivered, cold intolerance was evident only in Ogt-FKO mice 228

with the mice reaching critical hypothermia within 3 h (Figure 2C). During cold 229

exposure, there were no differences in blood glucose or plasma insulin levels between 230

the two genotypes (Figure 2D, E). eWAT and iWAT masses in Ogt-FKO mice were 231

reduced compared with those in control mice (Figures 2F, G). In contrast, BAT in 232

Ogt-FKO mice was heavier than that in control mice and displayed substantial lipid 233

droplet accumulation even after cold exposure (Figures 2F, G). Therefore, we focused 234

on the detailed molecular mechanism underlying O-GlcNAc modification 235

deficiency-mediated cold intolerance. 236

Interestingly, BAT Ucp1 gene expression was significantly low at room 237

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temperature in Ogt-FKO mice (Figure 2H). Moreover, Ogt-FKO mice displayed 238

impaired cold-stimulated Ucp1 mRNA expression compared with control mice (Figure 239

2H). UCP1 protein expression was also significantly decreased in Ogt-FKO mice 240

(Figure 2I). Similarly, immunohistochemical analysis revealed decreased UCP1 241

expression both at room temperature and following cold exposure (Figure 2J). To test 242

the differentiation level of BAT, we analyzed mRNA expression of Cox7a1, Cidea, and 243

Prdm16. There was no significant difference in the expression of these genes between 244

the two genotypes (Figure 2K). Taken together, Ogt-FKO mice displayed significant 245

intolerance to cold exposure compared with control mice, partially because of the lack 246

of Ucp1 expression. 247

248

PGC-1α and mitochondria-related protein expression in BAT are remarkably decreased 249

in Ogt-FKO mice compared with control mice 250

Induction of the cold-induced thermogenesis program in BAT in vivo is strongly 251

influenced by the sympathetic nervous system and the resulting action of the 252

β-adrenergic receptor/PKA/CREB axis. Thus, we next evaluated changes in the 253

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expression levels of genes associated with the β-adrenergic receptor/PKA/CREB axis. 254

First, we examined PKA, HSL, and CREB phosphorylations and found no significant 255

differences between Ogt-FKO and control mice (Supplementary Figure 2). However, 256

we observed a significant reduction in protein level of PGC-1α, a key regulator of 257

thermogenesis, although there were no significant changes in mRNA expression of the 258

gene encoding PGC-1α, Ppargc1a (Figure 3A, B). Because PGC-1α is also known as a 259

master regulator of mitochondrial biogenesis, we next evaluated the expression of genes 260

associated with mitochondria. Although the mtDNA copy number was similar in the 261

two genotypes (Figure 3C), mRNA expression of mtDNA-encoded genes was 262

significantly decreased in Ogt-FKO mice, suggesting decreased transcription of 263

mtDNA-encoded genes probably due to the decreased TFAM protein levels (Figure 3D). 264

Moreover, immunohistochemical analysis of COX4 in BAT revealed decreased 265

expression in Ogt-FKO mice (Figure 3E). In addition, the protein levels of MTCO1, 266

COX4, TFAM, and SDHA were significantly decreased in BAT (Figure 3F, G). These 267

results suggested that Ogt deletion in adipose tissues resulted in impaired cold-induced 268

thermogenesis in BAT through dysregulation of PGC-1α. 269

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Ogt deletion in mouse BAT significantly increases the BAT mass 271

To evaluate whether BAT is the sole organ responsible for the dysregulation of 272

cold-induced thermogenesis following Ogt deletion, Ogt-BKO mice were generated by 273

crossbreeding Ogt-flox mice with Ucp1-Cre mice (Figure 4A). In Ogt-BKO mice, OGT 274

protein expression was decreased only in BAT, resulting in decreased O-GlcNAc 275

modification as measured using the RL2 antibody (Figure 4B). Similar to Ogt-FKO 276

mice, Ogt-BKO mice developed normally (Figure 4C). There were no differences in 277

body composition, food intake, energy expenditure, resting energy expenditure, oxygen 278

consumption, carbon dioxide production, or locomotor activity between Ogt-BKO and 279

control mice (Figure 4D–H, Supplementary Figure 3). Although there were no 280

differences in eWAT or iWAT masses, a difference in BAT mass was observed (Figure 281

4I). Consistent with Ogt-FKO mice, Ogt-BKO mice displayed accumulation of enlarged 282

lipid droplets in BAT cells (Figure 4J, Supplementary Figure 1B). 283

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Ogt-BKO mice display marked intolerance to cold exposure similar to Ogt-FKO mice 285

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Similar to Ogt-FKO mice, cold intolerance was evident in Ogt-BKO mice with mice 286

reaching critical hypothermia within 3 h (Figure 5A). Ogt-BKO mice showed no 287

significant changes in expression levels of OGT or O-GlcNAc modification detected by 288

the RL2 antibody in iWAT after 3 h of cold exposure (Supplementary Figure 4A–C). 289

The BAT UCP1 protein level was severely diminished in Ogt-BKO mice compared with 290

control mice at room temperature (Figure 5B). Moreover, PGC-1α protein level was 291

significantly lower, although mRNA expression was unchanged compared with control 292

mice, suggesting post-translational modification (Figure 5C, D). To further explorer this 293

mechanism, we evaluated primary brown adipocytes from Ogt-BKO mice. PGC-1α 294

expression was significantly reduced in primary brown adipocytes from Ogt-BKO mice 295

compared with those from control mice. Treatment with proteasomal inhibitor MG132 296

partially but significantly increased PGC-1α protein levels in Ogt-BKO mice, 297

suggesting that O-GlcNAc modification protects PGC-1α from proteasomal degradation 298

(Figure 5E–G). Similar to Ogt-FKO mice, immunohistochemical analysis showed that 299

COX4 was decreased in BAT (Figure 5H), and other mitochondrial protein levels were 300

significantly decreased in Ogt-BKO mice (Figure 5I, J). There was no difference in 301

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blood glucose levels, but a significant decrease in plasma insulin levels in Ogt-BKO 302

mice (Figure 5K, L). In other glucose metabolism-related tissues such as liver, muscle, 303

and pancreas, no apparent difference was found in OGT or mitochondrial related protein 304

expression, and O-GlcNAc modification was observed in Ogt-BKO mice compared 305

with control mice (Supplementary Figure 5B). This phenomenon was consistent in 306

Ogt-FKO mice (Supplementary Figure 5A). 307

308

Ogt-BKO mice maintain their body temperature during cold exposure following glucose 309

administration 310

During cold exposure, increased glucose and fatty acid oxidation appears to maintain 311

core temperature by stimulating heat production in BAT. To evaluate glucose 312

metabolism during cold exposure, we performed IPGTTs in Ogt-BKO and control mice. 313

Surprisingly, body temperatures during an IPGTT at 4°C were sustained even in 314

Ogt-BKO mice (Figure 6A). However, Ogt-BKO mice displayed slightly but 315

significantly lower glucose levels compared with control mice at 30 and 60 min of cold 316

exposure (Figure 6B). Compared with glucose levels at room temperature, substantially 317

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lowered glucose levels were observed during cold exposure in both genotypes, 318

suggesting increased glucose utilization (Figure 6B). These data suggest that 319

diet-induced thermogenesis by glucose rescued hypothermia in Ogt-BKO mice. To test 320

this hypothesis, we performed glucose- and oil-loading tests during cold exposure. 321

There was no apparent difference in shivering between glucose- and oil-loaded groups 322

(data not shown). Oral administration of oil failed to maintain body temperature in 323

Ogt-BKO mice during cold exposure (Figure 6C). Following cold exposure, oil 324

administration in Ogt-BKO mice resulted in significantly lower glucose levels 325

compared with control mice (Figure 6D). In addition, Ogt-BKO mice fed a normal diet 326

could maintain body temperature, but Ogt-BKO mice fed a low carbohydrate ketogenic 327

diet could not, although there were no changes in blood glucose or ketone body levels 328

between Ogt-BKO and control mice (Figure 6E–G). Because a ketogenic diet is 329

deficient in carbohydrates but high in fat, these data suggest that Ogt-BKO mice 330

preferentially use glucose, and fat utilization is defective during cold exposure. To 331

further explorer the mechanism, we next investigated whether Ogt deletion affect 332

lipolysis and fatty acid oxidation in BAT. The protein expression of ATGL and HSL, 333

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key enzymes of lipolysis, were similar between Ogt-BKO and control mice 334

(Supplementary Figure 6A, B). Protein expression of LCAD and MCAD, fatty acid 335

oxidation gene known PGC-1α targets, were significantly lower in Ogt-BKO mice 336

(Figure 6H–J). These data strongly support the hypothesis that O-GlcNAc modification 337

deficiency impairs fatty acid oxidation in BAT. 338

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Discussion 340

The current study was designed to clarify the role of protein O-GlcNAc 341

modification in adipose tissues. We demonstrated that this modification was essential 342

for cold-induced thermogenesis in BAT. Advances in brown adipose cell biology during 343

the last decade have increased our understanding of the cellular origin (14-16), function 344

(4; 16; 17), and adult human tissue distribution (3; 18-20) of BAT. However, a 345

comprehensive overview of BAT biology has not been fully clarified. One of the most 346

prominent findings from this study was that Ogt-KO mice displayed impaired 347

thermogenesis during cold exposure. Compared with control mice, we identified an 348

increased number of large lipid droplets in the BAT of both Ogt-FKO and Ogt-BKO 349

mice. To reveal the detailed mechanism underlying impaired thermogenesis by 350

O-GlcNAc modification deficiency, we compared the expression levels of 351

mitochondrial proteins in the BAT of Ogt-KO and control mice. In the BAT of Ogt-FKO 352

mice, protein and mRNA expression levels of Ucp1 and mitochondrial DNA-encoded 353

proteins were decreased significantly and accompanied by decreased PGC-1α 354

expression. PGC-1α-dependent Ucp1 expression and mitochondrial biogenesis are 355

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essential for cold exposure-induced thermogenesis in BAT (21). Thus, O-GlcNAc 356

modification deficiency in BAT may impair cold-induced thermogenesis by modulating 357

PGC-1α. 358

PGC-1α is a key transcriptional co-activator that regulates mitochondrial 359

biogenesis (21). PGC-1α stability and activity are regulated by post-translational 360

mechanisms such as acetylation and phosphorylation (22-24). PGC-1α degradation can 361

also be regulated by ubiquitin-dependent proteasomal degradation (25; 26). We found 362

no difference in PGC-1α mRNA expression between Ogt-KO and control mice, whereas 363

PGC-1α protein levels were decreased in Ogt-KO mice (Figures 3B, 5D). In addition, 364

primary adipocytes from Ogt-BKO mice showed lower PGC-1α protein levels 365

compared with control mice (Figure 5E, G). Treatment with the proteasomal inhibitor 366

resulted in a partial but significant increase in PGC-1α protein levels, indicating the 367

ubiquitin-proteasomal pathway in the stability of PGC-1α (Figure 5E, G). These results 368

suggest that O-GlcNAc modification may regulate the PGC-1α degradation process in 369

BAT. This hypothesis is in agreement with a previous study that demonstrated that 370

O-GlcNAc modification of PGC-1α protein in the liver antagonizes ubiquitination and 371

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subsequent degradation (27). Taken together, O-GlcNAc modification of PGC-1α 372

protein may stabilize PGC-1α by inhibiting ubiquitination in BAT. 373

An alternative explanation for the decreased mitochondrial proteins is 374

activation of autophagy. It is known that mitochondrial homeostasis is regulated by the 375

balance between mitochondrial biogenesis and degradation (28). LC3, a regulatory 376

protein essential for induction of autophagy, localizes to autophagosome membranes 377

during autophagy activation. We found that LC3-I protein expression was decreased and 378

LC3-II protein expression was increased in Ogt-BKO mice, suggesting acceleration of 379

autophagy (Supplementary Figure 7A, B). Moreover, protein expressions of ATG5 and 380

ATG7, essential for autophagy induction, were increased in Ogt-BKO mice compared 381

with control mice (Supplementary Figure 7A, C and D). In transmission electron 382

microscopy, autophagosomes were found in Ogt-BKO mice, but not in control mice 383

(Supplementary Figure 7E). These data suggest that autophagy is enhanced in Ogt-BKO 384

mice. It has been supported that autophagy have a role in remodeling mitochondrial 385

contents and regulating adipose mass and differentiation (28; 29). It is also possible that 386

the enhancement of autophagy is a consequence of damaged mitochondria due to Ogt 387

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depletion. Further examinations are necessary to reveal the relationship of dysregulated 388

mitochondrial proteins and autophagy under Ogt deletion. 389

Another interesting finding of this study was that impaired thermogenesis in 390

Ogt-KO mice was observed under fasting conditions. This effect was recovered by oral 391

intake of a normal diet or following glucose injection, but not by consumption of a fatty 392

acid-rich ketogenic diet deficient in glucose (Figure 6C, E, Supplementary Figure 8). 393

Moreover, we found a marked decrease in the protein levels of acyl-CoA 394

dehydrogenases, such as MCAD and LCAD, in Ogt-BKO mice (Figure 6H–J). 395

Therefore, the metabolic rate of fatty acid was probably decreased. A previous study in 396

mice with adipose-specific deletion of carnitine palmitoyltransferase 2 demonstrated 397

similar changes because of the lack of fatty acid oxidation (30). In addition, it has been 398

reported that PGC-1α is related to regulation of gene expression involved in fatty acid 399

oxidation (31; 32), and acute RNA interference-mediated PGC-1α knockdown leads to 400

profound downregulation of fatty acid gene expression (33). Hence, we speculate that 401

fatty acid oxidation was impaired because of the decrease of PGC-1α protein in Ogt-KO 402

mice. Collectively, O-GlcNAc modification is essential for fat oxidation in BAT. 403

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Because glucose does not need a beta-oxidation process to form acetyl-CoA, glucose 404

may be preferentially used in Ogt-KO mice to maintain body temperature and generate 405

heat even under the reduced UCP1 condition. This hypothesis is partially supported by 406

our results showing which enzymes were intact in the glycolysis pathway 407

(Supplementary Figure 6C). In addition, blood glucose levels in Ogt-BKO mice during 408

cold exposure were lower than those in control mice (Figure 6B, D). Thus, enhanced 409

glucose uptake and use in BAT of Ogt-KO mice were likely to be associated with heat 410

production during cold exposure. Thus, O-GlcNAc modification may act as a 411

“metabolic switch” in BAT and plays a crucial role in whole body glucose and lipid 412

homeostasis in cold environments. 413

Ogt deletion from both WAT and BAT resulted in reduced WAT and increased 414

BAT masses (Figure 1D–F). In contrast, Ogt-BKO mice displayed similar WAT and 415

increased BAT masses compared with control mice (Figure 4D). Because both 416

Ogt-FKO and Ogt-BKO mice displayed intolerance to cold exposure, the differences in 417

WAT have little effect on this phenotype. 418

There are two limitations to this study. First, we did not identify the direct target of 419

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31

Ogt in BAT. Although we speculate that PGC-1α may be a potential direct target of Ogt, 420

further experiments are necessary to test this hypothesis. Second, energy expenditure 421

during hypothermia was not measured as a result of a technical issue. Because the 422

sympathetic nervous system plays a significant role in cold-induced thermogenesis, 423

further experiments such as β3 adrenergic stimulation may predict energy expenditure 424

during hypothermia. 425

In conclusion, the post-translational modification, O-GlcNAc modification 426

plays a pivotal role in cold-induced thermogenesis in BAT. The current findings provide 427

novel insights into BAT biology. 428

429

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Acknowledgements 430

We are indebted to Ms. Keiko Kosaka, Ms. Yoshiko Asano, Mr. Takefumi Yamamoto 431

and the Central Research Laboratory of Shiga University of Medical Science for their 432

expert technical assistance in this study. We thank Drs. Evan Rosen (Harvard 433

University), Wataru Ogawa and Tetsuya Hosooka (Kobe University) for providing the 434

Adipoq-Cre mice. We appreciate constructive discussion from Drs. Hiroshi Sakaue and 435

Shingo Kajimura. 436

This study was supported by Grants-in-Aid for Scientific Research (KAKENHI) from 437

the Japan Society for the Promotion of Science (No. 15K09383 to O.S. and No. 438

16K09743 to S.U.). This work was supported by a grant from the Bio & Medical 439

Technology Development Program of the National Research Foundation (NRF), which 440

is funded by the Ministry of Science, ICT & Future Planning 441

(NRF-2014M3A9D5A01073886). This study was funded by the Shiga University of 442

Medical Science. The Department of Medicine, Shiga University of Medical Science, 443

has received research promotion grants (Shogaku Kifukin) from Astellas, 444

Boehringer-Ingerheim, Daiichi-Sankyo, Kowa Pharmaceuticals, Kyowa-hakko-Kirin, 445

of 61Diabetes

33

Mitsubishi Tanabe, MSD, Ono Pharmaceutical, Sanofi, Sanwa Kagaku Kenkyusho, 446

Shionogi, Taisho-Toyama, Takeda and Teijin Pharma. However, the research topics of 447

these donation grants are not restricted. 448

N.O., S.I., K.M., S.U. and H.M. designed the study. N.O., S.I., S.Y.P., C.S.C. and L.M. 449

conducted the research. N.O. analyzed the data. N.O., K.M. and H.M. wrote the 450

manuscript. S.K. gave constructive comments regarding the study concept. O.S., K.M., 451

S.U, S.K. and H.M. reviewed and edited the manuscript. K.M. is the guarantor of this 452

work and, as such, had full access to all the data in the study and takes responsibility for 453

the integrity of the data and the accuracy of the data analysis. All authors read and 454

approved the final manuscript. 455

456

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Figure legends 554

Figure 1. Ogt deletion from adipose tissues in mice significantly reduces WAT mass, but 555

increases BAT mass. 556

A: Schematic diagram of Ogt-FKO mouse generation. B: Representative western blot 557

analysis of OGT and O-GlcNAcylation (RL2) in eWAT, iWAT, and BAT. C: Body 558

weights of Ogt-FKO mice and littermate controls (n=6 each). D: Representative images 559

and weights of eWAT from Ogt-FKO mice and littermate controls (n=8 each). E: 560

Representative images and weights of iWAT from Ogt-FKO mice and littermate 561

controls (n=8 each). F: Representative images and weights of BAT from Ogt-FKO mice 562

and littermate controls (n=8 each). G: Hematoxylin and eosin staining of Ogt-FKO and 563

control mice. H: Transmission electron microscopy of BAT from Ogt-FKO and control 564

mice. I–K: Fat cell area in Ogt-FKO and control mice. Data are means ± SEM. *P < 565

0.05. NS indicates not statistically significant. 566

567

Figure 2. Ogt-FKO mice display significant intolerance to cold exposure compared with 568

control mice. 569

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A and B: Glucose changes during intraperitoneal glucose tolerance tests (IPGTTs) and 570

intraperitoneal insulin tolerance tests (IPITTs) (n=6 each). C and D: Body temperatures 571

and blood glucose levels in Ogt-FKO and control mice during cold exposure (n=8 each). 572

E: Plasma insulin levels after 3 h of cold exposure (n=8 each). F: Adipose tissue weights 573

of Ogt-FKO and control mice after 3 h of cold exposure (n=6 each). G: Hematoxylin 574

and eosin staining of Ogt-FKO and control mice at room temperature and after 3 h cold 575

of exposure. H: qPCR analysis of BAT (n=6 each). I: Western blot analysis and 576

quantification (n=6 each). The Y-axis indicates that data is relative to controls. J: 577

Representative immunohistochemical analysis of UCP1 in BAT from Ogt-FKO and 578

control mice at room temperature and after 3 h of cold exposure. K: qPCR analysis of 579

iWAT (n=6 each). Data are means ± SEM. *P < 0.05. NS indicates not statistically 580

significant. RT: room temperature. 581

582

Figure 3. PGC-1α and mitochondria-related protein levels in BAT are remarkably 583

decreased in Ogt-FKO mice compared with control mice. 584

A: qPCR analysis of BAT (n=6 each). B: Western blot analysis and quantification (n=5–585

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6). The Y-axis indicates that data is relative to controls. C: mtDNA content was 586

analyzed by RT-qPCR. Expression of a mitochondrial genome coding gene (Cox2) and 587

nuclear genome coding gene (Ucp2) were analyzed using specific primers. Values 588

represent the ratio of the amount of mtCox2 to gUcp2 (n=4). D: qPCR analysis of BAT 589

(n=6 each). E: Representative immunohistochemical analysis of COX4 in BAT from 590

Ogt-FKO and control mice. F and G: Western blot analysis and quantification (n=5–6). 591

The Y-axis indicates that data is relative to controls. Data are means ± SEM. *P < 0.05. 592

NS indicates not statistically significant. RT: room temperature; ND: NADH 593

dehydrogenase; MTCO1: cytochrome c oxidase subunit 1; COX4: cytochrome oxidase 594

subunit 4; TFAM: mitochondrial transcription factor A; SDHA: succinate 595

dehydrogenase complex, subunit A; PDH: Pyruvate dehydrogenase. 596

597

Figure 4. Ogt deletion in BAT in mice significantly increases BAT mass. 598

A: Schematic diagram of Ogt-BKO mouse generation. B: Representative western blot 599

analysis of OGT and O-GlcNAcylation (RL2) in BAT. C and D: Body weights and 600

composition of Ogt-BKO mice (n=7) and littermate controls (n=6). E: Food intake, F: 601

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energy expenditure, G: respiratory exchange ratio (RER), and H: locomotor activity of 602

Ogt-BKO mice (n=7) and littermate controls (n=6). I: Adipose tissue weights of 603

Ogt-BKO mice and littermate controls (n=6 each). J: Hematoxylin and eosin staining of 604

Ogt-BKO mice and littermate controls. Data are means ± SEM. *P < 0.05. NS indicates 605

not statistically significant. 606

607

Figure 5. Ogt-BKO mice display marked intolerance to cold exposure similar to 608

Ogt-FKO mice. 609

A: Body temperatures of Ogt-BKO and control mice during cold exposure (n=6 each). 610

B: Western blot analysis and quantification (n=6 each). The Y-axis indicates that data is 611

relative to controls. C: qPCR analysis of BAT (n=6 each). D: Western blot analysis and 612

quantification (n=6 each). The Y-axis indicates that data is relative to controls. E–G: 613

Representative western blot analysis and quantification (n=4 each). H: Representative 614

immunohistochemical analysis of COX4 in BAT from Ogt-BKO and control mice. I and 615

J: Western blot analysis and quantification (n=6 each). The Y-axis indicates that data is 616

relative to controls. K: Blood glucose levels in Ogt-BKO and control mice during cold 617

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exposure (n=6 each). L: Plasma insulin levels after 3 h of cold exposure (n=6 each). 618

Data are means ± SEM. *P < 0.05. NS indicates not statistically significant. MTCO1: 619

cytochrome c oxidase subunit 1; COX4: cytochrome oxidase subunit 4; TFAM: 620

mitochondrial transcription factor A; SDHA: succinate dehydrogenase complex, subunit 621

A; PDH: pyruvate dehydrogenase. 622

623

Figure 6. Ogt-BKO mice maintain their body temperature during cold exposure 624

following glucose administration. 625

A: Body temperatures during IPGTTs at 4°C (n=6 each). B: Blood glucose level 626

changes during IPGTTs (n=6 each). C: Body temperatures of Ogt-BKO and control 627

mice during oral glucose and olive oil tolerance tests at 4°C (n=4 each). D: Blood 628

glucose levels in Ogt-BKO and control mice before and after 3 h of cold exposure (n=4 629

each). E: Body temperatures of Ogt-BKO and control mice at 4°C (n=5 each). F: Blood 630

glucose levels in Ogt-BKO and control mice before and after 3 h of cold exposure (n=5 631

each). G: Blood ketone body levels in Ogt-BKO and control mice before and after 3 h 632

of cold exposure (n=5 each). H–J: Western blot analysis and quantification (n=6 each). 633

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The Y-axis indicates that data is relative to controls. Data are means ± SEM. *P < 0.05. 634

NS indicates not statistically significant. RT: room temperature; ND: normal diet; KD: 635

ketogenic diet, MCAD: medium-chain acyl coenzyme A dehydrogenase; LCAD: 636

long-chain acyl coenzyme A dehydrogenase. 637

638

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C

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eWAT iWAT

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E

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J K

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te n

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Adipocyte area(𝜇𝑈) Adipocyte area(𝜇𝑈) Adipocyte area(𝜇𝑈)

Adiponectin-Cre

♂

Cre Adiponectin

exon1

♀

Ogt

Ogt(flox/flox)

Ogt(Y/flox)

Adiponectin-Cre(+)

♂

×

Ogt(Y/flox)

Adiponectin-Cre(-)

♂

A Ogt-flox

Ogt-FKO

Figure 1

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2

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n)

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0 1 2 3

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iWAT BAT

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gh

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Hours Hours

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Figure 2

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×

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A B

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Bod

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1.5

2

PGC-1α

β-tubulin

0

0.5

1

1.5

0

0.5

1

1.5

2

A B C

*

*

* *

N.S.

UCP1/β

-tu

bu

lin

Ogt-flox Ogt-BKO

PG

C-1α/β

-tu

bu

lin

D

Pp

arg

c1a/G

AP

DH

Figure 5

OGT

PGC-1α

Ubiquitin

β-tubulin 0

1

2OGT/β

-tu

bu

lin

0

1

2

PG

C-1α/β

-tu

bu

lin

Ogt-flox Ogt-BKO Ogt-flox Ogt-BKO

MG132(-) MG132(+) MG132(-) MG132(+) MG132(-) MG132(+)

E F G

*

Hours

Blo

od

glu

cose

(m

g/d

L)

K

0

0.2

0.4

0.6

Pla

sma

insu

lin

(n

g/m

L)

L

*

0

50

100

150

200

0 1 2 3

Ogt-flox

Ogt-BKO

MTCO1

COX4

PDH

SDHA

TFAM

β-tubulin

Ogt-flox Ogt-BKO

I

Ogt-flox Ogt-BKO

H

COX4

J

0

0.5

1

1.5

2

Ogt-flox

Ogt-BKO

Fold

ch

ange * * * * *

* *

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0

100

200

300

400

0 30 60 90 120

* *

20

25

30

35

40

0 1 2

Bod

y t

emp

erat

ure

(°C

)

Hours

Blo

od

glu

cose

(m

g/d

L)

Ogt-flox

Ogt-BKO

Ogt-flox (RT)

Ogt-BKO (RT)

Ogt-BKO (4 °C)

Ogt-flox (4 °C)

B A

20

25

30

35

40

0 1 2 3

*

Ogt-flox (ND)

Ogt-BKO (ND)

Ogt-BKO (KD)

Ogt-flox (KD)

Bod

y t

emp

erat

ure

(°C

)

Hours

Minutes after glucose injection

0

1

2

3K

eton

e b

od

ies

(μmol/

L)

E G

Og

t-fl

ox

(ND

)

Og

t-B

KO

(ND

)

Og

t-fl

ox

(KD

)

Og

t-B

KO

(KD

)

before

after 3-hour

C D

0

100

200

300

F

Blo

od

glu

cose

(m

g/d

L)

before

after 3-hour

Og

t-fl

ox

(ND

)

Og

t-B

KO

(ND

)

Og

t-fl

ox

(KD

)

Og

t-B

KO

(KD

)

Figure 6

0

10

20

30

40

0 1 2 3

Ogt-flox (glucose)

Ogt-BKO (glucose)

Ogt-BKO (olive oil)

Ogt-flox (olive oil)

Bod

y t

emp

erat

ure

(°C

)

Hours

* *

0

100

200

300

Ogt-

flox

(glu

cose

)

Ogt-

BK

O

(glu

cose

)

Ogt-

flox

(oli

ve

oil

)

Ogt-

BK

O

(oli

ve

oil

) Blo

od

glu

cose

(m

g/d

L)

before

after 3-hour

*

*

N.S.

0

0.5

1

1.5

LCAD/β

-tu

bu

lin

*

LCAD

MCAD

H

β-tubulin

I

0

0.5

1

1.5

MCAD/β

-tu

bu

lin

J

*

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Ogt-flox Ogt-FKO Ogt-flox Ogt-BKO

Supplementary Figure 1. Ogt-KO mice display an accumulation of enlarged lipid droplets in BAT cells. A: Representative

immunohistochemical analysis of perilipin in BAT of control and Ogt-FKO mice. B: Representative immunohistochemical

analysis of perilipin in BAT of control and Ogt-BKO mice.

A B

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pCREB

CREB

Ogt-flox Ogt-FKO

0

0.5

1

1.5

2N.S.

pC

RE

B/C

RE

B

pP

KA

sub

strate β

-tub

ulin

Ogt-flox Ogt-FKO

HSL

pHSL(serp563)

Ogt-flox Ogt-FKO Ogt-flox Ogt-FKO

HSL

pHSL(serp660)

0

0.5

1

1.5

2N.S.

pH

SL

(ser

p5

63)/

HS

L

0

0.5

1

1.5

2N.S.

pH

SL

(ser

p6

60)/

HS

L

A B C D

Supplementary Figure 2. Ogt-FKO mice show no significant changes in expression levels of proteins associated with the β-

AR/PKA/CREB axis. A-D: Western blot analysis and its quantification (n=6 each). The Y-axis indicates that data is relative to controls.

Data are means ± SEM. NS indicates not statistically significant.

CREB: cAMP response element-binding protein, pPKA: phosho-(Ser/Thr) protein kinase A substrate, HSL: hormone sensitive lipase.

of 61Diabetes

Supplementary Figure 3. VO2 and VCO2 show no difference in Ogt-BKO and control mice.

A and B: VO2 and VCO2 of Ogt-BKO (n=7) and control mice (n=6). Data are means ± SEM. NS indicates not statistically significant.

i

0

1000

2000

3000

4000N.S.

VO

2 (

ml/

kg/h

r)

A

0

1000

2000

3000

4000

N.S.

VC

O2

(m

l/k

g/h

r)

B

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Supplementary Figure 4. Ogt-BKO mice show no significant changes in expression levels of OGT, RL2 in iWAT after 3-hour cold

exposure. A-C: Representative western blot analysis and its quantification for OGT and O-GlcNAcylation (RL2) in iWAT at RT and after

cold exposure. (n=6 each). The Y-axis indicates that data is relative to controls. Data are means ± SEM. NS indicates not statistically

significant. RT: room temperature

A

Ogt-flox

（RT）

Ogt-BKO

（RT）

Ogt-BKO

(4 °C)

Ogt-flox

(4 °C)

OGT

β-tubulin

RL

2

(O-G

lcNA

cylatio

n)

0

0.5

1

1.5

2

B

Ogt-flox

Ogt-BKO

RT 4°C

OG

T/β

-tu

bu

lin

0

0.5

1

1.5

2

C

RL

2/β

-tu

bu

lin

RT 4°C

Ogt-flox

Ogt-BKO

N.S. N.S. N.S.

N.S.

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Supplementary Figure 5. Ogt-FKO and Ogt-BKO mice show no significant changes in expression of OGT, RL2, PGC-1α, MTCO1, and

TFAM. A: Representative western blot analysis of Ogt-FKO and control mice (n=3 each). B: Representative western blot analysis of Ogt-

BKO and control mice (n=3 each).

Pancreas Liver Muscle Pancreas Liver Muscle

RL

2

(O-G

lcNA

cylatio

n)

OGT

β-actin (Liver,Pancreas) /

pan-actin (Muscle)

PGC-1α

TFAM

A B

MTCO1 N/D N/D

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β-tubulin

HSL

ATGL

A B

Supplementary Figure 6. Ogt-BKO mice show no significant changes in expression of ATGL, HSL. A: Representative western blot

analysis of Ogt-BKO and control mice (n=6 each). B: Quantitative ratios of ATGL and HSL to β-tubulin (n=6 each). The Y-axis indicates

that data is relative to controls. C: qPCR analysis of BAT (n=4 each). Data are means ± SEM, NS indicates not statistically significant.

Glut4: Glucose transporter 4; Pfk1: Phosphofructokinase 1; Pgk1: Phosphoglycerate kinase 1, Pk: Pyruvate kinase

0

1

2

ATGL HSL

Ogt-flox

Ogt-BKO

N.S. N.S.

Fold

ch

ange

0

0.5

1

1.5

2

gen

e ex

pre

ssio

n/G

AP

DH

Glut4 Pfk1 Pgk1 Pk

Ogt-flox

Ogt-BKO

N.S. N.S. N.S.

N.S.

C

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ATG5

ATG7

β-tubulin

LC3-I

LC3-II

0

1

2

3

AT

G7

/β-t

ub

uli

n

*

Ogt-flox Ogt-BKO

Supplementary Figure 7. Ogt-BKO mice showed the enhancement of autophagy. A: Representative western blot analysis of Ogt-BKO and

control mice (n=4-6 each). B-D: Quantitative ratios of ATG5 and ATG7 to β-tubulin, and ratio of LC3II to LC3I (n=4-6 each). The Y-axis

indicates that data is relative to controls. E: Representative transmission electron microscopy of BAT from Ogt-BKO and control mice

(allow; autophagosome). Data are means ± SEM. *P < 0.05. NS indicates not statistically significant.

A

D E

0

0.5

1

1.5

2

AT

G5

/β-t

ub

uli

n *

B C

0

1

2

3

4

LC

3II

/LC

3I

*

Ogt-flox Ogt-BKO

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20

25

30

35

40

0 1 2 3

Bod

y t

emp

erat

ure

(°C

)

Hours

20

25

30

35

40

0 1 2 3

Bod

y t

emp

erat

ure

(°C

)

Hours

Ogt-flox (glucose)

Ogt-FKO (glucose)

Ogt-FKO (olive oil)

Ogt-flox (olive oil)

Ogt-flox (ND)

Ogt-FKO (ND)

Ogt-FKO (KD)

Ogt-flox (KD)

*

Supplementary Figure 8. Ogt-FKO mice fed a normal diet can maintain body temperature, but Ogt-FKO mice fed a ketogenic diet can not.

A and B: Body temperatures of Ogt-FKO and control mice at 4°C (n=5 each). Data are means ± SEM. *P < 0.05. NS indicates not

statistically significant. ND: normal diet; KD: ketogenic diet.

A B

*

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Supplementary Table 1. Primer sequences

Forward Reverse

Ucp1 5’-GTGAAGGTCAGAATGCAAGC-3’ 5’-AGGGCCCCCTTCATGAGGTC-3’

Ppargc1a

5’GTTTTATTTGCATCCAGAGC-3’ 5’AAGCTTGCGCAGGTAA-3’

ND1

5’CCATTTGCAGACCCCATAAA-3’ 5’GGTGTGGTATTGGTAGGGGAAC-3’

ND2 5’AGCATGAGGAGGACTTAACCAAAC-3’ 5’TGAGGGATGGATTGTAAGGAAGA-3’

ND3 5’GCATTCTGACTTCCCCAAATAAATC-3’ 5’GTGCAGAGCTTGTAGGGTCAAA-3’

ND4 5’CAAAAGCCCATGTTGAAGCTC-3’ 5’GGAGAGAAGGATGAAGGGGTATG-3’

ND5 5’ATCCTCACTTCAGCCAACAACA-3’ 5’GTTCGTCCATACCATCATCCAA-3’

Cidea 5’CGTGGTGGACACAGAGGAGTT-3’

5’TCCCGATTTCTTTGGTTGCT-3’

Cox7a1

5’CAGTACACTTGAAAGGCGGG-3’

5’CCAGCCCAAGCAGTATAAGC-3’

Prdm16 5’TCCCACCAGACTTCGAGCTA-3’

5’AAAGTCGGCCTCCTTCAGTG-3’

Glut4 5’ATCATCCGGAACCTGGAGG-3’ 5’CGGTCAGGCGCTTTAGACTC-3’

Pfk1 5’TGTGGTCCGAGTTGGTATCTT -3’

5’GCACTTCCAATCACTGTGCC -3’

Pgk1 5’GAGCCTCACTGTCCAAACTA -3’

5’CTTTAGCGCCTCCCAAGATA -3’

Pk 5’CTGTGGAGATGCTGAAGGAG -3’

5’CAACAGGACGGTAGAGAATGG -3’

GAPDH 5’ATGGCCTTCCGTGTTCCT-3’ 5’GCCTGCTTCACCACCTTCT-3’

Cox2 5’TTTTCAGGCTTCACCCTAGATGA-3’ 5’GAAGAATGTTATGTTATGTTTACTCCTA-3’

Ucp2 5’GCGACCAGCCCATTGTAGA-3’ 5’GCGTTCTGGGTACCATCCTAAC-3’

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