1 empagliflozin, via switching metabolism towards lipid ... · 39 these data suggest that...

1

Empagliflozin, via Switching Metabolism towards Lipid Utilization, Moderately 1

Increases LDL-cholesterol Levels through Reduced LDL Catabolism. 2

3

Running head: F. Briand et al. SGLT2 inhibition and LDL-C 4

François Briand1,#,*

Eric Mayoux2,#

, Emmanuel Brousseau1, Noémie Burr

1, Isabelle Urbain

1, 5

Clément Costard1, Michael Mark

2, Thierry Sulpice

1. 6

1Physiogenex SAS, Prologue Biotech, 516 Rue Pierre et Marie Curie, 31670 Labège, France. 7

2Boehringer Ingelheim Pharma, CardioMetabolic Diseases Research, BirkendorferStraße 65, 8

88397 Biberach an der Riss, Germany. 9

#Both authors contributed equally to this work. 10

This work has been funded by Boehringer Ingelheim Pharma. 11

*Corresponding author : François Briand, Ph.D, Physiogenex SAS, Prologue Biotech, 516 12

Rue Pierre et Marie Curie, 31670 Labège, France. 13

Tel.: +33 561 287 048 14

Fax: +33 561 287 043 15

E-mail: [email protected] 16

-17 pages, 1 table, 3 figures, 19 references 17

-abstract word count: 215 18

-manuscript total words count (excluding abstract, references and figure legends): 1999. 19

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Diabetes Publish Ahead of Print, published online April 5, 2016

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Abstract 21

In clinical trials, small increase in LDL cholesterol has been reported with sodium-glucose co-22

transporter type 2 (SGLT2) inhibitors. The mechanisms by which the SGLT2 inhibitor 23

empagliflozin increases LDL-cholesterol levels were investigated in diet-induced 24

dyslipidemic hamsters. 25

Compared with vehicle, empagliflozin 30mg/kg/day for 2 weeks significantly reduced fasting 26

blood glucose by 18%, with significant increase in fasting plasma LDL-cholesterol, free fatty 27

acids and total ketone bodies by 25, 49 and 116%, respectively. In fasting conditions, 28

glycogen hepatic levels were further reduced by 84% with empagliflozin, while HMG-CoA 29

reductase activity and total cholesterol hepatic levels were 31 and 10% higher, respectively 30

(both P<0.05 vs. vehicle). A significant 20% reduction in hepatic LDL-receptor protein 31

expression was also observed with empagliflozin. Importantly, none of these parameters were 32

changed by empagliflozin in fed conditions. Empagliflozin significantly reduced the 33

catabolism of 3H-cholesteryl oleate labelled LDL injected intravenously by 20%, indicating 34

that empagliflozin raises LDL levels through reduced catabolism. Unexpectedly, 35

empagliflozin also reduced intestinal cholesterol absorption in vivo, which led to a significant 36

increase in LDL- and macrophage-derived cholesterol fecal excretion (both P<0.05 vs. 37

vehicle). 38

These data suggest that empagliflozin, by switching energy metabolism from carbohydrate to 39

lipid utilization, moderately increases ketones production and LDL-cholesterol levels. 40

Interestingly, empagliflozin also reduces intestinal cholesterol absorption, which in turn 41

promotes LDL- and macrophage-derived cholesterol fecal excretion. 42

Key words- SGLT2 inhibition • empagliflozin • LDL • cholesterol metabolism • reverse 43

cholesterol transport 44

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Introduction 45

Specific sodium glucose co-transporter inhibitors represent an emerging and promising new 46

class of glucose lowering drugs in the management of type 2 diabetes mellitus. The unique 47

mode of action of this class of novel agents can effectively decrease blood glucose levels, 48

independently of the insulin pathway, via increasing glucose excretion in urine, i.e. glucosuria 49

(1, 2). Beside improved glycemic parameters, SGLT2 inhibitors have shown additional 50

benefits such as body weight loss and blood pressure lowering, with low risk of 51

hypoglycaemia (3). However, an increase in LDL-cholesterol (LDL-C) plasma levels has 52

been also observed in patients treated with SGLT2 inhibitors (1). The mechanism by which 53

SGLT2 inhibition raises LDL-C levels remains unclear. It has been suggested that the 54

increase in LDL-C, may be partly due to haemoconcentration, as SGLT2 inhibitors induce 55

volume contraction subsequent to increased urinary volume (4, 5). However, the transient 56

diuretic effect of SGLT2 inhibitors may not completely contribute to the observed LDL-C 57

increase. We therefore investigated the effects of the SGLT2 inhibitor empagliflozin in the 58

diet-induced insulin resistant dyslipidemic Golden Syrian hamster, a validated preclinical 59

model with similar cholesterol metabolism as compared with humans (6, 7). 60

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Methods 67

All animal protocols were approved by the local (Comité régional d’éthique de Midi-68

Pyrénées) and national (Ministère de l’Enseignement Supérieur et de la Recherche) ethical 69

committees. Male Golden Syrian hamsters (91-100g, 6 week-old, Elevage Janvier, Le Genest 70

Saint Isle, France) were fed ad libitum over 4 weeks with a high fat/high cholesterol diet 71

(HFHC, 0.5% cholesterol, 0.25% deoxycholate, 11.5% coconut oil, 11.5% corn oil) with 10% 72

fructose in the drinking water, as described (7). After 2 weeks of diet to induce dyslipidemia, 73

hamsters were randomized into 2 sets of non-radioactive (set 1) or radioactive (set 2) 74

experiments, according to their blood glucose and LDL-cholesterol levels in fed or overnight 75

fasting conditions (fasting starts at 5:00pm, blood collection at ~08:00am), and were then 76

treated orally for 2 weeks with vehicle or empagliflozin 30mg/kg once daily. The dose was 77

selected from a pilot study where glucose urine excretion was measured in this hamster model 78

treated acutely with empagliflozin 3, 10 and 30mg/kg. The 30mg/kg dose was found to 79

increase glucose urine excretion by 1200-fold vs. vehicle, while the 3 and 10mg/kg doses 80

showed slighter effect (80- and 200-fold respectively). At the end of the treatment period, a 81

first set of hamsters was used to measure biochemical parameters using commercial kits in fed 82

or overnight fasting conditions. Lipoprotein total cholesterol profile from was assessed using 83

Fast Protein Liquid Chromatography (FPLC) analysis using one pooled plasma sample (one 84

pool per treatment group), western blot analysis for LDL-receptor protein expression and 85

fecal cholesterol mass excretion were performed as described previously (7). A second set of 86

hamsters underwent radio-tracer based in vivo experiments to measure intestinal cholesterol 87

absorption, LDL-cholesteryl esters kinetics or macrophage-to-feces reverse cholesterol 88

transport, as described previously (6, 7). Intestinal cholesterol absorption was assessed after 89

administration of 14

C-cholesterol labeled olive oil by oral gavage and intraperitoneal injection 90

of poloxamer-407 (a lipase inhibitor) to measure 14

C-tracer plasma tracer appearance at time, 91

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3, 5 and 6 hour after oral gavage (6). Kinetics of LDL-cholesteryl oleate were performed by 92

injecting intravenously 3H-cholesteryl oleate labeled LDL in overnight fasted hamsters, 93

previously isolated from hamsters fed the same high fat/high cholesterol diet (7). Hamsters 94

were kept fasted for the first 6 hours of the kinetic experiment and were then kept in 95

individual cages with access to food and water for feces collection over 72 hours. Plasma 3H-96

tracer decay curve was monitored over 72 hours after injection to calculate 3H-cholesteryl 97

oleate LDL fractional catabolic rate (FCR) using the SAAMII software. Liver (collected after 98

72 hours) and feces were used to measure 3H-tracer recovery in cholesterol and bile acids 99

fraction after chemical extraction (6, 7). 100

Macrophage-to-feces reverse cholesterol transport was measured over 72 hours after injecting 101

intraperitoneally 3H-cholesterol labeled / oxidized-LDL loaded J774 macrophages (6, 7). In 102

this experiment, hamsters were not fasted and had constant access to food and water over 72 103

hours. Plasma 3H-tracer appearance was measured every 24 hours and liver (collected after 72 104

hours) and feces (collected over 72 hours) were used to measure 3H-tracer recovery in 105

cholesterol and bile acids fraction after chemical extraction. 106

Data are expressed as mean ± SEM. Unpaired Student t-test or 1-way ANOVA + Dunnett 107

post-test was used for statistical analysis. A p<0.05 was considered significant. 108

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Results 115

Empagliflozin treatment significantly triggered more biochemical parameter changes in 116

overnight fasting condition than in fed condition (table 1). 117

Plasma LDL-cholesterol levels were found higher by 25% in hamsters treated with 118

empagliflozin (P<0.05 vs. vehicle) only in fasting condition. Concomitantly, fasting blood 119

glucose was reduced by 18% (P<0.05 vs. vehicle) while plasma total ketone bodies and free 120

fatty acids were raised by 116% (P<0.001 vs. vehicle) and 49% (P<0.01 vs. vehicle), 121

respectively. Hepatic total cholesterol and fatty acids levels in overnight fasting conditions 122

were 10% and 8% higher in hamsters treated with empagliflozin (both P<0.05 vs. vehicle). As 123

well, hepatic total ketone bodies levels were 14% higher with empagliflozin, although not 124

significantly. Hepatic pyruvate levels and HMG-CoA reductase activity were 19% and 31% 125

higher, respectively, in overnight fasted hamsters treated with empagliflozin (both P<0.05 vs. 126

vehicle). Compared with vehicle, hepatic glycogen levels were dramatically blunted by 84% 127

with empagliflozin (P<0.001 vs. vehicle). In sharp contrast with the fasting condition, 128

empagliflozin showed limited effects on biochemical parameters measured in fed condition 129

with the exception of minor difference on haematocrit, liver weight and plasma free glycerol 130

compared with vehicle. 131

To further confirm the raise in plasma LDL-cholesterol levels total cholesterol lipoprotein 132

profile in overnight fasted hamsters was measured by FPLC (figure 1A). As expected, 133

empagliflozin led to higher total cholesterol levels in fractions corresponding to LDL. Since 134

higher plasma LDL-cholesterol may be linked to lower LDL-receptor expression, western blot 135

analysis was also performed using liver samples collected from overnight fasted hamsters. 136

Compared with vehicle, hepatic protein expression of the LDL-receptor was found to be 137

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reduced by 20% (figure 1B) in overnight fasted hamsters treated with empagliflozin (P<0.05 138

vs. vehicle). 139

As higher LDL-cholesterol levels could also be related to increased intestinal cholesterol 140

absorption, this mechanism was also measured in vivo after oral administration of 14

C-141

cholesterol labelled olive oil. Strikingly, hamsters treated with empagliflozin showed a 14

C-142

tracer plasma appearance reduced by up to 40% over 6 hours after 14

C-tracer administration, 143

indicating lower intestinal cholesterol absorption (figure 1C). In agreement with the lower 144

intestinal cholesterol absorption, fecal cholesterol mass excretion was 49% higher in hamsters 145

treated with empagliflozin (figure 1D), as compared with vehicle (P<0.01). 146

We next investigated LDL-cholesterol metabolism in vivo by injecting 3H-cholesteryl oleate 147

labelled LDL intravenously in hamsters. Empagliflozin treatment resulted in slowed 3H-tracer 148

decay curve over 72 hours, leading to a 20% reduction in LDL-cholesteryl ester catabolism 149

(figure 2A), as compared with vehicle (P<0.05). At 72 hours after 3H-cholesteryl oleate 150

labelled LDL, hepatic 3H-tracer recoveries in the whole liver and the hepatic cholesterol 151

fraction were respectively reduced by 11% (P<0.01 vs. vehicle) and 19% (P<0.001 vs. 152

vehicle) with empagliflozin treatment (figure 2B). As a result of reduced cholesterol 153

absorption in the intestine, LDL-derived 3H-cholesterol fecal excretion was 26% higher 154

(P<0.05 vs. vehicle) in hamsters treated with empagliflozin (figure 2C). 155

To investigate macrophage-to-feces reverse cholesterol transport in vivo, hamsters were 156

injected intraperitoneally with 3H-cholesterol labelled/oxidized LDL loaded macrophages. 157

Compared with vehicle, empagliflozin did not change plasma 3H-tracer appearance over 72 158

hours (figure 2D). Hepatic 3H-tracer recoveries in the whole liver and the hepatic cholesterol 159

fraction tended to be reduced with empagliflozin, although this was not significant (figure 160

2E). However, 3H-cholesterol fecal excretion (figure 2F) was increased by 29% in hamsters 161

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treated with empagliflozin (P<0.05). This data indicate that reduced intestinal cholesterol 162

absorption with empagliflozin treatment promotes fecal excretion of cholesterol deriving from 163

the macrophage. 164

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Discussion 181

The present study indicates that empagliflozin raises LDL-cholesterol levels only in fasting 182

conditions through reduction in LDL-cholesterol catabolism and alters cholesterol metabolism 183

at both the hepatic and intestinal levels in hamsters. 184

Overnight fasted hamsters treated with empagliflozin showed higher LDL-cholesterol levels 185

concomitant with higher free fatty acids and total ketone bodies plasma levels. The higher 186

level of total ketone bodies and fatty acids is in agreement with previous reports indicating 187

that chronic treatment with SGLT2 inhibitors induces ketogenesis and a metabolism switch 188

towards lipid oxidation to counter-balance the carbohydrate restriction in the fasting state (8-189

10). The excretion of glucose via urine and related calories loss with SGLT2 inhibition 190

therefore replicates starvation shift from carbohydrate to lipid utilization for energy in the 191

fasting state (11). Chronic SGLT2 inhibition also seems to mimic the LDL-raising effects of 192

ketogenic diet, in which LDL-cholesterol levels correlate with blood ketone bodies levels 193

(12). In the present study, evidence for a metabolic shift towards fat utilisation was also 194

observed at the liver level (e.g. hepatic glycogen and pyruvate levels) in fasted hamsters 195

treated with empagliflozin. The increased hepatic fatty acids levels, may fuel the pool of 196

acetyl-CoA, an important metabolic branch point, as a source for both ketone bodies 197

production and hepatic cholesterol synthesis (13), the later associated with a higher HMGCoA 198

reductase activity and hepatic total cholesterol levels. As hepatic levels of cholesterol 199

regulates LDL-receptor expression (14, 15) empagliflozin treatment lowered LDL-receptor 200

expression and plasma LDL-cholesterol catabolism, which in turns increased LDL-cholesterol 201

plasma levels. Although a raise in LDL-cholesterol levels is seen as an increase in 202

cardiovascular events risk (16), it is probably not so prominent with empagliflozin. Indeed, 203

the Empa Reg Outcome study recently delivered a spectacular 38% reduction in 204

cardiovascular mortality and 35% reduction in hospitalisation with heart failure, with no 205

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change in event rate of nonfatal myocardial infarction and non-fatal stroke (17). Moreover, 206

our study revealed that even after chronic treatment with empagliflozin, the increase in LDL-207

C was only observed in overnight fasted condition. In the clinical setting, LDL-C levels are 208

routinely assessed from plasma collected in the fasted state. Therefore, clinical investigations 209

evaluating the effects of empagliflozin on LDL-C levels in fed conditions would be of 210

interest. In addition, our in vivo experiments also highlighted potential anti-atherogenic 211

mechanisms induced by empagliflozin, such as LDL- and macrophage-derived fecal 212

cholesterol excretion. Macrophage-to-feces reverse cholesterol transport is known to be 213

inversely correlated with atherosclerosis (18) and an enhanced excretion of LDL-derived 214

cholesterol in the feces theoretically prevents its accumulation in the arterial wall. Whether 215

these mechanisms, besides body weight loss and blood pressure lowering, contribute to the 216

reduced cardiovascular risk in patients treated with empagliflozin (17) remains to be further 217

investigated. 218

Another point of investigation is the reduced intestinal cholesterol absorption observed in 219

hamsters treated with empagliflozin. Since a balance exists between hepatic cholesterol 220

synthesis and intestinal cholesterol absorption (19), the lower intestinal cholesterol absorption 221

may therefore result from the stimulation of hepatic cholesterol synthesis by empagliflozin. 222

However, the molecular mechanism by which empagliflozin alters intestinal cholesterol 223

metabolism remains to be elucidated. 224

In conclusion, the present study suggests that empagliflozin raises LDL-cholesterol levels 225

only in fasting condition by reducing LDL-receptor expression and LDL-cholesterol 226

catabolism. As illustrated in figure 3, the proposed mechanism leading to the LDL-C increase 227

originates from the metabolic switch toward lipid utilization, which triggers in parallel a 228

moderate activation of ketogenesis pathway and hepatic cholesterol synthesis within the liver. 229

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Future studies to test whether SGLT2 inhibitors have similar rhythmic effect in plasma from 230

patients, fasted versus fed would be required. 231

232

Authors’ contribution: F.B., E.M, M.M. and T.S. designed research. F.B., E.B., N.B., I.U. 233

and C.C. conducted research, F.B. and E.M. analyzed data and wrote the paper. T.S. had 234

primary responsibility for final content. All authors have read and approved the final 235

manuscript. 236

237

Acknowledgements 238

The authors thank Dominique Lopes for animal care, Marjolaine Quinsat and Hélène Lakehal 239

for technical assistance; and Aurélie Couderc for quality control. 240

Guarantor: Thierry Sulpice is the guarantor of this work and, as such, had full access to all 241

the data in the study and takes responsibility for the integrity of the data and the accuracy of 242

the data analysis. 243

Conflict of interest: Eric Mayoux and Michael Mark are employees of Boehringer Ingelheim. 244

All other authors are employees of Physiogenex 245

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References 251

1 Nauck MA. Update on developments with SGLT2 inhibitors in the management of 252

type 2 diabetes. Drug Des Devel Ther 2014;8:1335-1380 253

2 Vivian EM. Sodium-glucose co-transporter (SGLT2) inhibitors: a growing class of 254

antidiabetic agents. Drugs Context 2014;3:212264 255

3 Maliha G, Townsend RR. SGLT2 inhibitors: Their potential reduction in blood 256

pressure. J Am Soc Hypertens 2015;9:48-53 257

4 Pieber TR, Famulla S, Eilbracht J, Cescutti J, Soleymanlou N, Johansen OE, Woerle 258

HJ, Broedl UC, Kaspers S. Empagliflozin as adjunct to insulin in patients with type 1 259

diabetes: a 4-week, randomized, placebo-controlled trial (EASE-1). Diabetes Obes Metab 260

2015;17:928-935 261

5 Lund SS, Sattar N, Salsali A, Crowe S, Broedl UC, Ginsberg HN. Potential relevance 262

of changes in haematocrit to changes in lipid parameters with empagliflozin in patients with 263

type 2 diabetes (Abstract – P750). Diabetologia 2015;58(suppl 1):S360 264

6 Briand F, Thieblemont Q, Muzotte E, Sulpice T. Upregulating Reverse Cholesterol 265

Transport With Cholesteryl Ester Transfer Protein Inhibition Requires Combination With the 266

LDL-Lowering Drug Berberine in Dyslipidemic Hamsters. Arterioscler Thromb Vasc Biol 267

2013;33:01-11 268

7 Briand F, Thieblemont Q, Muzotte E, Sulpice T. High-fat and fructose intake induces 269

insulin resistance, dyslipidemia, and liver steatosis and alters in vivo macrophage-to-feces 270

reverse cholesterol transport in hamsters. J Nutr 2012;142:704-709 271

8 Taylor SI, Blau JE, Rother KI. SGLT2 Inhibitors May Predispose to Ketoacidos. J 272

Clin Endocrinol Metab 2015;100:2849-528 273

274

of 20Diabetes

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9 Yokono M, Takasu T, Hayashizaki Y, Mitsuoka K, Kihara R, Muramatsu Y, Miyoshi 275

S, Tahara A, Kurosaki E, Li Q, Tomiyama H, Sasamata M, Shibasaki M, Uchiyama Y. 276

SGLT2 selective inhibitor ipragliflozin reduces body fat mass by increasing fatty acid 277

oxidation in high-fat diet-induced obese rats. Eur J Pharmacol 2014;727:66-74 278

10 Ferrannini E, Muscelli E, Frascerra S, Baldi S, Mari A, Heise T, Broedl UC, Woerle 279

HJ. Metabolic response to sodium-glucose cotransporter 2 inhibition in type 2 diabetic 280

patients. J Clin Invest 2014;124, 499-508 281

11 Aoki TT. Metabolic adaptations to starvation, semistarvation, and carbohydrate 282

restriction. Prog Clin Biol Res 1981;67:161-177 283

12 Johnston CS, Tjonn SL, Swan PD, White A, Hutchins H, Sears B. Ketogenic low-284

carbohydrate diets have no metabolic advantage over nonketogenic low-carbohydrate diets. 285

Am J Clin Nutr 2006;83:1055-1061 286

13 Coffee CJ. Branch point in Metabolism, p163. Book: Metabolism, Hayes Barton Press, 287

2004. ISBN-13: 978-1593771928 288

14 Brown MS, Golstein JL. A proteolytic pathway that controls the cholesterol content of 289

membranes, cells, and blood. Proc Natl Acad Sci 1999;96:11041-11048 290

15 Singh AB, Kan CF, Shende V, Dong B, Liu J. A novel posttranscriptional mechanism 291

for dietary cholesterol-mediated suppression of liver LDL receptor expression. J Lipid Res 292

2014;55:1397-1407 293

16 Ferrières J. Effects on coronary atherosclerosis by targeting low-density lipoprotein 294

cholesterol with statins. Am J Cardiovasc Drugs 2009;9:109-115 295

17 Zinman B, Wanner C, Lachin JM, Fitchett D, Bluhmki E, Hantel S, Mattheus M, 296

Devins T, Johansen OE, Woerle HJ, Broedl UC, Inzucchi SE; EMPA-REG OUTCOME 297

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Investigators. Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes. 298

New Engl J Med 2015;373:2117-2128 299

18 Rader DJ, Alexander ET, Weibel GL, Billheimer J, Rothblat GH. The role of reverse 300

cholesterol transport in animals and humans and relationship to atherosclerosis. J Lipid Res 301

2009;50:S189-S194 302

19 Miettinen TA, Gylling H, Viikari J, Lehtimäki T, Raitakari OT. Synthesis and 303

absorption of cholesterol in Finnish boys by serum non-cholesterol sterols: the cardiovascular 304

risk in Young Finns Study. Atherosclerosis 2008;200:177-183 305

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Figures legends 306

307

Figure 1. Lipoprotein total cholesterol profiles assessed by Fast Protein Liquid 308

Chromatography from pooled plasma samples (A), representative western blots and 309

hepatic LDL-receptor protein expression after densitometry analysis (B), in vivo 310

intestinal 14C-cholesterol absorption (C) and fecal cholesterol mass excretion (D) in 311

hamsters treated with vehicle (white bars or open circles) or empagliflozin 30mg/kg/day 312

(black dashed bars or closed circles). *P<0.05, †P<0.01 and ‡P<0.001 vs. vehicle, n=9-10 313

hamsters/group. 314

315

Figure 2. 3H-cholesteryl oleate labeled LDL plasma decay curve over 72 hours and LDL-316

cholesteryl esters fractional catabolic rate (A), 3H-tracer recoveries in whole liver 317

homogenate, cholesterol and bile acids fractions (B), 3H-tracer recoveries in fecal 318

cholesterol and bile acids fractions (C) at time 72 hours after 3H-cholesteryl oleate 319

labeled LDL intravenous injection. 3H-tracer appearance in plasma over 72 hours (D), 320

3H-tracer recoveries in whole liver homogenate, cholesterol and bile acids fractions (E), 321

3H-tracer recoveries in fecal cholesterol and bile acids fractions (F) at time 72 hours 322

after 3H-cholesterol labeled/oxidized LDL loaded macrophages intraperitoneal injection. 323

Hamsters treated with vehicle or empagliflozin 30mg/kg/day are represented with white 324

bars, open circles or black dashed bars, closed circles). *P<0.05, †P<0.01 and ‡P<0.001 325

vs. vehicle, n=9-10 hamsters/group. 326

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Figure 3. Proposed mechanisms for the alteration of cholesterol metabolism by 328

empagliflozin. SGLT2 inhibition switches from carbohydrate to fat oxidation and 329

stimulates ketone bodies production and hepatic cholesterol synthesis in fasting 330

conditions. These metabolic alterations result in lower LDL-receptor expression and 331

moderate increase in LDL-cholesterol levels. The reduced intestinal cholesterol 332

absorption, which leads to higher macrophage- and LDL-derived cholesterol fecal 333

excretion remain to be further investigated. HMGCS1 & HMGCS2: 3-Hydroxy-3-334

Methylglutaryl-CoA synthases; HMGCoA red: 3-Hydroxy-3-methylglutaryl-CoA 335

reductase. 336

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TABLE 1. Body weight and biochemical parameters in fed or overnight fast conditions. Data are mean ± SEM. *P<0.05 vs. vehicle;

†P<0.01 vs. vehicle; ‡P<0.001 vs. vehicle, n=9-10 hamsters/group.

fed conditions overnight fasting conditions

parameters vehicle empagliflozin 30mg/kg vehicle empagliflozin 30mg/kg

body weight (g) 110 ± 2 114 ± 2 110 ± 2 111 ± 1

hematocrit (%) 49.8 ± 0.7 47.9 ± 0.6* 48.3 ± 0.5 49.4 ± 0.6

plasma total protein (g/L) 81.2 ± 1.8 81.9 ± 1.8 79.6 ± 2.5 76.0 ± 1.0

blood glucose (mg/dL) 86.0 ± 5.5 88.6 ± 2.6 73.4 ± 4.0 59.9 ± 2.5*

Plasma total cholesterol (g/L) 4.0 ± 0.2 4.0 ± 0.2 3.0 ± 0.1 2.9 ± 0.2

Plasma LDL-cholesterol (g/L) 1.8 ± 0.1 1.6 ± 0.1 1.2 ± 0.1 1.5 ± 0.1*

Plasma ketone bodies (µM) 773 ± 76 909 ± 124 3094 ± 171 6685 ± 510‡

Plasma free fatty acids (mM) 0.62 ± 0.06 0.70 ± 0.05 0.45 ± 0.03 0.67 ± 0.05†

Plasma free glycerol (g/L) 0.023 ± 76 0.033 ± 0.004* 0.009 ± 0.001 0.011 ± 0.001

Liver weight (g) 5.61 ± 0.13 6.04 ± 0.13* 4.90 ± 0.13 4.75 ± 0.06

Hepatic triglycerides (mg/g liver) 15.1 ± 0.9 16.9 ± 0.1 16.6 ± 1.3 15.3 ± 0.7

Hepatic cholesterol (mg/g liver) 38.9 ± 0.8 40.2 ± 1.7 43.1 ± 1.9 47.7 ± 1.1*

Hepatic fatty acids (µmol/g liver) 362 ± 9 352 ± 12 386 ± 11 418 ± 8*

Hepatic ketone bodies

(µmol/g liver) 12.4 ± 0.5 12.1 ± 0.5

14.7 ± 0.6 16.8 ± 0.8

Hepatic pyruvate (µmol/g liver) 6.2 ± 0.5 6.4 ± 0.3 6.7 ± 0.4 8.0 ± 0.3*

Hepatic HMGCoAred activity

(mU/mg protein) 0.302 ± 0.034 0.357 ± 0.040

0.255 ± 0.019 0.334 ± 0.028*

Hepatic glycogen (mg/g liver) 39.1 ± 3.9 37.3 ± 2.2 4.31 ± 0.64 0.7 ± 0.4‡

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Figure 2.

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Figure 3.

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