Empagliflozin, Via Switching Metabolism Toward Lipid Utilization, Moderately Increases LDL Cholesterol Levels Through Reduced LD

2032 Diabetes Volume 65, July 2016

François Briand,1 Eric Mayoux,2 Emmanuel Brousseau,1 Noémie Burr,1 Isabelle Urbain,1 Clément Costard,1 Michael Mark,2 and Thierry Sulpice1

Empagliﬂozin, via Switching Metabolism Toward Lipid Utilization, Moderately Increases LDL Cholesterol Levels Through Reduced LDL Catabolism

Diabetes 2016;65:2032–2038 | DOI: 10.2337/db16-0049

In clinical trials, a small increase in LDL cholesterol has Specific sodium glucose cotransporter (SGLT) inhibitors been reported with sodium–glucose cotransporter 2 represent an emerging and promising new class of glucose- (SGLT2) inhibitors. The mechanisms by which the SGLT2 in- lowering drugs in the management of type 2 diabetes. hibitor empagliflozin increases LDL cholesterol levels were Theuniquemodeofactionofthisclassofnovelagents investigated in hamsters with diet-induced dyslipidemia. can effectively decrease blood glucose levels, independently Compared with vehicle, empagliflozin 30 mg/kg/day for of the insulin pathway, via increasing glucose excretion in 2 weeks significantly reduced fasting blood glucose by urine, i.e., glucosuria (1,2). Besides improved glycemic pa- 18%, with significant increase in fasting plasma LDL rameters, SGLT2 inhibitors have shown additional benefits cholesterol, free fatty acids, and total ketone bodies by such as body weight loss and blood pressure–lowering, with 25, 49, and 116%, respectively. In fasting conditions, low risk of hypoglycemia (3). However, an increase in LDL glycogen hepatic levels were further reduced by 84% with cholesterol (LDL-C) plasma levels has also been observed in empagliflozin, while 3-hydroxy-3-methylglutaryl-CoA patients treated with SGLT2 inhibitors (1). The mechanism reductase activity and total cholesterol hepatic levels by which SGLT2 inhibition raises LDL-C levels remains were 31 and 10% higher, respectively (both P < 0.05 vs. unclear. It has been suggested that the increase in LDL-C vehicle). A significant 20% reduction in hepatic LDL

PHARMACOLOGY AND THERAPEUTICS receptor protein expression was also observed with may be partly due to hemoconcentration, as SGLT2 inhib- empagliflozin. Importantly, none of these parameters were itors induce volume contraction subsequent to increased changed by empagliflozin in fed conditions. Empagliflozin urinary volume (4,5). However, the transient diuretic effect significantly reduced the catabolism of 3H-cholesteryl of SGLT2 inhibitors may not completely contribute to oleate–labeled LDL injected intravenously by 20%, in- theobservedLDL-Cincrease.Wethereforeinvestigated dicating that empagliflozin raises LDL levels through the effects of the SGLT2 inhibitor empagliflozin in the reduced catabolism. Unexpectedly, empagliflozin also re- diet-induced insulin-resistant dyslipidemic golden Syrian duced intestinal cholesterol absorption in vivo, which led hamster, a validated preclinical model with cholesterol to a significant increase in LDL- and macrophage-derived metabolism similar to that of humans (6,7). cholesterol fecal excretion (both P < 0.05 vs. vehicle). These data suggest that empagliflozin, by switching RESEARCH DESIGN AND METHODS energy metabolism from carbohydrate to lipid utilization, moderately increases ketone production and LDL choles- All animal protocols were approved by the local (Comité terol levels. Interestingly, empagliflozin also reduces intes- régional d’éthique de Midi-Pyrénées) and national (Ministère tinal cholesterol absorption, which in turn promotes de l’Enseignement Supérieur et de la Recherche) ethics LDL- and macrophage-derived cholesterol fecal excretion. committees.MalegoldenSyrianhamsters(91–100 g,

1Physiogenex SAS, Prologue Biotech, Labège, France F.B. and E.M. contributed equally to this study. 2 Cardiometabolic Diseases Research, Boehringer Ingelheim, Biberach an der © 2016 by the American Diabetes Association. Readers may use this article as Riss, Germany long as the work is properly cited, the use is educational and not for proﬁt, and Corresponding author: François Briand, [email protected]. the work is not altered. Received 11 January 2016 and accepted 31 March 2016. diabetes.diabetesjournals.org Briand and Associates 2033

6 weeks old; Elevage Janvier, Le Genest Saint Isle, France) kinetics, or macrophage-to-feces reverse cholesterol transport were fed ad libitum over 4 weeks with a high-fat/high- as previously described (6,7). Intestinal cholesterol absorp- cholesterol diet (0.5% cholesterol, 0.25% deoxycholate, tion was assessed after administration of 14C-cholesterol– 11.5% coconut oil, and 11.5% corn oil) with 10% fructose labeled olive oil by oral gavage and intraperitoneal injection in the drinking water as previously described (7). After of poloxamer-407 (a lipase inhibitor) to measure 14C-tracer 2 weeks of diet to induce dyslipidemia, hamsters were plasma tracer appearance at time 3, 5, and 6 h after oral ga- randomized into two sets of nonradioactive (set 1) or vage (6). Kinetics of LDL cholesteryl oleate were performed radioactive (set 2) experiments, according to blood glu- by intravenously injecting 3H-cholesteryl oleate–labeled cose and LDL-C levels in fed or overnight fasting condi- LDL in overnight fasted hamsters, previously isolated tions (fasting starting at 5:00 P.M. and blood collection at from hamsters fed the same high-fat/high-cholesterol ;8:00 A.M.), and were then treated orally for 2 weeks diet (7). Hamsters were kept fasted for the first 6 h of with vehicle or empagliflozin 30 mg/kg once daily. The the kinetic experiment and were then kept in individual dose was selected from a pilot study where glucose urine ex- cages with access to food and water for feces collection cretion was measured in this hamster model treated acutely over 72 h. Plasma 3H-tracer decay curve was monitored with empagliflozin 3, 10, and 30 mg/kg. The 30 mg/kg over 72 h after injection to calculate 3H-cholesteryl ole- dose was found to increase glucose urine excretion by ate LDL fractional catabolic rate using Simulation Anal- 1,200-fold versus vehicle, while the 3 and 10 mg/kg doses ysis and Modeling (SAAM II) software. Liver (collected showed a slighter effect (80- and 200-fold, respectively). At after 72 h) and feces were used to measure 3H-tracer the end of the treatment period, a first set of hamsters recovery in cholesterol and bile acid fraction after chem- was used to measure biochemical parameters using com- ical extraction (6,7). mercial kits in fed or overnight fasting conditions. Lipo- Macrophage-to-feces reverse cholesterol transport was protein total cholesterol profile was assessed using fast measured over 72 h after intraperitoneally injecting protein liquid chromatography analysis using one pooled 3H-cholesterol–labeled/oxidized LDL–loaded J774 mac- plasma sample (one pool per treatment group); Western rophages (6,7). In this experiment, hamsters were not blot analyses for LDL receptor protein expression and fecal fasted and had constant access to food and water over cholesterol mass excretion were performed as previously 72 h. Plasma 3H-tracer appearance was measured every described (7). A second set of hamsters underwent radio- 24 h, and liver (collected after 72 h) and feces (collected active tracer–based in vivo experiments to measure in- over 72 h) were used to measure 3H-tracer recovery in testinal cholesterol absorption, LDL cholesteryl esters cholesterol and bile acid fraction after chemical extraction.

Table 1—Body weight and biochemical parameters in fed or overnight fast conditions Fed conditions Overnight fasting conditions Empagliﬂozin Empagliﬂozin Parameters Vehicle 30 mg/kg Vehicle 30 mg/kg Body weight (g) 110 6 2 114 6 2 110 6 2 111 6 1 Hematocrit (%) 49.8 6 0.7 47.9 6 0.6* 48.3 6 0.5 49.4 6 0.6 Plasma total protein (g/L) 81.2 6 1.8 81.9 6 1.8 79.6 6 2.5 76.0 6 1.0 Blood glucose (mg/dL) 86.0 6 5.5 88.6 6 2.6 73.4 6 4.0 59.9 6 2.5* Plasma total cholesterol (g/L) 4.0 6 0.2 4.0 6 0.2 3.0 6 0.1 2.9 6 0.2 Plasma LDL-C (g/L) 1.8 6 0.1 1.6 6 0.1 1.2 6 0.1 1.5 6 0.1* Plasma ketone bodies (mmol/L) 773 6 76 909 6 124 3,094 6 171 6,685 6 510‡ Plasma free fatty acids (mmol/L) 0.62 6 0.06 0.70 6 0.05 0.45 6 0.03 0.67 6 0.05† Plasma free glycerol (g/L) 0.023 6 76 0.033 6 0.004* 0.009 6 0.001 0.011 6 0.001 Liver weight (g) 5.61 6 0.13 6.04 6 0.13* 4.90 6 0.13 4.75 6 0.06 Hepatic triglycerides (mg/g liver) 15.1 6 0.9 16.9 6 0.1 16.6 6 1.3 15.3 6 0.7 Hepatic cholesterol (mg/g liver) 38.9 6 0.8 40.2 6 1.7 43.1 6 1.9 47.7 6 1.1* Hepatic fatty acids (mmol/g liver) 362 6 9 352 6 12 386 6 11 418 6 8* Hepatic ketone bodies (mmol/g liver) 12.4 6 0.5 12.1 6 0.5 14.7 6 0.6 16.8 6 0.8 Hepatic pyruvate (mmol/g liver) 6.2 6 0.5 6.4 6 0.3 6.7 6 0.4 8.0 6 0.3* Hepatic HMG-CoAred activity (mU/mg protein) 0.302 6 0.034 0.357 6 0.040 0.255 6 0.019 0.334 6 0.028* Hepatic glycogen (mg/g liver) 39.1 6 3.9 37.3 6 2.2 4.31 6 0.64 0.7 6 0.4‡ Data are mean 6 SEM. n =9–10 hamsters/group. HMG-CoAred, HMG-CoA reductase. *P , 0.05 vs. vehicle. †P , 0.01 vs. vehicle. ‡P , 0.001 vs. vehicle. 2034 SGLT2 Inhibition and LDL-C Diabetes Volume 65, July 2016

Data are expressed as mean 6 SEM. Unpaired Stu- P , 0.05 vs. vehicle). In addition, hepatic total ketone dent t test or one-way ANOVA plus Dunnett posttest was body levels were 14% higher with empagliflozin, al- used for statistical analysis. A P , 0.05 was considered though not significantly. Hepatic pyruvate levels and significant. 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity were 19 and 31% higher, respectively, in overnight fasted RESULTS hamsters treated with empagliflozin (both P , 0.05 vs. Empagliflozin treatment significantly triggered more bio- vehicle). Compared with vehicle, hepatic glycogen levels chemical parameter changes in the overnight fasting con- were dramatically blunted by 84% with empagliflozin dition than in the fed condition (Table 1). (P , 0.001 vs. vehicle). In sharp contrast with the fasting Plasma LDL-C levels were found to be higher by 25% in condition, empagliflozin showed limited effects on bio- hamsters treated with empagliflozin (P , 0.05 vs. vehicle) chemical parameters measured in the fed condition with only in the fasting condition. Concomitantly, fasting blood the exception of minor differences on hematocrit, liver glucose was reduced by 18% (P , 0.05 vs. vehicle), while weight, and plasma free glycerol compared with vehicle. plasma total ketone bodies and free fatty acids were raised For further confirmation of the raise in plasma LDL-C by 116% (P , 0.001 vs. vehicle) and 49% (P , 0.01 vs. levels, total cholesterol lipoprotein profile in overnight vehicle), respectively. Hepatic total cholesterol and fatty fasted hamsters was measured by fast protein liquid acid levels in overnight fasting conditions were 10 and chromatography (Fig. 1A). As expected, empagliflozin led 8% higher in hamsters treated with empagliflozin (both to higher total cholesterol levels in fractions corresponding

Figure 1—Lipoprotein total cholesterol profiles assessed by fast protein liquid chromatography from pooled plasma samples (A), representative Western blots and hepatic LDL receptor protein expression after densitometry analysis (B), in vivo intestinal 14C-cholesterol absorption (C), and fecal cholesterol mass excretion (D) in hamsters treated with vehicle or empagliflozin 30 mg/kg/day. *P < 0.05 and ***P < 0.001. n =9–10 hamsters/group. diabetes.diabetesjournals.org Briand and Associates 2035 to LDL. Since higher plasma LDL-C may be linked to lower As higher LDL-C levels could also be related to increased LDL receptor expression, Western blot analysis was also intestinal cholesterol absorption, this mechanism was also performed using liver samples collected from overnight measured in vivo after oral administration of 14C-cholesterol– fasted hamsters. Compared with vehicle, hepatic protein labeled olive oil. Strikingly, hamsters treated with empagli- expression of the LDL receptor was found to be reduced flozin showed a 14C-tracer plasma appearance reduced by by 20% (Fig. 1B) in overnight fasted hamsters treated with up to 40% over 6 h after 14C-tracer administration, indi- empagliflozin (P , 0.05 vs. vehicle). cating lower intestinal cholesterol absorption (Fig. 1C). In

Figure 2—3H-cholesteryl oleate–labeled LDL plasma decay curve over 72 h and LDL cholesteryl ester fractional catabolic rate (A); 3H-tracer recoveries in whole liver homogenate, cholesterol, and bile acid fractions (B); and 3H-tracer recoveries in fecal cholesterol and bile acids fractions (C) at time 72 h after 3H-cholesteryl oleate–labeled LDL intravenous injection. 3H-tracer appearance in plasma over 72 h (D); 3H-tracer recoveries in whole liver homogenate, cholesterol, and bile acid fractions (E); and 3H-tracer recoveries in fecal cholesterol and bile acids fractions (F) at time 72 h after 3H-cholesterol–labeled/oxidized LDL–loaded macrophage intraperitoneal injection. Hamsters treated with vehicle or empagliflozin 30 mg/kg/day are represented with white bars, open circles, or black dashed bars, closed circles, respectively. *P < 0.05, **P < 0.01, and ***P < 0.001. n =9–10 hamsters/group. 2036 SGLT2 Inhibition and LDL-C Diabetes Volume 65, July 2016 agreement with the lower intestinal cholesterol absorption, empagliflozin, although this was not significant (Fig. 2E). fecal cholesterol mass excretion was 49% higher in hamsters However, 3H-cholesterol fecal excretion (Fig. 2F)was treated with empagliflozin (Fig. 1D) compared with vehicle increased by 29% in hamsters treated with empagliflozin (P , 0.01). (P , 0.05). These data indicate that reduced intestinal We next investigated LDL-C metabolism in vivo by cholesterol absorption with empagliflozin treatment pro- injecting 3H-cholesteryl oleate–labeled LDL intravenously motes fecal excretion of cholesterol deriving from the in hamsters. Empagliflozin treatment resulted in slowed macrophage. 3H-tracer decay curve over 72 h, leading to a 20% reduction in LDL cholesteryl ester catabolism (Fig. 2A), compared with DISCUSSION vehicle (P , 0.05). At 72 h after 3H-cholesteryl oleate– The current study indicates that empagliflozin raises LDL-C labeled LDL, hepatic 3H-tracer recoveries in the whole liver levels only in fasting conditions through reduction in LDL-C and the hepatic cholesterol fraction were respectively reduced catabolism and alters cholesterol metabolism at both the by 11% (P , 0.01 vs. vehicle) and 19% (P , 0.001 vs. hepatic and intestinal levels in hamsters. vehicle) with empagliflozin treatment (Fig. 2B). As a result Overnight fasted hamsters treated with empagliflozin of reduced cholesterol absorption in the intestine, LDL- showed higher LDL-C levels concomitant with higher derived 3H-cholesterol fecal excretion was 26% higher free fatty acids and total ketone body plasma levels. The (P , 0.05 vs. vehicle) in hamsters treated with empagli- higher level of total ketone bodies and fatty acids is in flozin (Fig. 2C). agreement with previous reports indicating that chronic For investigation of macrophage-to-feces reverse cho- treatment with SGLT2 inhibitors induces ketogenesis lesterol transport in vivo, hamsters were injected intraper- and a metabolism switch toward lipid oxidation to counter- itoneally with 3H-cholesterol–labeled/oxidized LDL–loaded balance the carbohydrate restriction in the fasting state macrophages. Compared with vehicle, empagliflozin did not (8–10). The excretion of glucose via urine and related change plasma 3H-tracer appearance over 72 h (Fig. 2D). calorie loss with SGLT2 inhibition therefore replicate Hepatic 3H-tracer recoveries in the whole liver and the starvation shift from carbohydrate to lipid utilization for hepatic cholesterol fraction tended to be reduced with energy in the fasting state (11). Chronic SGLT2 inhibition

Figure 3—Proposed mechanisms for the alteration of cholesterol metabolism by empagliflozin. SGLT2 inhibition switches from carbohydrate to fat oxidation and stimulates ketone body production and hepatic cholesterol synthesis in fasting conditions. These metabolic alterations result in lower LDL receptor (LDL-r) expression and moderate increase in LDL-C levels. The reduced intestinal cholesterol absorption, which leads to higher macrophage- and LDL-derived cholesterol fecal excretion, remains to be further investigated. HMGCS1, HMG-CoA synthase 1; HMGCS2, HMG-CoA synthase 2; HMGCoA red, HMG-CoA reductase. diabetes.diabetesjournals.org Briand and Associates 2037 also seems to mimic the LDL-raising effects of ketogenic switch toward lipid utilization, which triggers in parallel diet, in which LDL-C levels correlate with blood ketone a moderate activation of ketogenesis pathway and hepatic body levels (12). In the current study, evidence for a met- cholesterol synthesis within the liver. Future studies to abolic shift toward fat utilization was also observed at the test whether SGLT2 inhibitors have a similar rhythmic liver level (e.g., hepatic glycogen and pyruvate levels) in effect in plasma from patients fasted versus fed would be fasted hamsters treated with empagliflozin. The increased required. hepatic fatty acids levels may fuel the pool of acetyl-CoA, an important metabolic branch point, as a source for both ketone body production and hepatic cholesterol synthesis Acknowledgments. The authors thank Dominique Lopes for animal care, Marjolaine Quinsat and Hélène Lakehal for technical assistance, and Aurélie (13), with the latter associated with higher HMG-CoA Couderc for quality control (all of Physiogenex). reductase activity and hepatic total cholesterol levels. Duality of Interest. This work has been funded by Boehringer Ingelheim. As hepatic levels of cholesterol regulate LDL receptor E.M. and M.M. are employees of Boehringer Ingelheim. F.B., E.B., N.B., I.U., C.C., fl expression (14,15), empagli ozin treatment lowered LDL and T.S. are employees of Physiogenex. receptor expression and plasma LDL-C catabolism, which Author Contributions. F.B., E.M., M.M., and T.S. designed research. in turn increased LDL-C plasma levels. Although a raise in F.B., E.B., N.B., I.U., and C.C. conducted research. F.B. and E.M. analyzed data LDL-C levels is seen as an increase in cardiovascular event and wrote the manuscript. T.S. had the primary responsibility for the final risk (16), it is probably not so prominent with empagliflozin. content. All authors read and approved the final manuscript. T.S. is the Indeed, the EMPA-REG OUTCOME study (BI 10773 guarantor of this work and, as such, had full access to all the data in the study [Empagliflozin] Cardiovascular Outcome Event Trial in and takes responsibility for the integrity of the data and the accuracy of the Type 2 Diabetes Mellitus Patients) recently delivered a data analysis. Prior Presentation. Parts of this study were presented in abstract form at spectacular 38% reduction in cardiovascular mortality the 51st Annual Meeting of the European Association for the Study of Diabetes, and 35% reduction in hospitalization with heart failure, Stockholm, Sweden, 14–18 September 2015. with no change in event rate of nonfatal myocardial in- farction and nonfatal stroke (17). Moreover, our study References revealed that even after chronic treatment with empagli- 1. Nauck MA. Update on developments with SGLT2 inhibitors in the man- flozin, the increase in LDL-C was only observed in the agement of type 2 diabetes. Drug Des Devel Ther 2014;8:1335–1380 overnight fasted condition. In the clinical setting, LDL-C 2. Vivian EM. Sodium-glucose co-transporter 2 (SGLT2) inhibitors: a growing levels are routinely assessed from plasma collected in the class of antidiabetic agents. Drugs Context 2014;3:212264 3. Maliha G, Townsend RR. 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