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Effects of the sodium-glucose cotransporter 2 inhibitor dapagliflozin on substrate metabolism in prediabetic insulin resistant individuals: A randomized, double-blind crossover trial

  • Anna Veelen
    Affiliations
    Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands
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  • Charlotte Andriessen
    Affiliations
    Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands
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  • Yvo Op den Kamp
    Affiliations
    Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands
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  • Edmundo Erazo-Tapia
    Affiliations
    Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands
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  • Marlies de Ligt
    Affiliations
    Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands
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  • Julian Mevenkamp
    Affiliations
    Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands

    Department of Radiology and Nuclear Medicine, Maastricht University Medical Center, Maastricht, the Netherlands
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  • Johanna A. Jörgensen
    Affiliations
    Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands
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  • Esther Moonen-Kornips
    Affiliations
    Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands
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  • Gert Schaart
    Affiliations
    Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands
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  • Russell Esterline
    Affiliations
    BioPharmaceuticals R&D, Late-Stage Development, Cardiovascular, Renal and Metabolism, AstraZeneca, Gaithersburg, MD, USA
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  • Bas Havekes
    Affiliations
    Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands

    Department of Internal Medicine, Division of Endocrinology, Maastricht University Medical Center, Maastricht, the Netherlands
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  • Jan Oscarsson
    Affiliations
    BioPharmaceuticals R&D, Late-Stage Development, Cardiovascular, Renal and Metabolism, AstraZeneca, Gothenburg, Sweden
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  • Vera B. Schrauwen-Hinderling
    Affiliations
    Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands

    Department of Radiology and Nuclear Medicine, Maastricht University Medical Center, Maastricht, the Netherlands
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  • Esther Phielix
    Affiliations
    Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands
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  • Patrick Schrauwen
    Correspondence
    Corresponding author.
    Affiliations
    Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands
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Open AccessPublished:December 30, 2022DOI:https://doi.org/10.1016/j.metabol.2022.155396

      Highlights

      • SGLT2i treatment results in beneficial metabolic adaptations in non-diabetic, insulin resistant individuals
      • SGLT2i treatment led to lower 24-h glucose levels and higher free fatty acid and ß-hydroxybutyrate levels
      • SGLT2i treatment improved skeletal muscle mitochondrial oxidative capacity
      • SGLT2i treatment did not significantly affect hepatic glycogen levels, but seemed to affect muscle glycogen levels

      Abstract

      Aims/hypothesis

      Sodium-glucose cotransporter 2 inhibitor (SGLT2i) treatment in type 2 diabetes mellitus patients results in glucosuria, causing an energy loss, and triggers beneficial metabolic adaptations. It is so far unknown if SGLT2i exerts beneficial metabolic effects in prediabetic insulin resistant individuals, yet this is of interest since SGLT2is also reduce the risk for progression of heart failure and chronic kidney disease in patients without diabetes.

      Methods

      Fourteen prediabetic insulin resistant individuals (BMI: 30.3 ± 2.1 kg/m2; age: 66.3 ± 6.2 years) underwent 2-weeks of treatment with dapagliflozin (10 mg/day) or placebo in a randomized, placebo-controlled, cross-over design. Outcome parameters include 24-hour and nocturnal substrate oxidation, and twenty-four-hour blood substrate and insulin levels. Hepatic glycogen and lipid content/composition were measured by MRS. Muscle biopsies were taken to measure mitochondrial oxidative capacity and glycogen and lipid content.

      Results

      Dapagliflozin treatment resulted in a urinary glucose excretion of 36 g/24-h, leading to a negative energy and fat balance. Dapagliflozin treatment resulted in a higher 24-hour and nocturnal fat oxidation (p = 0.043 and p = 0.039, respectively), and a lower 24-hour carbohydrate oxidation (p = 0.048). Twenty-four-hour plasma glucose levels were lower (AUC; p = 0.016), while 24-hour free fatty acids and nocturnal β-hydroxybutyrate levels were higher (AUC; p = 0.002 and p = 0.012, respectively) after dapagliflozin compared to placebo. Maximal mitochondrial oxidative capacity was higher after dapagliflozin treatment (dapagliflozin: 87.6 ± 5.4, placebo: 78.1 ± 5.5 pmol/mg/s, p = 0.007). Hepatic glycogen and lipid content were not significantly changed by dapagliflozin compared to placebo. However, muscle glycogen levels were numerically higher in the afternoon in individuals on placebo (morning: 332.9 ± 27.9, afternoon: 368.8 ± 13.1 nmol/mg), while numerically lower in the afternoon on dapagliflozin treatment (morning: 371.7 ± 22.8, afternoon: 340.5 ± 24.3 nmol/mg).

      Conclusions/interpretation

      Dapagliflozin treatment of prediabetic insulin resistant individuals for 14 days resulted in significant metabolic adaptations in whole-body and skeletal muscle substrate metabolism despite being weight neutral. Dapagliflozin improved fat oxidation and ex vivo skeletal muscle mitochondrial oxidative capacity, mimicking the effects of calorie restriction.

      Trial registration

      ClinicalTrials.gov NCT03721874.

      Keywords

      Abbreviations:

      AU (arbitrary units), AUC (area under the curve), EGP (endogenous glucose production), ESM (electronic supplementary material), MRS (magnetic resonance spectroscopy), MTPRT (Macronutrient and Taste Preference Ranking Task), MUFA (monounsaturated fatty acids), NAFLD (non-alcoholic fatty liver disease), OGIS (oral glucose insulin sensitivity), PUFA (polyunsaturated fatty acids), RER (respiratory exchange ratio), SFA (saturated fatty acids), SGLT2 (sodium-glucose cotransporter 2), SGLT2i (sodium-glucose cotransporter 2 inhibitor), T2DM (type 2 diabetes mellitus)

      1. Introduction

      The sodium-glucose cotransporter 2 (SGLT2) inhibitor (SGLT2i) dapagliflozin is approved for treatment of type 2 diabetes mellitus (T2DM) as well as heart failure and chronic kidney disease irrespective of T2DM [
      • McMurray J.J.V.
      • Solomon S.D.
      • Inzucchi S.E.
      • et al.
      Dapagliflozin in patients with heart failure and reduced ejection fraction.
      ,
      • Wiviott S.D.
      • Raz I.
      • Bonaca M.P.
      • et al.
      Dapagliflozin and cardiovascular outcomes in type 2 diabetes.
      ,
      • Heerspink H.J.L.
      • Stefánsson B.V.
      • Correa-Rotter R.
      • et al.
      Dapagliflozin in patients with chronic kidney disease.
      ]. SGLT2i treatment results in the excretion of approximately 60–90 g of glucose per day in patients with T2DM. This glucosuria has been shown to induce a clinically relevant reduction in HbA1c and fasting glucose in patients with T2DM [
      • Zaccardi F.
      • Webb D.R.
      • Htike Z.Z.
      • Youssef D.
      • Khunti K.
      • Davies M.J.
      Efficacy and safety of sodium-glucose co-transporter-2 inhibitors in type 2 diabetes mellitus: systematic review and network meta-analysis.
      ,
      • Shyangdan D.S.
      • Uthman O.A.
      • Waugh N.
      SGLT-2 receptor inhibitors for treating patients with type 2 diabetes mellitus: a systematic review and network meta-analysis.
      ]. In addition, SGLT2i reduces body weight and blood pressure [
      • Shyangdan D.S.
      • Uthman O.A.
      • Waugh N.
      SGLT-2 receptor inhibitors for treating patients with type 2 diabetes mellitus: a systematic review and network meta-analysis.
      ,
      • Mazidi M.
      • Rezaie P.
      • Gao H.-K.
      • Kengne A.P.
      Effect of sodium-glucose Cotransport-2 inhibitors on blood pressure in people with type 2 diabetes mellitus: a systematic review and meta-analysis of 43 randomized control trials with 22 528 patients.
      ,
      • Muzurović E.
      • Mikhailidis D.P.
      Impact of glucagon-like peptide 1 receptor agonists and sodium-glucose transport protein 2 inhibitors on blood pressure and lipid profile.
      ], and restores water and sodium balance [
      • Lambers Heerspink H.J.
      • de Zeeuw D.
      • Wie L.
      • Leslie B.
      • List J.
      Dapagliflozin a glucose-regulating drug with diuretic properties in subjects with type 2 diabetes.
      ,
      • Ohara K.
      • Masuda T.
      • Morinari M.
      • et al.
      The extracellular volume status predicts body fluid response to SGLT2 inhibitor dapagliflozin in diabetic kidney disease.
      ]. We [
      • Op den Kamp Y.J.M.
      • de Ligt M.
      • Dautzenberg B.
      • et al.
      Effects of the SGLT2 inhibitor dapagliflozin on energy metabolism in patients with type 2 diabetes: a randomized, double-blind crossover trial.
      ] and others [
      • Daniele G.
      • Xiong J.
      • Solis-Herrera C.
      • et al.
      Dapagliflozin enhances fat oxidation and ketone production in patients with type 2 diabetes.
      ,
      • Ferrannini E.
      • Muscelli E.
      • Frascerra S.
      • et al.
      Metabolic response to sodium-glucose cotransporter 2 inhibition in type 2 diabetic patients.
      ,
      • Ferrannini E.
      • Baldi S.
      • Frascerra S.
      • et al.
      Shift to fatty substrate utilization in response to sodium-glucose cotransporter 2 inhibition in subjects without diabetes and patients with type 2 diabetes.
      ] have shown that SGLT2i treatment reduces carbohydrate oxidation and increases fat oxidation in patients with T2DM. Additionally, SGLT2i decreases diurnal plasma insulin levels and increases plasma free fatty acid, β-hydroxybutyrate, and glucagon levels [
      • Op den Kamp Y.J.M.
      • de Ligt M.
      • Dautzenberg B.
      • et al.
      Effects of the SGLT2 inhibitor dapagliflozin on energy metabolism in patients with type 2 diabetes: a randomized, double-blind crossover trial.
      ,
      • Yamada K.
      • Nakayama H.
      • Yoshinobu S.
      • et al.
      Effects of a sodium glucose co-transporter 2 selective inhibitor, ipragliflozin, on the diurnal profile of plasma glucose in patients with type 2 diabetes: a study using continuous glucose monitoring.
      ], and reduces hepatic lipid content in patients with T2DM [
      • Op den Kamp Y.J.M.
      • de Ligt M.
      • Dautzenberg B.
      • et al.
      Effects of the SGLT2 inhibitor dapagliflozin on energy metabolism in patients with type 2 diabetes: a randomized, double-blind crossover trial.
      ,
      • Latva-Rasku A.
      • Honka M.J.
      • Kullberg J.
      • et al.
      The SGLT2 inhibitor dapagliflozin reduces liver fat but does not affect tissue insulin sensitivity: a randomized, double-blind, placebo-controlled study with 8-week treatment in type 2 diabetes patients.
      ,
      • Eriksson J.W.
      • Lundkvist P.
      • Jansson P.A.
      • et al.
      Effects of dapagliflozin and n-3 carboxylic acids on non-alcoholic fatty liver disease in people with type 2 diabetes: a double-blind randomised placebo-controlled study.
      ,
      • Kahl S.
      • Gancheva S.
      • Straßburger K.
      • et al.
      Empagliflozin effectively lowers liver fat content in well-controlled type 2 diabetes: a randomized, double-blind, phase 4,placebo-controlled trial.
      ].
      The loss of urinary glucose by SGLT2i also leads to a compensatory increase in endogenous glucose production (EGP) in the overnight fasted state [
      • Op den Kamp Y.J.M.
      • de Ligt M.
      • Dautzenberg B.
      • et al.
      Effects of the SGLT2 inhibitor dapagliflozin on energy metabolism in patients with type 2 diabetes: a randomized, double-blind crossover trial.
      ,
      • Daniele G.
      • Xiong J.
      • Solis-Herrera C.
      • et al.
      Dapagliflozin enhances fat oxidation and ketone production in patients with type 2 diabetes.
      ,
      • Ferrannini E.
      • Muscelli E.
      • Frascerra S.
      • et al.
      Metabolic response to sodium-glucose cotransporter 2 inhibition in type 2 diabetic patients.
      ,
      • Merovci A.
      • Solis-Herrera C.
      • Daniele G.
      • et al.
      Dapagliflozin improves muscle insulin sensitivity but enhances endogenous glucose production.
      ,
      • Muscelli E.
      • Astiarraga B.
      • Barsotti E.
      • et al.
      Metabolic consequences of acute and chronic empagliflozin administration in treatment-naive and metformin pretreated patients with type 2 diabetes.
      ]. We hypothesized that – next to gluconeogenesis – increased glycogenolysis is responsible for the higher EGP. Such increased glycogenolysis could lead to a reduction of hepatic glycogen levels overnight, which could explain beneficial metabolic adaptations such as improvements in mitochondrial function and increased fat oxidation [
      • Izumida Y.
      • Yahagi N.
      • Takeuchi Y.
      • et al.
      Glycogen shortage during fasting triggers liver–brain–adipose neurocircuitry to facilitate fat utilization.
      ,
      • Schrauwen P.
      • van Marken Lichtenbelt W.D.
      • Saris W.H.
      • Westerterp K.R.
      Role of glycogen-lowering exercise in the change of fat oxidation in response to a high-fat diet.
      ], similar to those observed after caloric restriction [
      • Op den Kamp Y.J.M.
      • de Ligt M.
      • Dautzenberg B.
      • et al.
      Effects of the SGLT2 inhibitor dapagliflozin on energy metabolism in patients with type 2 diabetes: a randomized, double-blind crossover trial.
      ].
      Not only T2DM patients, but also prediabetic insulin resistant individuals are characterized by metabolic disturbances [
      • Kelley D.E.
      • Goodpaster B.
      • Wing R.R.
      • Simoneau J.A.
      Skeletal muscle fatty acid metabolism in association with insulin resistance, obesity, and weight loss.
      ,
      • Corpeleijn E.
      • Mensink M.
      • Kooi M.E.
      • Roekaerts P.M.H.J.
      • Saris W.H.M.
      • Blaak E.E.
      Impaired skeletal muscle substrate oxidation in glucose-intolerant men improves after weight loss.
      ]. Therefore, investigating potential effects of SGLT2i in prediabetic insulin resistant individuals on energy and substrate metabolism is of interest since restoring the 24-hour energy metabolism may be one of the mechanisms of SGLT2i contributing to reduced progression of chronic kidney disease and heart failure in patients without T2DM [
      • McMurray J.J.V.
      • Solomon S.D.
      • Inzucchi S.E.
      • et al.
      Dapagliflozin in patients with heart failure and reduced ejection fraction.
      ,
      • Wiviott S.D.
      • Raz I.
      • Bonaca M.P.
      • et al.
      Dapagliflozin and cardiovascular outcomes in type 2 diabetes.
      ,
      • Heerspink H.J.L.
      • Stefánsson B.V.
      • Correa-Rotter R.
      • et al.
      Dapagliflozin in patients with chronic kidney disease.
      ], who are, however, insulin resistant [
      • Sit D.
      • Kadiroglu A.K.
      • Kayabasi H.
      • Yilmaz M.E.
      The prevalence of insulin resistance in nondiabetic nonobese patients with chronic kidney disease.
      ,
      • Swan J.W.
      • Anker S.D.
      • Walton C.
      • et al.
      Insulin resistance in chronic heart failure: relation to severity and etiology of heart failure.
      ].
      Therefore, for the first time, we have investigated if SGLT2i treatment improves 24-hour energy- and substrate metabolism in prediabetic insulin resistant individuals and additionally, examine the subsequent SGLT2i effects on skeletal muscle mitochondrial oxidative capacity as a potential underlying mechanism of benefit. Furthermore, we test the hypothesis that the beneficial effects of SGLT2i on metabolic health include a reduction in overnight fasted hepatic glycogen levels as compensation for the urinary glucose loss.

      2. Research design and methods

      2.1 Study design and participants

      The study had a double-blinded, randomized, placebo-controlled, cross-over design and was conducted at Maastricht University, the Netherlands, between April 2019 and July 2021. Due to the COVID-19 pandemic, the study completion was delayed. The Ethics Committee of Maastricht University Medical Center approved the study, which was registered at clinicaltrials.gov (NCT03721874) and conducted conform the declaration of Helsinki [
      World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. Adopted by the 18th WMA General Assembly, Helsinki, Finland, June 1964 and amended by the: 29th WMA General Assembly, Tokyo, Japan, October 1975, 35th WMA General Assembly, Venice, Italy, October 1983, 41st WMA General Assembly, Hong Kong, September 1989, 48th WMA General Assembly, Somerset West, Republic of South Africa, October 1996, 52nd WMA General Assembly, Edinburgh, Scotland, October 2000, 53rd WMA General Assembly, Washington DC, USA, October 2002 (Note of Clarification added), 55th WMA General Assembly, Tokyo, Japan, October 2004 (Note of Clarification added), 59th WMA General Assembly, Seoul, Republic of Korea, October 2008, and 64th WMA General Assembly, Fortaleza, Brazil, October 2013.
      ]. Male and female individuals between 40 and 75 years and BMI of 27–38 kg/m2 without T2DM were eligible for participation. Moreover, the eligible participants should have a sedentary lifestyle and an impaired glucose homeostasis based on one or a combination of criteria including impaired fasting glucose, impaired glucose tolerance, HbA1c ≥ 5.7 and ≤6.4 % (≥39 and ≤46 mmol/mol) and reduced glucose clearance rate ≤ 360 mL/min/m2 indicating insulin resistance calculated by the Oral Glucose Insulin Sensitivity (OGIS) model [
      • Mari A.
      • Pacini G.
      • Murphy E.
      • Ludvik B.
      • Nolan J.J.
      A model-based method for assessing insulin sensitivity from the oral glucose tolerance test.
      ]. Detailed eligibility criteria are presented in electronic supplementary material (ESM) Table 1.
      Table 1Participant characteristics.
      CharacteristicMeans ± SD or number

      Total (n = 14)
      Sex, female/male6/8
      Age, years66.3 ± 6.2
      Weight, kg89.1 ± 10.3
      BMI, kg/m230.0 ± 2.1
      Fat mass, %35.6 ± 7.4
      eGFR, mL/min99 ± 19
      Fasting plasma glucose, mmol/L5.8 ± 0.6
      2-hour plasma glucose, mmol/L7.7 ± 1.9
      Fasting plasma insulin, pmol/L100 ± 49
      HbA1c, %/mmol/mol5.5 ± 0.5/37.1 ± 5.3
      Glucose clearance, mL/min/m2310 ± 36
      n = 13.
      HOMA-IR4.3 ± 0.6
      Impaired glucose homeostasis (n)
       Impaired fasting glucose, 6.1–6.9 mmol/L3
       Impaired glucose tolerance, 7.8–11.1 mmol/L5
       Elevated HbA1c, 5.7–6.4 %6
       Impaired insulin sensitivity, OGIS ≤ 360 mL/min/m213
      Participant characteristics at baseline. Values are given as means ± SD or number (n = 14).
      a n = 13.

      2.2 Procedures

      After informed consent, participants were randomly assigned to two 14-day intervention periods in which each participant received either dapagliflozin (10 mg/day) or placebo, separated by a 6–8 weeks washout. Main measurements were performed on day 12–14 of each intervention period during which participants stayed at the research facility. Study medication was taken once daily in the morning after awakening, and was continued during the end-of-treatment measurements. A detailed overview of the schedule of measurements can be seen in ESM Fig. 1.
      Two days prior to their stay, participants were instructed to refrain from strenuous physical activities and alcohol consumption. On day 12, participants arrived at the research facility at 5 pm and entered a respiration chamber at 6.30 pm where they received a standardized dinner at 7 pm. Detailed information about the provided meals can be found in the ESM. The respiration chamber is a small room equipped with whole-chamber indirect calorimetry [Omnical, Maastricht Instruments, Maastricht, the Netherlands [
      • Schoffelen P.F.
      • Westerterp K.R.
      • Saris W.H.
      • Ten Hoor F.
      A dual-respiration chamber system with automated calibration.
      ]] to continuously measure oxygen consumption and carbon dioxide production. At 9 pm, participants completed the Macronutrient and Taste Preference Ranking Task [MTPRT [
      • de Bruijn S.E.M.
      • de Vries Y.C.
      • de Graaf C.
      • Boesveldt S.
      • Jager G.
      The reliability and validity of the macronutrient and taste preference ranking task: a new method to measure food preferences.
      ]] to determine their food preferences and at 10.30 pm the lights of the respiration chamber were switched off and participants were instructed to sleep.
      On day 13, participants were woken up at 6.45 am. During the day, participants stayed in the respiration chamber and followed a strict protocol consisting of subsequent blood draws, meals, and low-intensity activity exercises. Participants followed activity exercises to mimic physical activity levels in free-living conditions. Blood samples were collected at 17 time points spread over 24 h, see ESM for an exact overview of the blood samples. Standardized meals were provided at 8 am, 12 pm, 4 pm, and 6 pm directly after the corresponding blood draw. Lights were switched off at 10.30 pm and participants were instructed to sleep. Urine was collected for 24 h in 2 different aliquots of 16 or 8 h each to determine differences in daytime and night-time. Urinary nitrogen was measured to calculate protein oxidation and urinary glucose was measured to correct for glucose loss.
      On day 14, participants were woken up at 6.30 am. After leaving the respiration chamber, magnetic resonance spectroscopy (MRS) was performed at 7.30 am to measure hepatic glycogen, lipid content, and lipid composition. Subsequently at 9 am, blood pressure was measured and a skeletal muscle biopsy was taken according to the Bergström method [
      • Bergstrom J.
      Percutaneous needle biopsy of skeletal muscle in physiological and clinical research.
      ]. Breakfast was provided at 10 am and lunch at 12 pm. At 4 pm, a second skeletal muscle biopsy was performed which was followed by a second hepatic glycogen MRS examination at 5 pm. After the final MRS measurement was completed, the study protocol ended. A more detailed description of the measurement methods can be found in the ESM.

      2.3 Biochemical analyses

      Biochemical analyses are described in the ESM.

      2.4 Statistics

      Results are presented as mean ± SEM unless stated otherwise. Shapiro–Wilk normality test was performed to evaluate normal distribution. To compare differences between dapagliflozin and placebo treatment, a 2-tailed paired Student's t-test was used for normally distributed data and a Wilcoxon paired signed-rank for not normally distributed data. Pearson's correlation coefficient analyses were performed to identify associations between outcome measures. Analyses were performed for n = 14 unless specified otherwise. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 27.0.

      3. Results

      Fourteen men and women completed the study (ESM Fig. S2). Baseline characteristics are presented in Table 1. Out of the 14 participants, 13 participants were randomized based on OGIS ≤360 mL/min/m2 [
      • Mari A.
      • Pacini G.
      • Murphy E.
      • Ludvik B.
      • Nolan J.J.
      A model-based method for assessing insulin sensitivity from the oral glucose tolerance test.
      ], and one participant had a value of 363 mL/min/m2. This individual was randomized based on the HbA1c criteria (≥ 5.7 % / 39 mmol/mol) and HOMA-IR was 2.7, indicating insulin resistance [
      • Gayoso-Diz P.
      • Otero-González A.
      • Rodriguez-Alvarez M.X.
      • et al.
      Insulin resistance (HOMA-IR) cut-off values and the metabolic syndrome in a general adult population: effect of gender and age: EPIRCE cross-sectional study.
      ]. Among the 14 participants, 8 had prediabetes based on their fasting plasma glucose values (6.1–6.9 mmol/L) or their 2-hour plasma glucose values (7.8–11.1 mmol/L), and 6 had HbA1c levels ≥ 5.7 % (39 mmol/mol).
      After 14 days of treatment, body weight was similar between dapagliflozin and placebo periods (dapagliflozin: 88.5 ± 2.8, placebo: 88.1 ± 2.7 kg, p = 0.397, ESM Fig. 3A). No differences were found in HbA1c (dapagliflozin: 5.5 ± 0.1 (37.1 ± 1.5), placebo: 5.6 ± 0.1 (37.3 ± 1.2) % (mmol/mol), p = 0.780, ESM Fig. 3B). Systolic blood pressure (dapagliflozin: 141.6 ± 3.4, placebo: 147.9 ± 3.2 mmHg, p = 0.028, ESM Fig. 3C) and diastolic blood pressure (dapagliflozin: 87.3 ± 2.3, placebo: 91.1 ± 2.4 mmHg, p = 0.023, ESM Fig. 3D) were lower after dapagliflozin treatment compared to placebo, whereas heart rate was similar (dapagliflozin: 64.7 ± 4.0, placebo: 64.6 ± 4.3 beats/min, p = 0.556, ESM Fig. 3E).

      3.1 24-hour energy metabolism

      Twenty-four-hour urinary glucose excretion rate was higher after dapagliflozin compared to placebo (dapagliflozin: 1.52 ± 0.159, placebo: 0.004 ± 0.0002 g/h, p < 0.0001, Fig. 1A ), resulting in a total dapagliflozin-induced glucose excretion of ~36 g in 24-h. Glucose excretion rate after dapagliflozin was twice as high during daytime compared to night-time (daytime: 1.865 ± 0.190, night-time: 0.889 ± 0.127 g/h, Fig. 1B and C). No changes were observed in urine volume produced (dapagliflozin: 2060 ± 177, placebo: 2143 ± 145 mL/24-h, p = 0.571, Fig. 1D).
      Fig. 1
      Fig. 1Effect of dapagliflozin on 24-hour energy metabolism. A: 24-hour glucose excretion; B: daytime glucose excretion; C: night-time glucose excretion; D: urine volume; E: 24-hour energy expenditure; F: sleeping metabolic rate; G: 24-hour energy balance; H: 24-hour respiratory exchange ratio; I: 24-hour fat oxidation; J: 24-hour carbohydrate oxidation; K: 24-hour protein oxidation; L: 24-hour substrate balance, for carbohydrate (CHO) balance only the values corrected for glucose excretion are presented; M: day- and night-time respiratory exchange ratio; N: day- and night-time fat oxidation; O: day- and night-time carbohydrate oxidation. White bars represent placebo (P), and black bars represent dapagliflozin (D). * indicates statistical significance (p < 0.05). Data are expressed as mean ± SEM. CHO; carbohydrate.
      Twenty-four-hour energy expenditure was similar between dapagliflozin and placebo (dapagliflozin: 10.2 ± 0.3, placebo: 10.1 ± 0.4 MJ/24-h, p = 0.444, Fig. 1E). Similarly, sleeping metabolic rate did not differ between the treatment periods (dapagliflozin: 7.1 ± 0.3, placebo: 7.0 ± 0.3 MJ/24-h, p = 0.728, n = 11, Fig. 1F). During the stay in the respiration chamber, energy intake was kept similar during the two study periods, and energy balance was not different between the treatment conditions (dapagliflozin: −0.11 ± 0.15, placebo: -0.07 ± 0.14 MJ/24-h, p = 0.654, Fig. 1G). However, after correction for the glucose loss, energy balance was significantly different between dapagliflozin and placebo treatment (dapagliflozin: −0.71 ± 0.16, placebo: -0.07 ± 0.14 MJ/24-h, p < 0.0001, Fig. 1G).
      Twenty-four-hour respiratory exchange ratio (RER), reflecting the relative contribution of fat and carbohydrate oxidation, was numerically lower after dapagliflozin treatment (dapagliflozin: 0.814 ± 0.006, placebo: 0.827 ± 0.004, p = 0.051, Fig. 1H). Similarly, 24-h fat oxidation was significantly higher (dapagliflozin: 136.1 ± 7.6, placebo: 124.3 ± 6.0 g/24-h, p = 0.043, Fig. 1I) and carbohydrate oxidation was significantly lower after dapagliflozin treatment (dapagliflozin: 185.6 ± 12.0, placebo: 211.7 ± 10.3 g/24-h, p = 0.048, Fig. 1J). Protein oxidation was numerically higher after dapagliflozin treatment but did not reach statistical significance (dapagliflozin: 80.0 ± 3.7, placebo 76.8 ± 3.7 g/24-h, p = 0.100, Fig. 1K). As a result, fat balance tended to be more negative (dapagliflozin: −16.0 ± 5.9, placebo: −4.6 ± 4.1 g/24-h, p = 0.052, Fig. 1L) and carbohydrate balance was more positive after dapagliflozin treatment (dapagliflozin: 33.0 ± 10.4, placebo 6.1 ± 9.8 g/24-h, p = 0.048). However, after correction for glucosuria, the carbohydrate balance was not significantly different between dapagliflozin and placebo (dapagliflozin: −3.4 ± 9.8, placebo: 6.0 ± 9.8 g/24-h, p = 0.551, Fig. 1L), illustrating the tight regulation of 24-h carbohydrate balance. Protein balance was numerically lower after dapagliflozin compared to placebo, but this difference was not statistically significant (dapagliflozin: 10.5 ± 2.4, placebo: 13.5 ± 2.0 g/24-h, p = 0.079, Fig. 1L).

      3.2 Energy metabolism measured during daytime and night-time

      Daytime RER (from 7 am to 10.30 pm) was significantly lower after dapagliflozin treatment compared to placebo (dapagliflozin: 0.818 ± 0.005, placebo: 0.831 ± 0.004, p = 0.046, Fig. 1M). RER measured during the first night in the respiration chamber (night 1, from 12 am to 5.30 am) was significantly lower after dapagliflozin treatment (dapagliflozin: 0.809 ± 0.008, placebo: 0.826 ± 0.007, respectively, p = 0.019), whereas RER during the second night in the respiration chamber (night 2, from 12 am to 5.30 am) was not statistically significantly different between dapagliflozin and placebo (dapagliflozin: 0.803 ± 0.009, placebo: 0.816 ± 0.006, p = 0.125, n = 11), probably due to the higher fat content (45 energy%) of the meals provided in the respiration chamber during the day before night 2. Fat oxidation during daytime and night 1 were significantly higher, whereas during night 2 fat oxidation was numerically higher, but did not reach statistical significance, after dapagliflozin treatment versus placebo (daytime: 156.8 ± 9.1 versus 141.7 ± 7.4 g/24-h, p = 0.031, n = 14; night 1: 89.5 ± 4.8 versus 79.7 ± 4.2 g/24-h, p = 0.039, n = 14; night 2: 95.3 ± 6.2 versus 87.3 ± 4.1 g/24-h in dapagliflozin versus placebo, p = 0.110, n = 11, Fig. 1N). Carbohydrate oxidation during daytime and night 1 were significantly lower, whereas the lower night 2 carbohydrate oxidation did not reach statistical significance, after dapagliflozin treatment versus placebo (daytime: 224.5 ± 13.1 versus 254.4 ± 11.9 g/24-h, p = 0.050, n = 14; night 1: 114.8 ± 12.6 versus 140.3 ± 13.9 g/24-h, p = 0.018, n = 14; night 2: 106.9 ± 14.0 versus 124.7 ± 10.8 g/24-h in dapagliflozin versus placebo, p = 0.120, n = 11, Fig. 1O).

      3.3 Hepatic and skeletal muscle glycogen content

      Overnight fasting total hepatic glycogen, corrected for liver volume, was numerically lower after dapagliflozin treatment, but did not reach statistical significance between treatment periods (dapagliflozin: 0.169 ± 0.015, placebo: 0.188 ± 0.016 arbitrary units (AU), p = 0.129, n = 12, Fig. 2C , see Fig. 2A also showing the results when liver volume is not taken into account). Liver volume measured in the morning (p = 0.638, n = 12, Fig. 2B), or the afternoon was not affected by dapagliflozin (p = 0.282, n = 7, Fig. 2E). When comparing the change in total hepatic glycogen (corrected for liver volume), measured in the morning (7.30 am) versus afternoon (5 pm), there was no difference between the dapagliflozin and placebo periods (dapagliflozin: −12.2 ± 5.0, placebo: −11.7 ± 7.3 %, p = 0.966, n = 7, Fig. 2F, see Fig. 2D also showing the results when liver volume is not taken into account).
      Fig. 2
      Fig. 2Effect of dapagliflozin on hepatic and skeletal muscle glycogen content. A: Hepatic glycogen content, not corrected for liver volume, measured in the morning; B: liver volume measured in the morning; C: total hepatic glycogen, corrected for liver volume, measured in the morning; D: relative difference between morning and afternoon in hepatic glycogen content, not corrected for liver volume; E: relative difference between morning and afternoon in liver volume; F: relative difference between morning and afternoon in total hepatic glycogen, corrected for liver volume; G: skeletal muscle glycogen measured in the morning and afternoon; H: delta (afternoon minus morning) skeletal muscle glycogen. For A–C, n = 11; for D–F, n = 7; for G–H, n = 10. White bars represent placebo (P), and black bars represent dapagliflozin (D). * indicates statistical significance (p < 0.05). Data are expressed as mean ± SEM.
      Morning and afternoon skeletal muscle glycogen levels did not differ between dapagliflozin and placebo (morning: 371.7 ± 22.8 versus 332.9 ± 27.9 nmol/mg, p = 0.250, n = 10; afternoon: 340.5 ± 24.3 versus 368.8 ± 13.1 nmol/mg in dapagliflozin versus placebo, p = 0.191, n = 10; Fig. 2G). However, after dapagliflozin treatment, skeletal muscle glycogen levels numerically declined from morning to afternoon (morning: 371.7 ± 22.8, afternoon: 340.5 ± 24.3 nmol/mg, p = 0.058, n = 10, Fig. 2G), whereas after placebo skeletal muscle glycogen levels numerically increased from morning to afternoon (morning: 332.9 ± 27.9, afternoon: 368.8 ± 13.1 nmol/mg, p = 0.116, n = 10, Fig. 2G). Delta glycogen levels (afternoon minus morning) were negative upon dapagliflozin (−31.1 ± 14.3 nmol/mg) and positive upon placebo (35.9 ± 20.6 nmol/mg), and there was a trend toward a significant difference between the changes in glycogen during the day comparing dapagliflozin and placebo periods (p = 0.055, n = 10, Fig. 2H).

      3.4 24-hour blood values

      During the stay in the respiration chamber, blood samples were taken every 2 h during daytime (8 am – 10 pm) and hourly during night-time (11 pm – 7 am). Fasting plasma glucose at 8 am after an overnight fast was not different between treatment periods (dapagliflozin: 5.6 ± 0.1, placebo: 5.7 ± 0.2 mmol/L, p = 0.346, Fig. 3A ). However, the area under the curve (AUC) for 24-h plasma glucose profiles was lower after dapagliflozin treatment (p = 0.017) and tended to be lower during daytime and night-time (p = 0.064 and p = 0.056, respectively). Overnight fasting free fatty acid levels were not different between dapagliflozin and placebo (dapagliflozin: 331.1 ± 31.0, placebo: 307.3 ± 23.7 μmol/L, p = 0.463, Fig. 3B). However, AUC for 24-hour free fatty acid profiles, as well as day- and night-time profiles, were significantly higher after dapagliflozin (p = 0.002, p = 0.014, and p = 0.005 for 24-h, daytime, and night-time, respectively). Overnight fasting plasma insulin levels measured at 8 am were similar between dapagliflozin and placebo (dapagliflozin: 10.1 ± 1.0, placebo: 10.3 ± 1.2 mU/L, p = 0.749, Fig. 3C), whereas AUC for plasma insulin during daytime was numerically lower on dapagliflozin versus placebo (p = 0.074). Fasting free glycerol levels measured at 8 am after an overnight fast were not different between treatments (dapagliflozin: 25.2 ± 3.3, placebo: 30.5 ± 2.8 μmol/L, p = 0.109, n = 12, Fig. 3D), and similarly, AUC for 24-hour free glycerol profiles, as well as day- and night-time profiles, were not different between dapagliflozin and placebo (p = 0.781, p = 0.946, and p = 0.847 for 24-h, daytime, and night-time, respectively, n = 12). Plasma β-hydroxybutyrate levels during night-time are shown since β-hydroxybutyrate levels were below the detection limit during the daytime; AUC for plasma β-hydroxybutyrate was significantly higher after dapagliflozin treatment as compared to placebo (p = 0.012, Fig. 3E). Also, AUC for serum urea levels night-time was significantly higher after dapagliflozin treatment as compared to placebo (p = 0.016, Fig. 3F).
      Fig. 3
      Fig. 3Effect of dapagliflozin 24-hour blood profiles. A: Glucose; B: Free fatty acids; C: insulin; D: free glycerol; E: β-hydroxybutyrate; F: urea. White squares represent placebo, black dots represent dapagliflozin. Grey area represents the sleeping period and the arrows indicate meals. * indicates statistical significance (p < 0.05). Data are expressed as mean ± SEM.

      3.5 Skeletal muscle mitochondrial oxidative capacity

      Mitochondrial oxidative capacity was assessed at 9.15 am in permeabilized skeletal muscle fibers. ADP-stimulated (state 3) respiration on a lipid-derived substrate (malate + octanoyl-carnitine (MO): 30.5 ± 2.1 versus 29.0 ± 2.0 pmol/mg/s in dapagliflozin versus placebo, p = 0.237, Fig. 4A ) and upon addition of glutamate (MOG: 44.3 ± 2.3 versus 42.6 ± 2.3 pmol/mg/s in dapagliflozin versus placebo, p = 0.209, Fig. 4C) were not different between treatment periods. After the addition of succinate, state 3 respiration was numerically higher after dapagliflozin compared to placebo (MOGS: 68.2 ± 3.2 versus 64.4 ± 3.2 pmol/mg/s in dapagliflozin versus placebo, p = 0.071, Fig. 4C). No differences were observed in state 3 respiration without octanoyl-carnitine (MG: 40.4 ± 1.9 versus 41.3 ± 2.5 pmol/mg/s, p = 0.598; MGS: 72.7 ± 2.7 versus 73.0 ± 3.7 pmol/mg/s in dapagliflozin versus placebo, p = 0.881, Fig. 4B and C). Maximal FCCP-induced uncoupled respiration, which reflects the maximal oxidative capacity of the electron transport chain, was significantly higher after dapagliflozin treatment compared to placebo (dapagliflozin: 87.6 ± 5.4, placebo: 78.1 ± 5.5 pmol/mg/s, p = 0.007, Fig. 4D). State 4o respiration, reflecting a proton leak, was not different between treatment periods (dapagliflozin: 22.0 ± 1.3, placebo: 21.2 ± 1.4 pmol/mg/s, p = 0.560, Fig. 4E).
      Fig. 4
      Fig. 4Effect of dapagliflozin on ex vivo skeletal muscle mitochondrial respiration measured in the morning. A: ADP-stimulated state 3 respiration upon a lipid-derived substrate (MO); B: ADP-stimulated state 3 respiration upon Complex I substates (MG); C: ADP-stimulated state 3 respiration upon parallel electron input to both Complex I and Complex II (MOG, MOGS, and MGS); D: maximal uncoupled respiration upon FCCP; E: oligomycin induced respiration; F: mitochondrial protein expression of oxidative phosphorylation (OXPHOS) complex I, complex II, complex III, complex IV, and complex V; G: protein expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α); H: correlation of change in PGC-1α protein expression and change in maximal uncoupled respiration upon FCCP. White bars represent placebo, black bars represent dapagliflozin. * indicates statistical significance (p < 0.05). Data are expressed as mean ± SEM. C, complex; FCCP, carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone; M, malate; O, octanoyl-carnitine; G, glutamate; S, succinate; Oxphos, oxidative phosphorylation.
      The protein content of OXPHOS complex III was significantly higher after dapagliflozin compared to placebo (dapagliflozin: 1.19 ± 0.06, placebo: 0.98 ± 0.06 AU, p = 0.047, Fig. 4F), whereas OXPHOS complex V was numerically higher after dapagliflozin, but did not reach statistical significance (dapagliflozin: 1.20 ± 0.05, placebo: 1.03 ± 0.06 AU, p = 0.067). None of the other complexes were statistically different between the treatment arms (Complex I: 1.33 ± 0.12 versus 1.08 ± 0.12 AU, p = 0.137; Complex II: 1.29 ± 0.10 versus 1.11 ± 0.10 AU, p = 0.133; Complex IV: 1.15 ± 0.07 versus 1.06 ± 0.09 AU, p = 0.363 in dapagliflozin versus placebo).
      Since mitochondrial respiration has been shown to be controlled by PGC-1α [
      • Wu Z.
      • Puigserver P.
      • Andersson U.
      • et al.
      Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1.
      ], levels of PGC-1α protein were measured in the muscle biopsies. PGC-1α protein content did not differ between dapagliflozin and placebo (1.14 ± 0.18 versus 1.00 ± 0.15 AU, p = 0.239, n = 12, Fig. 4G). No significant correlation was observed between change in PGC-1α levels and change in maximal FCCP-induced uncoupled respiration comparing the dapagliflozin and placebo periods (r = 0.476, p = 0.118, n = 12, Fig. 4H).

      3.6 Hepatic lipid content and composition

      Five out of 14 participants had a hepatic lipid content ≥ 5.0 %, which is generally used as the cut-off to define non-alcoholic fatty liver disease (NAFLD). Dapagliflozin did not affect hepatic lipid content (dapagliflozin: 6.0 ± 2.4, placebo: 6.4 ± 2.6 %, p = 0.510, ESM Fig. 4A). Similarly, the fraction of SFA, MUFA, and PUFA were not different after dapagliflozin treatment (SFA: 41.0 ± 1.4 versus 40.7 ± 2.6 %, p = 0.890, n = 10; MUFA: 43.6 ± 1.9 versus 42.1 ± 2.5 %, p = 0.483, n = 7; PUFA: 16.4 ± 1.8 versus 18.0 ± 2.6 %, p = 0.517, n = 7 in dapagliflozin versus placebo, ESM Fig. 4B, C and D).

      3.7 Intramyocellular lipid content

      Dapagliflozin did not affect IMCL content in all fibers, type 1 fibers, or type 2 fibers (All: 0.51 ± 0.22 versus 0.46 ± 0.13 %, p = 0.972; Type 1: 0.61 ± 0.25 versus 0.56 ± 0.17 %, p = 0.650; Type 2: 0.43 ± 0.20 versus 0.39 ± 0.11 %, p = 0.701 in dapagliflozin versus placebo, n = 13, ESM Fig. 5).

      3.8 Food preferences

      To assess whether there were differences in food preferences, the participants filled out MTPRT on the evening of day 12. The relative preference for sweet products did not differ between treatment periods (dapagliflozin: 2.64 ± 0.8, placebo: 2.65 ± 0.9, p = 0.809, ESM Fig. 6A). Similarly, the relative preference for high-carbohydrate, high-fat, high-protein, or low-energy products was similar (p > 0.05, ESM Fig. 6B). No differences were found in liking ratings (VAS-scale 0–100) for sweet, savory, high-carbohydrate, high-fat, high-protein, or low-energy products (p > 0.05, ESM Fig. 6C).

      3.9 Safety

      No serious adverse events or events of diabetic ketoacidosis were reported.

      4. Discussion

      We here show that dapagliflozin can exert similar adaptive metabolic changes in energy metabolism in prediabetic insulin resistant individuals as in T2DM patients after only 14 days of treatment and no detectable effect on body weight. To compensate for the loss of glucose, it has been shown that SGLT2i treatment increase EGP in the overnight fasted state in patients with T2DM [
      • Op den Kamp Y.J.M.
      • de Ligt M.
      • Dautzenberg B.
      • et al.
      Effects of the SGLT2 inhibitor dapagliflozin on energy metabolism in patients with type 2 diabetes: a randomized, double-blind crossover trial.
      ,
      • Daniele G.
      • Xiong J.
      • Solis-Herrera C.
      • et al.
      Dapagliflozin enhances fat oxidation and ketone production in patients with type 2 diabetes.
      ,
      • Ferrannini E.
      • Muscelli E.
      • Frascerra S.
      • et al.
      Metabolic response to sodium-glucose cotransporter 2 inhibition in type 2 diabetic patients.
      ,
      • Merovci A.
      • Solis-Herrera C.
      • Daniele G.
      • et al.
      Dapagliflozin improves muscle insulin sensitivity but enhances endogenous glucose production.
      ,
      • Muscelli E.
      • Astiarraga B.
      • Barsotti E.
      • et al.
      Metabolic consequences of acute and chronic empagliflozin administration in treatment-naive and metformin pretreated patients with type 2 diabetes.
      ]. Until now only one study investigated the direct contribution of gluconeogenesis and glycogenolysis on SGLT2i-induced EGP [
      • Wolf P.
      • Fellinger P.
      • Pfleger L.
      • et al.
      Gluconeogenesis, but not glycogenolysis, contributes to the increase in endogenous glucose production by SGLT-2 inhibition.
      ]. In that study, a single dose of dapagliflozin increased EGP in healthy control individuals, mainly by increasing gluconeogenesis, while the rate of glycogenolysis was not affected. However, the plasma levels of the gluconeogenic substrates were not affected by dapagliflozin. Here, we investigated if the glucosuria induced by dapagliflozin was compensated by a reduction in overnight hepatic glycogen levels. We found that after 14 days of treatment, overnight hepatic glycogen was numerically lower after dapagliflozin treatment as compared to placebo but the difference did not reach statistical significance. The lack of a significant effect on hepatic glycogen content after dapagliflozin treatment may indicate that gluconeogenesis is predominantly responsible for increased EGP. In line with this suggestion, we observed an increase in urea which is reflective of enhanced gluconeogenesis. On the other hand, we did not observe changes in levels of free glycerol, which is a precursor for gluconeogenesis. Alternatively, considering that the glucose loss overnight only summed up to a total of ~7–8 g, it may be difficult to detect a corresponding change in hepatic glycogen levels, and the lack of effect on hepatic glycogen content may also indicate that the power of our study was insufficient to detect changes in hepatic glycogen content. Furthermore, it is possible that an increase in gluconeogenesis can contribute to both retaining glycogen content (or blunting the decline), as well as maintaining plasma glucose levels. Further investigations are needed to elucidate the exact contribution of gluconeogenesis and glycogenolysis to EGP after SGLT2i treatment.
      Also skeletal muscle glycogen content was not different between dapagliflozin and placebo in the overnight fasted state. Interestingly, skeletal muscle glycogen content increased from morning to afternoon by ~18 % after placebo, which is in line with previously observed diurnal changes in skeletal muscle glycogen levels due to postprandial glycogen storage [
      • Macauley M.
      • Smith F.E.
      • Thelwall P.E.
      • Hollingsworth K.G.
      • Taylor R.
      Diurnal variation in skeletal muscle and liver glycogen in humans with normal health and Type 2 diabetes.
      ]. However, there was an opposite trend from morning to afternoon, an ~8 % decrease in glycogen content, after dapagliflozin treatment. These findings may suggest that upon excess urinary glucose loss, dietary carbohydrates may be preferentially directed toward the liver, as hepatic glycogen levels may be more crucial for glucose homeostasis. This suggestion is in line with the similar change in hepatic glycogen levels from the morning to the afternoon comparing placebo and dapagliflozin periods. However, it must be noted that we could only measure hepatic glycogen both in the morning and afternoon in a subset of the volunteers. Furthermore, it could be speculated that skeletal muscle glycogen levels are restored by the enhanced EGP during the night, which is in line with our previous observation that overnight fasted non-oxidative glucose disposal (mainly glycogen storage) was increased in T2DM patients treated with dapagliflozin [
      • Op den Kamp Y.J.M.
      • de Ligt M.
      • Dautzenberg B.
      • et al.
      Effects of the SGLT2 inhibitor dapagliflozin on energy metabolism in patients with type 2 diabetes: a randomized, double-blind crossover trial.
      ]. Larger studies with glycogen measurements around the clock are needed to investigate the effect of SGLT2i on substrate fluxes in muscle and liver.
      We hypothesized that SGLT2i treatment would lead to an improvement in nocturnal fat oxidation in prediabetic insulin resistant individuals. Indeed, nocturnal fat oxidation was increased while carbohydrate oxidation was decreased during the first night in the respiration chamber. During the second night in the respiration chamber, the difference in fat oxidation between dapagliflozin treatment and placebo was blunted. Most likely, the higher fat oxidation in the placebo arm during the second night was due to the diet provided during the stay in the respiration chamber, which was relatively low in carbohydrates (38 energy%) and high in fat (45 energy%) and may have further stimulated fat oxidation affecting both treatment periods. Interestingly, despite the relative high-fat diet, fat balance was more negative during dapagliflozin treatment.
      In line with improved fat oxidation in vivo, especially in the nocturnal state, we also observed an improved ex vivo skeletal muscle mitochondrial oxidative capacity after dapagliflozin treatment. An improved mitochondrial oxidative capacity may be clinically relevant as a decreased mitochondrial oxidative capacity is associated with insulin resistance and T2DM [
      • Abdul-Ghani M.A.
      • DeFronzo R.A.
      Mitochondrial dysfunction, insulin resistance, and type 2 diabetes mellitus.
      ]. There are (pre)clinical results showing that both caloric restriction and SGLT2i can activate the AMPK-SIRT1-PGC-1α signalling pathway [
      • Xu L.
      • Nagata N.
      • Nagashimada M.
      • et al.
      SGLT2 inhibition by empagliflozin promotes fat utilization and browning and attenuates inflammation and insulin resistance by polarizing M2 macrophages in diet-induced obese mice.
      ,
      • Yang X.
      • Liu Q.
      • Li Y.
      • et al.
      The diabetes medication canagliflozin promotes mitochondrial remodelling of adipocyte via the AMPK-Sirt1-Pgc-1α signalling pathway.
      ,
      • Zhou H.
      • Wang S.
      • Zhu P.
      • Hu S.
      • Chen Y.
      • Ren J.
      Empagliflozin rescues diabetic myocardial microvascular injury via AMPK-mediated inhibition of mitochondrial fission.
      ,
      • Civitarese A.E.
      • Carling S.
      • Heilbronn L.K.
      • et al.
      Calorie restriction increases muscle mitochondrial biogenesis in healthy humans.
      ], which is involved in many processes, including mitochondrial biogenesis [
      • Wu Z.
      • Puigserver P.
      • Andersson U.
      • et al.
      Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1.
      ]. However, in our study, PGC-1α levels in the morning were not significantly influenced by dapagliflozin treatment and we did not observe a significant correlation between changes in PGC-1α levels and changes in maximal mitochondrial oxidative capacity comparing the dapagliflozin and placebo periods. In conclusion, more studies are needed in order to conclude the importance of the AMPK-SIRT1-PGC-1α pathway for mitochondrial function after SGLT2i treatment.
      The glucose loss reported in this study was ~36 g/24-h, resulting in an energy deficit of ~600 kJ/24-h, which represents only 6 % of the daily energy intake. Interestingly, this mild glucose loss did result in lower 24-hour plasma glucose levels. These results illustrate that mild glucose loss can exert strong metabolic adaptations in substrate metabolism in organs well distal to the direct pharmacological impact of the drug. It is well known that in the insulin resistant state, metabolic disturbances in energy and substrate metabolism and impaired mitochondrial function are present [
      • Wefers J.
      • Connell N.J.
      • Fealy C.E.
      • et al.
      Day-night rhythm of skeletal muscle metabolism is disturbed in older, metabolically compromised individuals.
      ]. Therefore, our results highlight the potential of SGLT2i to systemically restore such metabolic disturbances under insulin resistant conditions.
      A limitation of our study is the short duration of dapagliflozin treatment and the low number of participants. Although we observed positive metabolic adaptations without changes in body weight, the outcomes might be different after several months of SGLT2i treatment. It has been shown that SGLT2i increase caloric intake after 6 months of treatment [
      • Matsuba I.
      • Kanamori A.
      • Takihata M.
      • et al.
      Canagliflozin increases calorie intake in type 2 diabetes without changing the energy ratio of the three macronutrients: CANA-K study.
      ], possibly as a compensatory mechanism to overcome the urinary glucose losses. Here we also investigated whether dapagliflozin-induced glucose loss would lead to a change in food preferences in favour of carbohydrate intake, but we could not confirm previous findings [
      • Horie I.
      • Abiru N.
      • Hongo R.
      • et al.
      Increased sugar intake as a form of compensatory hyperphagia in patients with type 2 diabetes under dapagliflozin treatment.
      ]. Regardless of changes in caloric intake, it should be noted that interventions that increase energy turnover, such as cold exposure [
      • Hanssen M.J.
      • Hoeks J.
      • Brans B.
      • et al.
      Short-term cold acclimation improves insulin sensitivity in patients with type 2 diabetes mellitus.
      ] and exercise [
      • Brouwers B.
      • Schrauwen-Hinderling V.B.
      • Jelenik T.
      • et al.
      Exercise training reduces intrahepatic lipid content in people with and people without nonalcoholic fatty liver.
      ], generally are associated with improvements in metabolic health, also when no weight loss is achieved.

      5. Conclusion

      In conclusion, we show that 2 weeks of dapagliflozin treatment in prediabetic insulin resistant individuals lowered 24-hour glucose levels, and improved 24-h and nocturnal fat oxidation, as well as ex vivo mitochondrial oxidative capacity without significant changes in hepatic glycogen stores. Future long-term clinical trials should investigate if the calorie restriction-like effects of SGLT2i on energy and substrate metabolism may underlie the reported organ protective effects of SGLT2i that are also observed in people with chronic kidney disease and heart failure without T2DM. Furthermore, the exact role of overnight changes in hepatic glycogen and/or gluconeogenesis in regulating energy metabolism needs further investigation.

      CRediT authorship contribution statement

      A.V., M.d.L., R.E., J.O., V.B.S.-H., E.P., and P.S. designed the experiments. A.V., C.A., Y.O.d.K., E.E.-T., J.J., E.M.-K., and G.S. performed the measurements. A.V., J.M., J.J., V.B.S.-H., E.P., and P.S. were involved in the data analysis. A.V., J.O., and P.S. drafted the manuscript. All authors reviewed and edited the manuscript. P.S. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

      Data availability

      The datasets that were obtained in this study can be made available by the corresponding author upon reasonable request.

      Declaration of competing interest

      V.B.S.-H. was supported by an ERC starting grant (grant no. 759161 “MRS in diabetes”). P.S. previously received research funding from AstraZeneca. R.E. and J.O. are AstraZeneca employees and stockholders.

      Acknowledgements

      The authors thank all study participants for their participation.

      Funding

      AstraZeneca funded the study and provided study medication. The study funder was involved in the design of the study, interpretation of the data; and in writing the report. The study funder was not involved in the collection and analysis of the data; and did not impose any restrictions regarding the publication of the report.

      Appendix A. Supplementary data

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