SGLT2 Inhibitors, Ketogenesis, and Ketoacidosis

In this study, we will test the hypothesis that distinct mechanisms account for the SGLT2i-induced stimulation of ketogenesis and lipolysis versus endogenous (hepatic) glucose production in patients with type 2 diabetes (T2D) and type 1 diabetes (T1D), and that the increases in ketone production and lipolysis can be prevented by concomitant administration of the thiazolidinedione pioglitazone. We will conduct five distinct experiments to test this hypothesis in patients with T2D and T1D.

STUDY 1: To examine the effect of empagliflozin versus empagliflozin/pancreatic clamp on EGP (6,6, D2-glucose), gluconeogenesis (D2O), lipolysis (U-2H-glycerol), ketogenesis (13C-palmitate conversion to 3-betahydroxybuyrate), and norepinephrine turnover (3H-NE) in type 2 diabetes subjects.

STUDY 2. To examine the role of the SNS on the empagliflozin-induced stimulation of EGP, lipolysis, and ketone production in T2D by comparing the effect of empagliflozin versus empagliflozin plus propranolol.

STUDY 3. To examine the 2-HIT hypothesis that the SGLT2i-induced stimulation of EGP, lipolysis, and ketone production requires the combination of volume depletion plus insulinopenia in T2D individuals.

STUDY 4. To examine whether the empagliflozin-induced stimulation of EGP, lipolysis, and ketone production in T2D individuals can be blocked by pioglitazone (which has direct hepatic and adipose tissue effects).

STUDY 5. To examine whether the empagliflozin-induced stimulation of EGP, lipolysis, and ketone production in T1D individuals can be blocked by pioglitazone (which has direct hepatic and adipose tissue effects).

STUDY 1

Participants: 30 T2D subjects, age = 30-75 y, BMI = 23-38 kg/m2, HbA1c = 7.0-10%, eGFR \> 60 ml/min/1.73m2, BP \< 145/85 mmHg. Participants must be in general good health based on medical history, physical exam, screening blood chemistries, CBC, TSH/T4, EKG, and urinalysis. Patients must have stable body weight (±1.5 kg) over the last 3 months and must not participate in an excessively heavy exercise program. Patients treated with diet, SU, metformin, or SU/MET are eligible. Patients treated with GLP-1 RA, DPP-4i, TZD, or insulin are excluded. Patients taking medications (other than SU/MET) known to affect glucose metabolism are excluded. Statin therapy is permissible if the dose has been stable for at least 3 months. Subjects with evidence of proliferative retinopathy or eGFR \< 60 are excluded. Women of childbearing potential are excluded unless they are taking/using appropriate contractive medications/devices.

Protocol: Subjects will be randomized to receive empagliflozin (n=20) or placebo (n=10) in 2:1 ratio. Subject stratification will be done according to the following parameters: age (\> or \< 50 y), BMI (\> or \< 30 kg/m2), eGFR (\> or \< 80 ml/min/1.73 m2), HbA1c (\> or \< 8.5%). Each subject will participate in two studies performed in random order with 7-10 day interval between studies. In Study 1a, EGP will be measured with a prime-continuous 6,6, D2-glucose infusion and lipolysis will be measured with prime-continuous infusion of U-2H-glycerol. The rate of ketogenesis will be determined by infusion of 13C palmitate and quantitating the enrichment of 13C in 3-hydroxybutyrate (BHB). Total body NE turnover will be measured with 3H-norepinephrine (3H-NE) infusion before and after empagliflozin administration. Study 1b will be similar to Study 1a with one exception. EGP, lipolysis, and ketogenesis, and NE turnover will be measured under pancreatic clamp conditions.

STUDY 2

Participants: 22 T2D subjects with identical characteristics as in Study 1.

Protocol Study 2a: Following a 10-hour overnight fast, a prime-continuous infusions of 6,6,D2-glucose and U-14C-glycerol are started and continued to study end to measure rates of HGP and lipolysis. At 8:00AM a prime-continuous infusion of 3H-norepinephrine is started and continued to 9:00 AM at which time it will be stopped. At 11:00PM on the night prior to study subjects will ingest D2O (3 grams/kg ffm) to quantitate gluconeogenesis and de novo lipogenesis as previously described. At 7:30AM (2.5 hours after the start of tracer infusions) arterialized blood samples will be obtained at -30, -20, -10, 5, and 0 minutes for determination of plasma glucose, FFA, glycerol, AcAc, BHB, insulin, glucagon, norepinephrine, and epinephrine concentrations, 6,6D2-glucose enrichment, and 3H-NE and 14C-glycerol specific activities. At -30 and -10 minutes plasma glucose and VLDL samples are obtained to measure gluconeogenesis and de novo lipogenesis. At time zero (9:00 AM) subjects will ingest empagliflozin (25 mg) and arterialized blood samples are obtained every 10-30 minutes for 300 minutes (study end) for all of the preceding plasma substrate, hormone, and isotope specific activity/enrichment measurements. At 240 minutes (1:00 PM) a second prime-continuous 3H-NE infusion is started for 60 minutes. Plasma samples for norepinephrine concentration and specific activity are obtained every 5-10 minutes over the 30 minutes prior to the start of 3H-NE infusion and from 260-300 minutes.

Protocol Study 2b: This study will be identical to Study 2a above with one exception. 30 minutes prior to the ingestion of empagliflozin (8:30 AM), a prime (200 ug/kg over 20 minutes)-continuous (80 ug/min) infusion of propranolol is started and continued to study end. We previously have shown the steady state propranolol concentration achieved by this infusion rate is sufficient to significantly inhibit insulin-mediated glucose disposal.

STUDY 3

Participants: 29 T2D subjects with the same inclusion and exclusion criteria as in Study 1. Subjects will participate in 4 studies that will be performed in random order on the CRC at TDI at 6 AM.

Protocol Study 3a: Infusions of 3-3H-glucose and U-14C-glycerol will be started as per Study 2 and continued until the end of study at 2:00 PM. Plasma concentrations of glucose, FFA, glycerol, AcAc, BHB, insulin, glucagon, epinephrine, norepinephrine, cortisol, renin and angiotensin (index of volume status) and 3H-glucose and 14C-glycerol specific activities will be measured at -30, -20, -10, -5 and 0 minutes. At time zero (9:00 AM) subjects will ingest empagliflozin (25 mg). Plasma substrate and hormone concentrations and 3H-glucose/14C-glycerol specific activities will be measured every 10-30 minutes for 300 minutes (2:00 PM) as per Study 2. Plasma norepinephrine, epinephrine, cortisol, renin, and angiotensin will be measured at 270, 280, 290, and 300 minutes.

Protocol Study 3b: At 7 AM an infusion of normal saline will be started at the rate of 150 ml/hour and continued to the end of study at 2 PM (total volume = 1050 ml; total Na and Cl = 154 meq). This amount of volume and NaCl administration is approximately twice the amount of urinary NaCl and volume that occurs over the initial 24 hours when dapagliflozin is initially administered as a single dose to T2D patients, as previously shown by us. In addition, all blood loss will quantitatively be replaced by an equivalent amount of normal saline.

Protocol Study 3c: This will be similar to Study 3a with one exception: at 9 AM somatostatin with insulin and glucagon replacement will be given to create volume depletion without insulinopenia.

Protocol Study 3d: This will be similar to Study 3a with two exceptions: (i) volume repletion will be given as in Study 3b and (ii) somatostatin will be infused with insulin and glucagon replacement as in Study 3c.

STUDY 4

Participants: 64 T2D subjects with the same inclusion and exclusion criteria as Protocol 1. Subjects with hematuria are excluded.

Protocol: Infusions of 3-3H-glucose and 14C-glycerol are started and continued to study end (2 PM). Baseline blood samples for HbA1c (x2) and for plasma insulin, glucagon, glucose, FFA, BHB, AcAc, glycerol, and plasma 3-3H-glucose and 14C-glycerol specific activities are drawn at -30, -20, -10, -5, and 0 minutes for measurement of lipolysis, ketone production (plasma ketone levels), and EGP. Empagliflozin (25 mg) is ingested at time zero (9AM) and plasma samples for the above are obtained every 10-20 minutes.

Following completion of the above study, subjects will be randomized to one of four groups (16 per group) for 10 weeks: (1) empagliflozin, 25 mg/day, plus pioglitazone placebo; (2) pioglitazone, 15 mg/day, increased to 30 mg after 2 weeks plus empagliflozin placebo; (3) empagliflozin (25 mg/d) plus pioglitazone (15/30 mg/d); (4) empa placebo plus pio placebo. Subjects will return to the CRC every 1-2 weeks for interim medical history, to check medication compliance, and to measure plasma insulin, glucagon, glucose, FFA, glycerol, BHB, and AcAc levels. At week 10, subjects will return to the CRC at 6AM and the baseline study will be repeated. HbA1c will be measured twice during week 10.

STUDY 5

Participants: 24 T1D subjects will comprise the study population. Inclusion criteria are: (1) age \> 18 years; (2) T1D with positive GAD antibody; (3) subjects must be in good general health (except for diabetes) as determined by physical exam, medical history, Chem 20, CBC, T4/TSH, urinalysis, and EKG; (4) fasting C-peptide concentration \<0.7 ng/ml; (5) poor glycemic control (HbA1c = 7.0-11.0%); (6) treatment with multiple daily insulin injections (basal plus prandial) or insulin pump; (7) stable insulin dose (±4 units in the preceding three months; (8) eGFR ≥ 60 ml/min/1.73m2; (9) weight stable over the preceding 3 months (±4 pounds); (10) do not participate in an excessively heavy exercise program. Exclusion criteria: (1) T2D; (2) HbA1c \<7.0% or \> 11.0%; (3) eGFR \< 60 ml/min/1.73m2; (4) hematuria in urinalysis;( 5) pregnancy or lactating; (6) major organ system disease other than diabetes; (7) evidence of proliferative diabetic retinopathy; (8) patients on ketogenic diet; (9) history of hospitalization for DKA, hypoglycemia or uncontrolled hyperglycemia in preceding 6 months.

Subjects will receive fasting measurement of plasma glucose, insulin, C-peptide, glucagon, GLP1, GIP, HbA1c, FFA, glycerol, BHB, AcAc, lactate and started on a 4 week run-in period during which they will be instructed to continue their daily insulin regimen unchanged unless instructed otherwise by the study team and to continue 4 times/day home blood glucose monitoring (HBGM) (before each meal and bed time). To determine the baseline rate of hypoglycemia, CGM (Dexcom G6) will be performed during the last week of the run in period. Subjects with evidence of hypoglycemia or who have FPG \< 120 mg/dl will have their daily insulin dose adjusted to achieve a mean FPG \> 120 mg/dl as confirmed by CGM. Since the primary aim of the study is to examine the efficacy of SGLT2i with and without pioglitazone, no adjustment in insulin dose will be done during the run in period or at the time of starting empagliflozin (see below) in patients with FPG \> 120 mg/dl in order not to increase the risk of ketoacidosis.

Protocol: Infusions of 3-3H-glucose and U-14C-glycerol will be started as described in the "BASELINE" study of Study 4. All blood sampling will be performed as in Study 4 with empagliflozin (25 mg) ingested at 9 AM (time zero). Following completion of this "BASELINE" study, subjects will be randomized to: (1) empagliflozin, 25 mg/day, plus placebo for 10 weeks or (2) empagliflozin (25 mg/d) plus pioglitazone (15 mg/d for 2 weeks, then increased to 30 mg/d for 8 weeks for a total treatment period of 10 weeks). Changes in daily insulin dose will be allowed after starting empagliflozin to avoid hypoglycemia and will be made based upon HBGM and CGM at the investigator's discretion. Plasma ketone concentration will be measured by the patient with a Keto-Mojo Meter for 3 days following any insulin dose adjustment before making any additional changes in insulin dose.

Following the start of empa/placebo or empa/pioglitazone, subjects will return to the CRC every 1-2 weeks for review of CGM and HBGM results and blood will be drawn for fasting plasma insulin, glucagon, glucose, FFA, glycerol, AcAc, and BHB. Patients will be instructed to measure plasma ketone conc (Keto-Mojo Meter) each morning. If the plasma ketone conc rises to \> 1 mM, subjects will be instructed to immediately contact the study team, and all fasting measurements will be repeated . After 10 weeks the "BASELINE" study will be repeated. HbA1c will be measured twice during week 10.

Eligibility Criteria:
Patients with T2D

Inclusion Criteria:

* Ages 30-75 years
* Body Mass Index (BMI) 21-45 kg/m2
* Hemoglobin A1C (HbA1c) = 7.0-10%
* Estimated glomerular filtration rate (eGFR) \> 60 ml/min/1.73m2
* Blood Pressure (BP) \< 145/85 mmHg
* Participants must be in general good health based on medical history, physical exam, screening blood chemistries, complete blood chemistry (CBC), thyroid stimulating hormone/thyroxine (TSH/T4), electrocardiogram (EKG), and urinalysis
* Stable body weight (±1.5 kg) over the last 3 months and must not participate in an excessively heavy exercise program
* Patients treated with diet, sulfonylurea (SU), metformin (MET), or SU/MET
* Statin therapy is permissible if the dose has been stable for at least 3 months

Exclusion Criteria:

* Patients treated with Glucagon-like peptide 1 receptor agonists (GLP-1 RA), Dipeptidyl Peptidase IV inhibitors (DPP-4i), Thiazolidinediones (TZD), or insulin are excluded
* Patients taking medications (other than SU/MET) known to affect glucose metabolism are excluded
* Subjects with evidence of proliferative retinopathy or eGFR \< 60 are excluded
* Women of childbearing potential are excluded unless they are taking/using appropriate contractive medications/devices

Patients with T1D

Inclusion Criteria:

* Age \> 18 years
* T1D with positive GAD antibody
* Subjects must be in good general health (except for diabetes) as determined by physical exam, medical history, Chem 20, CBC, T4/TSH, urinalysis, and EKG
* Fasting C-peptide concentration \<0.7 ng/ml
* Poor glycemic control (HbA1c = 7.0-11.0%)
* Treatment with multiple daily insulin injections (basal plus prandial) or insulin pump
* Stable insulin dose (±4 units in the preceding three months
* eGFR ≥ 60 ml/min/1.73m2; (9) weight stable over the preceding 3 months (±4 pounds)
* Not participating in an excessively heavy exercise program.

Exclusion criteria:

* Type 2 diabetes
* HbA1c \<7.0% or \> 11.0%
* eGFR \< 60 ml/min/1.73m2
* Hematuria in urinalysis
* Pregnancy or lactating
* Major organ system disease other than diabetes
* Evidence of proliferative diabetic retinopathy
* Patients on ketogenic diet
* History of hospitalization for DKA, hypoglycemia or uncontrolled hyperglycemia in preceding 6 months.