Effect of Empagliflozin on Hepatic Glucose Metabolism: Role of Autonomic Nervous System
Phase: PHASE4
Conditions: Hepatic Glucose Metabolism
Sponsor: The University of Texas Health Science Center at San Antonio

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
1 
 
 
 
 
 
 
 
 
Official Study Title: Effect of Empagliflozin on Hepatic Glucose Metabolism: Role of 
Autonomic Nervous System 
 
NCT number: NCT03193684 
 
IRB Approval Date: 05.23.2020 
 
 
Unique Protocol ID: HSC20170214H 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
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Protocol Template Form 
 
Item 1 UTHSCSA 
Tracking Number 
HSC20170214H 
 
Item 2 Abstract / Project Summary 
 
 
Provide a succinct and accurate description of the proposed 
research. State the purpose/aims. Describe concisely the research 
design and methods for achieving the stated goals. This section 
should be understandable to all members of the IRB, scientific and 
non-scientific.   
DO NOT EXCEED THE SPACE PROVIDED. 
Purpose/Objectives: To investigate the effect of empagliflozin, an SGLT2 inhibitor on hepatic glucose production and 
the role of autonomic nervous system in mediating the increase in hepatic glucose production in response glucosuria   
Research Design/Plan: the role of autonomic nervous system in the increase in hepatic glucose production caused by 
empagliflozin will be examined with norepinephrine (NE) turnover in two protocols. The first protocol is cross sectional, in 
which 36 T2DM patients will receive hepatic glucose production (HGP) and NE turnover will be measured before and 
after empagliflozin or placebo administration. In protocol 2, diabetic and non-diabetic subjects will receive baseline HGP, 
NE turnover,) and liver fat measurement before at 2 days after the start and 12 weeks after empagliflozin or placebo 
treatment.  
Methods: the following techniques will be employed (1) Measurement of hepatic glucose production with 3H-glucose 
infusion, with and without glucose clamp, (2) substrate oxidation with indirect calorimetry and plasma 
ketone/lactate/insulin/glucagon concentrations; (; (3) Measurement of whole body norepinephrine turnover with 3H-
norepinephrine infusion; (4) Measurement of heart rate variability; (5) Measurement of liver fat content with 1H-MRS 
Clinical Relevance: The results of the present studies will help identify the mechanism responsible for the increase in 
HGP caused by empagliflozin and the increase in ketone production. The first action of the drug ameliorates its clinical 
efficacy while the second increases the risk of adverse events (ketoacidosis). Identifying the mechanisms underlying 
these actions will help developing therapeutic strategies which increase the drug clinical efficacy and mitigates its 
adverse events.    
 
 
 
Item 3 
Background 
 
Describe past experimental 
and/or clinical findings 
leading to the formulation 
of your study.   
For research involving 
unapproved drugs, 
describe animal and 
human studies.   
For research that involves 
approved drugs or devices, 
describe the FDA approved 
uses of this drug/device in 
relation to your protocol. 
Insert background: Effect of SGLT2 Inhibitors in T2DM Patients: inhibition of renal 
sodium-glucose contransport is a novel strategy for the treatment of T2DM (1). 
Currently 3 agents are approved by the FDA for the treatment of T2DM; dapagliflozin, 
canagliflozin and empagliflozin. Members of this class of drugs lower the plasma 
glucose concentration by inhibiting renal glucose uptake and causing glucosuria. 
Clinical studies have demonstrated that in drug naïve T2DM patients and in patients 
treated with stable dose of metformin and/or sulfonylurea, the addition of SGLT2 
inhibitor causes a 25-35 mg/dl decrease in fasting plasma glucose (FPG) concentration 
(2-4). Because the primary determinant of the FPG concentration is hepatic glucose 
production (HGP) (5,6), and SGLT2i consistently reduce the FPG, we anticipated that 
dapagliflozin treatment would be associated with reduced hepatic glucose output. 
Surprisingly, when administrated to T2DM patients following an overnight fast, HGP 
increased markedly (7,8). 
In a double blind, placebo controlled study in 18 T2DM males (age=52±3, BMI=52±3, 
FPG=164±10, and HbA1c=8.6±0.5), 2 weeks of treatment with dapagliflozin (10 
mg/day) caused 38 mg/dl reduction in fasting plasma glucose concentration (7). 
However, despite the decrease in FPG concentration, dapagliflozin caused 17% 
increase in the basal rate of HGP (bHGP) (from 2.17±0.11 to 2.53±0.16, p<0.05, Figure 
1). A similar increase in bHGP following empagliflozin treatment in T2DM was reported 
by Ferrannini and colleagues (8). The increase in bHGP caused by dapagliflozin can 
be viewed as a compensatory mechanism to compensate for urinary glucose loss, 
maintain the plasma glucose concentration at the normal level, and prevent 

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
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hypoglycemia. However, in T2DM individuals, the rise in HGP took place while the 
plasma glucose concentration was well within the hyperglycemic range (FPG ~130 
mg.dl). Thus, it is paradoxical. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The increase in bHGP following dapagliflozin administration lasted for the entire 
treatment period (2-weeks, and 4 weeks in the study by Ferrannini [8]). This 
paradoxical increase in bHGP has important clinical significance, since it returns to the 
systemic circulation approximately one half of the amount of glucose lost in the urine. 
Thus, it offsets urinary glucose loss caused by dapagliflozin and attenuates the 
decrease in plasma glucose concentration caused by the drug. 
The rise in bHGP following dapagliflozin administration raises an important question 
about the signal which links SGLT2 inhibition/glucosuria to the liver. To investigate the 
cause of this paradoxical rise in bHGP, we measured plasma insulin and glucagon 
concentrations following dapagliflozin administration. Figure 2 demonstrates that the 
increase in bHGP cause by dapagliflozin is accompanied by a small decrease in 
plasma insulin concentration and a large increase in plasma glucagon concentration 
(32%) (Figure 2). Thus, the ratio of plasma glucagon to insulin, the principal 
determinant of the rate of HGP, markedly increased (from 12±2 to 28±7, p<0.01) 
following dapagliflozin administration (Figure 2) and this increase lasted for the entire 
treatment period (2 weeks). It should be noted that, because the increase in bHGP was 
associated with a reduction in fasting plasma insulin concentration, the increase in 
bHGP produced by dapagliflozin does not reflect worsening of hepatic insulin 
resistance. The hepatic insulin resistance index (the product of bHGP and fasting 
plasma insulin) was slightly reduced by dapagliflozin. 
Dapagliflozin also decreased the postload plasma glucose concentration (9). 
Administration of 10 mg dapagliflozin caused 60% reduction in the incremental urea 
under the plasma glucose concentration curve during the OGTT (9). Since the disposal 
of the majority of an exogenous glucose load takes place in skeletal muscle (10), we 
measured whole bod insulin-mediated glucose uptake with the euglycemic insulin 
clamp. Dapagliflozin caused a 16% increase in insulin-mediated tissue glucose uptake 
(from 4.3±0.4 to 5.0±0.6 mg/kg.min, p<0.05) (7), suggesting improved peripheral 
insulin sensitivity. Further, dapagliflozin caused a doubling of beta cell function in T2DM 
patients (9). Since the primary site of SGLT2i action is on the kidney, and since SGLT2 
transporters have not been demonstrated in skeletal muscle or beta cells, the 
improvements in insulin sensitivity and insulin secretion can be attributed to reversal of 
glucotoxicity (2,11). 
We previously have shown that improving insulin sensitivity in T2DM individuals by 
lowering plasma free fatty acid concentration with acipimox (removal of lipotoxicity) 
resulted in an increase in glucose oxidation and decrease in fat oxidation and 50% 
increase in mitochondrial ATP synthesis rate (12). Unexpectedly, the improvement in 
insulin sensitivity following dapagliflozin treatment was associated with opposite effect 
on mitochondrial ATP synthesis and substrate oxidation (13). Dapagliflozin treatment 
Change in FPI (%)
0
25
50
75
100
  Change in Fasting 
Plasma Glucagon (%)
60
80
100
120
140
Glucagon/Insulin Ratio
0
10
20
30
40
Baseline Day 2 Day 3
Day 14
*
*
*
*
*
*
**
**
Figure 1: Acute Effect of 
dapagliflozin and placebo 
on HGP in T2DM 
patients 
Figure 2: Effect 
of dapagliflozin 
and placebo on 
plasma insulin, 
glucagon and 
glucagon to 
insulin ratio in 
T2DM patients 
Time (min)
0
60
120
180
240
EGP (mg/kg.min)
1.2
1.6
2.0
2.4
Drug
Ingestion
Dapagliflozin
Placebo
Time (min)
0
60
120
180
240
Plasma Insulin (uU/ml)
0
3
6
9
12
15
Placebo
Dapagliflozin
Time (min)
0
60
120
180
240
Plasma Glucagon (pg/ml) 
50
60
70
80
90
Dapagliflozin
Placebo
Drug
Ingestion

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
4 
(2 weeks) caused a 20% reduction in glucose oxidation during the fasting state with 
concomitant a 14% increase in fat oxidation (Figure 3). Moreover, although 
dapagliflozin significantly increased insulin-stimulated glucose disposal (by 16%), 
glucose oxidation during insulin infusion was reduced by approximately 50% in 
association with a 15% increase in fat oxidation (13). Of note, no significant change in 
plasma FFA concentration or suppression of plasma FFA concentration during the 
insulin clamp was observed following dapagliflozin administration. Thus, the increase 
in fat oxidation can not be explained by oversupply of fat to peripheral tissue; rather, it 
must be due to an effect of the drug on metabolic pathway(s) regulating fat oxidation. 
Regardless of the mechanism for the increase in fat oxidation caused by SGLT2 
inhibitors, we hypothesize that the increase in fat oxidation leads to depletion of 
intramyocellular and intrahepatic fat content and can explain the improvement in insulin 
sensitivity measured with the insulin clamp. In the present grant proposal, we will 
measure the effect of SGLT2 inhibitor, dapagliflozin on hepatic fat content measured 
with MRI spectroscopy. 
 
                                                  
 
 
 
 
 
 
 
The increase in fat oxidation following dapagliflozin treatment was associated with a 
surprising 55% reduction in mitochondrial ATP synthesis rate (Figure 4) and a modest, 
but significant increase in plasma ketone concentration (Figure 5) (13). These changes 
in substrate oxidation, together with the increase in bHGP caused by dapagliflozin, 
suggest that dapagliflozin shifts substrates away from the TCA cycle (acetyl Co-A 
towards ketone production and oxaloacetate towards gluconeogenesis; Figure 6) and 
are consistent with the reduction in ATP synthesis rate caused by dapagliflozin (Figure 
4). The increase in fat oxidation, decrease in ATP synthesis rate and increase in 
plasma glucagon and decrease in plasma insulin concentration explain the increased 
conversion of acetyl-CoA to ketones and increase in plasma ketone concentration, 
which under stress conditions can lead to the development of clinically significant 
ketoacidosis, as has been reported in patients treated with SGLT2 inhibitors (14,15). 
                                                                           
 
Pathogenesis of Impaired Fasting Glucose 
Impaired fasting glucose (FPG=100-125 mg/dl) (IFG) was introduced by the American 
Diabetes Association in 1997 as an intermediate stage in glucose tolerance between 
normal glucose tolerance and overt T2DM (16), and it was meant to identify subjects 
at increased future T2DM risk without the need for an OGTT. Compared to NGT, IFG 
Plasma Ketones
   (mmol/L)
0.00
0.05
0.10
0.15
0.20
0.25
Placebo
Dapaglifloizn
Figure 4: Effect of dapagliflozin and placebo 
on mitochondrial ATP synthesis rate 
measured ex vivo in isolated mitochondria 
from skeletal muscle. G/M=glutamate; 
pyr=pyruvate; suc=succinate; PC=palmitoyl 
carnitine  
 
 
 
 
 
 
 
 
Figure 6: Scheme illustrating the 
metabolic and hormonal effects of 
dapgliflozin on glucose and fat 
metabolism in T2DM patients 
Lipid Oxidation (mg/kg.min)
1.0
1.5
2.0
2.5
3.0
Fasting
Insulin Infusion
*
*
* p<0.05
*
*
Placebo
Dapaglifloizn
 
 
 
 

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
5 
subjects manifest an increase in the fasting plasma glucose concentration and 
excessive rise in plasma glucose concentration following glucose load, e.g. at 60 and 
75 minutes after OGTT and mixed meal (17). We (17-21) and others (22-26) have 
previously demonstrated that IFG subjects manifest moderate to severe insulin 
resistance in the liver with normal/near normal insulin sensitivity in skeletal muscle and 
impaired first phase but normal second phase insulin secretion (17-26). These 
metabolic defects in IFG subjects explain the shape of plasma glucose concentration 
following a glucose load. The normal/near normal insulin sensitivity in skeletal muscle 
and normal second phase insulin secretion in IFG subjects explains why the 2-hour 
plasma glucose concentration returns to the starting fasting, albeit elevated, level in 
IFG subjects. 
Despite a wealth of previous studies (17-32) which have examined the pathophysiology 
of IFG and which have identified multiple metabolic defects, including hepatic insulin 
resistance and impaired first phase insulin secretion, none of these defects can explain 
the elevated FPG concentration in IFG subjects. This question was the aim of my 
currently funded NIH R01 grant. 
The basal rate of hepatic glucose production (bHGP) is the principal determinant of the 
FPG concentration (5,6). We (18) and others (23-25) previously have demonstrated 
that IFG subjects manifest moderate to severe hepatic insulin resistance. However, the 
elevated fasting plasma insulin concentration in IFG subjects offsets the hepatic insulin 
resistance and maintains the basal rate of HGP in the normal range (18, 23-25). Indeed 
the vast majority (18,22-25, 27, 28), though not all (29,32), of previous studies have 
failed to document a significant rise in bHGP in IFG compared to NGT subjects, and 
the investigators who reported an elevated bHGP in IFG in some studies (29,32) also 
reported a normal bHGP in other studies (22,23,28). A normal rate of bHGP in IFG 
subjects in the presence of an elevated FPG concentration suggests a decreased basal 
glucose clearance rate. Consistent with this hypothesis, we (33) previously have 
demonstrated that IFG subjects manifest a decreased basal rate of glucose clearance 
during the fasting state. The aim of the currently funded NIH grant was to directly 
quantitate the basal rate of glucose uptake in IFG subjects and compare the results to 
those in NGT individuals. 
Whole Body Basal Glucose Uptake in NGT and IFG Subjects: Because the basal 
rate of glucose uptake is insulin independent (34,35) (driven by the mass action of 
glucose), we utilized the stepped hypeglycemic clamp in combination with the 
pancreatic clamp technique to quantitate whole body glucose-mediated glucose uptake 
in IFG and NGT subjects. 12 NGT (age 38±5, sex (f/m) 6/6, BMI=28.2±1.1, FPG=90±2) 
and 12 IFG (age 32±2, sex (m/f) 7/5, BMI=31.2.0±1.4, FPG=109±2) subjects 
participated in the study. Subjects received somatostatin infusion with replacement of 
basal plasma insulin and glucagon levels while the plasma glucose concentration was 
raised and maintained at +100 mg/dl (i.e. from ~100 mg/dl to 200 mg/dl) for 100 
minutes (0-100 minutes). At 100 minutes, the plasma glucose concentration was raised 
and maintained by an additional 100 mg/dl (i.e, from 200 to 300 mg/dl). 120 minutes 
before the start of glucose infusion a prime (40 µCi) continuous (0.4 µCi/min) infusion 
of 3H-glucose was started and continued until the end of the study to quantitate the rate 
of total body glucose appearance (Ra). 
Consistent with previous studies (33), the basal rate of HGP was slightly but not 
significantly reduced in IFG compared to NGT subjects; 2.15±0.14 vs 2.30±0.16 
mg/kg.min. However, the basal glucose clearance was significantly reduced in IFG 
subjects (2.61±0.11 vs 3.6±0.2 ml/kg.min). Compared to NGT, glucose-mediated 
glucose uptake was significantly reduced during both hyperglycemic clamp steps in 
IFG subjects (Figure 7). The rate of glucose disposal in IFG and NGT subjects was 
2.89±0.10 vs 3.39±0.29 (p=0.02) at 200 mg/dl and 3.73±0.27 vs 4.15±0.21 mg/kg.min 
(p<0.05) at 300 mg/dl, respectively. Thus, the slope of line relating total body glucose-
mediated glucose disposal and the plasma glucose concentration was 17% lower in 

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
6 
IFG compared to NGT. These results demonstrate impaired glucose-mediated glucose 
uptake (glucose resistance) in IFG subjects. 
 
 
 
 
 
 
 
 
 
 
 
 
 
Metabolic Effect of SGLT2 Inhibitor in NGT and IFG Subjects: To examine the 
effect of SGLT2 inhibition on FPG concentration in NGT, and IFG subjects compared 
to T2DM, we treated 8 NGT subjects (age= 58±2, m/f=6/2, BMI= 27.1±1.1, FPG=95±2, 
eGFR=120±10), 8 IFG subjects (age= 59±3, m/f=5/3, BMI= 30.3±0.8, FPG=110±2, 
eGFR=99±6) and 15 T2DM subjects (age= 55±2, m/f=12/3, BMI= 31.1±2.1, 
HbA1c=7.8±0.2, eGFR=107±7) with empagliflozin 25 mg/day for 2 weeks. 
Empagliflozin caused 45±4, 50±5 and 97±11 grams glucosuria per day in NGT, IFG 
and T2DM subjects, respectively. As anticipated, empagliflozin caused 32 mg/dl 
reduction in the FPG concentration in T2DM patients. Conversely, the fasting plasma 
glucose concentration in NGT subjects was not affected by empagliflozin treatment (95 
mg/dl vs 94 mg/dl) despite 45 grams per day of urinary glucose loss. Although urinary 
glucose loss was comparable in IFG subjects and NGT subjects, empagliflozin caused 
a modest but significant reduction in the FPG concentration (from 110±2 to 103±3 
mg/dl, p<0.01) in IFG subjects (Figure 10). Further, the magnitude of decrease in the 
FPG concentration in IFG subjects strongly and inversely correlated with the FPG 
concentration (r=-0.61, p<0.01) (Figure 11). These results are consistent with a recent 
study which demonstrated 6 mg/dl decrease in the FPG concentration (from 105 to 99 
mg/dl) accompanied by a 34% increase in bHGP after 4 weeks empagliflozin treatment 
in non-diabetic obese IGT subjects (40). Collectively, these results suggest that: (1) 
HGP, though not measured in our study, similar to the previous study (40), and must 
have been markedly elevated in both NGT and IFG to compensate for the large urinary 
glucose loss and maintain the FPG concentration at or close to the normal fasting level 
(~90-100 mg/dl).  
 
 
 
 
 
 
 
 
 
 
 
 
 
As anticipated, empagliflozin caused a small decrease in the fasting plasma insulin 
concentration and a large increase in the fasting plasma glucagon concentration in 
T2DM patients. Thus, consistent with our previous study (7), the fasting plasma 
glucagon to insulin ratio was doubled (from 5 to 11) by empagliflozin in T2DM patients. 
Plasma Glucose Conc. (mg/dl)
100
150
200
250
300
Total Glucose Disposal
        (mg/kg.min)
2
3
4
5
NGT
IFG
Splanchnic Glucose Uptake
          (mg/kgLBM.min)
0.00
0.15
0.30
0.45
0.60
0.75
NGT
IFG
*
* P=0.02
FPG Conc. (mg/dl)
90
95
100
105
110
115
NFG
IFG
BL
Day 1
Day 2
Day 3
Day 14
*
*
*
*
Fasting Plasma Glucose Conc.
               (mg/dl)
80
90
100
110
120
130
Change in FPG (mg/dl)
-20
-15
-10
-5
0
5
10
15
20
r=-0.61
p<0.01
FPI (mU/ml)
0
5
10
15
20
NFG
IFG
*
#
#
#
#
*
Baseline
Day 1
Day 14
p<0.05 vs IFG
p<0.05 vs BL
 
 
 
 
Figure 8: Hepatic and non-hepatic 
splanchnic glucose uptake 
measured with the Oral-IV double 
tracer technique in IFG and NGT 
subjects during the fasting state  
 
 
 
 
 
 
 
 
 
Figure 11: Relationship between the 
decrease in FPG conc. caused by 
empagliflozin treatment (25 mg/day) and 
the FPG conc.  
Figure 12: Fasting plasma insulin 
concentration before and at day 1 
to day 14 during empagliflozin 
treatment (25 mg/day) in IFG and 
NGT subjects  
Fasting Plasma Glucose Con.
               (mg/dl)
80
90
100
110
HGU (mg/kg LBM.min)
0.0
0.3
0.6
0.9
1.2
1.5
r=-0.72
p<0.001

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Surprisingly, however, empagliflozin did not affect the fasting plasma insulin and 
glucagon concentrations in NGT subjects and caused only a modest reduction in 
fasting plasma insulin concentration (Figure 12) with a small, transient, non-significant 
increase in fasting plasma glucagon in IFG subjects (Figure 13). Consistent with our 
previous study (13), empagliflozin caused a significant increase in plasma ketone 
concentration in T2DM patients (from 0.52±0.04 to 0.68±0.3, p=0.03), while plasma 
ketone concentration was not affected by empagliflozin treatment in either IFG 
(0.43±0.03 vs 0.43±0.03) or NGT (0.53±0.04 vs 0.45±0.07) subjects following 
empagliflozin treatment (Figure 14). The lack of increase in plasma ketone 
concentration in NGT subjects is surprising in light of the large shift in fuel consumption 
during the fasting state caused by empagliflozin in non-diabetic subjects (decrease in 
RQ from 0.81 to 0.74). Collectively, these results demonstrate that: (1) Although 
SGLT2 has been shown to be present in the alpha cell and SGLT2i have been shown 
to directly stimulate glucagon secretion from the alpha cell (41), it is likely that, in man 
in vivo, the decrease in plasma glucose concentration in T2DM subjects following 
SGLT2 inhibition is the principal signal causing the decrease in plasma insulin 
concentration and increase in plasma glucagon concentration, (2) mechanism(s) other 
than the increase in plasma glucagon and decrease in plasma insulin concentration 
(likely neuronal) stimulates HGP and cause a shift in substrate oxidation following 
SGLT2 inhibition in NGT subjects and IFG subjects, and (3) the increase in plasma 
glucagon concentration and/or decrease in plasma insulin concentration is an 
important signal for the increase in ketone production following SGLT2 inhibition. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
A recent in vitro study (42) suggested that canagliflozin and dapagliflozin inhibit 
mitochondrial complex 1 activity in cultured HEK-293 cells, suggesting an OFF-target 
action of the drug and this could explain the decrease in glucose oxidation in in vivo 
studies in man. To examine a possible direct effect of SGLT2 inhibitors on glucose 
oxidation in hepatocytes, we examined the effect of dapagliflozin, empagliflozin and 
canagliflozin (3 µM) on pyruvate oxidation in cultured hepatocytes. Figure 15 
demonstrates that 3 µM canagliflozin caused a 56% reduction in pyruvate oxidation in 
cultured hepatocytes, and similar results were obtained with dapagliflozin and 
empagliflozin. Of note, none of the 3 SGLT2 inhibitors affected ketone or glucose 
production in cultured hepatocytes. Inhibition of citrate synthase by SGLT2 inhibitor 
could explain the reduction in pyruvate oxidation and shift of OAA toward 
gluconeogenesis resulting in increased glucose production as was observed in in vivo 
studies in man. However, hepatic citrate synthase activity was not affected by 
canagliflozin. Inhibition of complex 1 in hepatocytes by SGLT2 inhibitor (42) could 
explain the inhibition of pyruvate oxidation in hepatocytes in vitro. A similar in vivo 
action of SGLT2 inhibitors potentially could explain the shift in substrate oxidation 
(decreased glucose and increased fat oxidation). The increase in fat oxidation provides 
an alternatve energy source to the cell by generating FADH which generates ATP by 
Plasma Ketone
   Conc. (mM)
0.3
0.4
0.5
0.6
0.7
0.8
Baseline
Day 1
Day 14
NGT
IFG
T2DM
*
* p<0.05
*
Figure 13: Fasting plasma 
glucagon concentration before 
(BL) and at day 1 to day 14 
during empagliflozin treatment 
(25 mg/day) in IFG and NGT 
Figure 14: Effect of 
empagliflozin treatment (25 
mg/day) on plasma ketone 
concentration in  NGT, IFG 
and T2DM subjects  
Pyruvate Oxidation
(nmol/mgprotein.h)
0
5
10
15
20
Vehicle
Canagliflozin
*
*p=0.02
Figure 15: Effect of dapagliflozin 3 
µM on pyruvate oxidation in 
hepatocytes in culture  
Plasma Glucagon Conc.
            (ng/ml)
0
20
40
60
80
100
NFG
IFG
Baseline
Day 1
Day 14

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
8 
contributing electrons directly to mitochondrial complex II, bypassing the inhibition of 
complex I by SGLT2 inhibitor. Such an action could cause an increase in plasma lactate 
concentration. A recent study has reported small, though non-significant increase in 
plasma lactate concentration in obese IGT individuals following empagliflozin treatment 
(40). An increase in plasma lactate concentration, in addition to the increase in ketone 
production, could contribute to the euglycemic acidosis reported in some patients 
receiving treatment with SGLT2 inhibitors (14,15). In the present study, we will 
measure the effect of dapagliflozin on plasma lactate, ketone, bicarbonate 
concentrations and plasma pH in NGT, IFG and T2DM patients.   
Our recent findings about the effect of empagliflozin on glucose metabolism in NGT 
and IFG subjects (Figures 10-14) and the effect of SGLT2 inhibitors on pyruvate 
oxidation in hepatocytes in vitro (Figure 15) require an update of our previous 
scheme (13) (Figure 6) describing the mechanism(s) responsible for the metabolic 
actions of SGLT2 inhibitors in T2DM patients. We suggest the following working 
hypothesis (Figure 16) which we will be testing in the current grant request: (1) 
Because empagliflozin did not stimulate glucagon secretion or inhibit insulin secretion 
in NGT subjects, it is likely that glucosuria stimulates HGP, at least initially, via 
mechanisms other than the increase in plasma glucagon concentration and decrease 
in plasma insulin concentration. Of note, the increase in HGP begins rapidly following 
dapagliflozin administration and before there is a significant increase in plasma 
glucagon concentration (Figure 2). This suggests a neurogenic origin, which results 
from activation of the renal nerves which communicate directly with the liver via 
neural connections in the portal circulation or indirectly via connections to the CNS. 
Consistent with this hypothesis, a recent study has demonstrated that the increase in 
HGP in dogs following hypoglycemia is markedly influenced by the brain (43). 
Further, renal denervation in experimental animals caused a significant increase in 
hepatic norepinephrine content (44). In the present grant proposal, we will measure 
the effect of empagliflozin  on sympathetic activity with 3H-NE infusion; (2) In addition 
to inhibition of renal glucose absorption, SGLT2 inhibitors likely exert a direct 
inhibitory action on mitochondrial metabolism (possibly by inhibiting complex I[42]) 
causing inhibition of pyruvate oxidation and reduction in glucose oxidation. (3) The 
inhibition of glucose oxidation results in a reciprocal increase in fat oxidation and 
acetyl Co-A levels (4) The increase in fat oxidation aims to provide an alternative 
energy source to meet the metabolic demands of the cell. Because fat oxidation is 
associated with generation of FADH which can generate ATP via donating electrons 
to complex 2, thereby, bypassing the block at complex 1. (5) the decrease in the 
fasting plasma glucose concentration in T2DM patients following glucosuria 
stimulates glucagon secretion and inhibits insulin secretion resulting in an increase in 
plasma glucagon to insulin ratio; (6) the increase in glucagon to insulin ratio further 
stimulates and maintains the increase in HGP and stimulates the conversion of acetyl 
Co-A to ketones (possibly by stimulating the expression of hydroxyl methylglutaryl 
Co-A synthase [HMGS], the rate limiting step for the conversion of acetyl Co-A to 
beta hydroxybuterate [45,46]); (7) in subjects with NGT, SGLT2 inhibition does not 
affect the FPG concentration. Thus, plasma glucagon and insulin concentrations are 
not affected by SGLT2 inhibition, and the plasma ketone concentration does not 
increase (Figure 14). In the present grant request, we will test these hypotheses by: 
(1) examining the effect of empagliflozin on HGP, substrate oxidation, plasma 
ketone/lactate/insulin/glucagon concentrations, hepatic fat content, basal hepatic 
glucose uptake and sympathetic nervous system activity in NGT, IFG and T2DM 
patients with FPG concentration <160 mg/dl using state-of-the-art techniques. 
Because empagliflozin does not affect the FPG, insulin and glucagon concentrations 
in NGT subjects, this allows us to provide definitive evidence for the role of 
sympathetic activation in the increase in HGP caused by empagliflozin, and (2) 
examination of the effect of empagliflozin on substrate oxidation, hepatic fat content, 
FPG concentration, and hepatic glucose uptake during the fasting state in IFG 

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
9 
subjects will provide insight about the pathophysiology of IFG and the therapeutic 
potential of this class of drugs in reversing IFG to NGT and reducing their risk of 
progression to T2DM. 
                                                                                  
Item 4 
Purpose and rationale 
Insert purpose, objectives 
and research 
questions/hypotheses 
here. 
 If you cut and paste from 
another document, make 
sure the excerpted 
material answers the 
question 
Insert purpose: Sodium-Glucose cotransport inhibitors (SGLT2i) are a novel class of 
antidiabetic agents which lower the plasma glucose concentration by inhibiting renal 
glucose reuptake and producing glucosuria. Hepatic glucose production (HGP) is the 
primary determinant of the fasting plasma glucose concentration. Although SGLT2i 
lower the fasting plasma glucose concentration in T2DM patients, we have 
demonstrated that SGLT2i produce a paradoxical increase in the rate of hepatic 
glucose production. Studies which have examined the mechanisms responsible for the 
increase in HGP caused by SGLT2i demonstrated a small decrease in plasma insulin 
concentration and a robust increase in plasma glucagon concentration. Thus, the 
plasma glucagon to insulin ratio was markedly increased and could explain, at least in 
part, the increase in HGP, while the decrease in plasma glucose concentration 
produced by SGLT2i improves insulin sensitivity by removal of glucotoxicity. The 
decrease in plasma glucose concentration results in decreased glucose influx to the 
cell which shifts fuel consumption from glucose to fat oxidation and cause an increase 
in ketone production. We and others recently have demonstrated that, despite the 
improvement in insulin sensitivity, SGLT2i inhibit mitochondrial ATP synthesis, 
suggesting a possible direct off-target effect of the drug on substrate oxidation 
independent of SGLT2 inhibition and glucosuria.  
Impaired fasting glucose (FPG=100-125 mg/dl) is an intermediate stage in the 
transition from normal glucose tolerance to overt diabetes. We have demonstrated that 
bHGP, which is the primary determinant of the FPG, is modestly decreased in IFG 
while the rate of basal glucose disposal is reduced in IFG subjects compared to NGT. 
Since approximately one half of basal glucose disposal takes place in the splanchnic 
area and skeletal muscle in an insulin-independent mechanism, we quantitated the rate 
of basal glucose disposal in IFG subjects and demonstrated that during the 
postabsorptive state total body glucose uptake is significantly reduced in IFG subjects. 
Interestingly, SGLT2i reduced the FPG in IFG subjects with no effect on the FPG in 
NGT subjects despite producing a similar amount of glucosuria in both groups. 
Moreover, SGLT2i inhibited glucose oxidation increased fat oxidation without affecting 
ketone production in IFG and NGT subjects. Further, SGLT2i did not affect the plasma 
insulin or glucagon concentrations in NGT subjects and caused only modest decrease 
in the fasting plasma insulin concentration and a small non-significant and transient 
increase in plasma glucagon concentration in IFG subjects. Lastly, we have 
demonstrated that in in vitro studies, all 3 SGLT2i inhibit pyruvate oxidation in 
hepatocytes in culture. 
Because SGLT2i did not affect the FPG concentration in NGT and caused only modest 
decrease in FPG in IFG despite large glucosuria, it has to cause a robust increase in 
HGP to maintain the plasma glucose concentration. Since SGLT2i increased 
decreased glucose oxidation and increased fat oxidation without increase in plasma 
ketone level or significant change in the plasma glucagon to insulin ratio in NGT and 
Figure 16: Working hypothesis for the 
mechanism(s) responsible for the metabolic 
actions of SGLT2 inhibitors in T2DM  

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
10 
IFG individuals, we hypothesize that: (1) the change in plasma insulin and glucagon 
concentrations in T2DM individuals is likely the effect of the decrease in plasma 
glucose concentration; (2) signals other than the change in glucagon to insulin ratio 
(likely neuronal) are activated by glucosuria and stimulate HGP in NGT and IFG 
individuals; (3) SGLT2i cause an increase in basal hepatic glucose uptake in IFG 
subjects and lead to a decrease in FPG; and (4) to examine these hypotheses, we 
suggest the following specific Aims:    
Specific Aim 1: We will measure FPG, bHGP (3H-glucose infusion), whole body fat 
oxidation (indirect calorimetry), plasma insulin, glucagon, FFA, ketone and lactate 
concentrations, and hepatic fat content (1H-MRS) in IFG, NGT and T2DM patients 
(drug naïve with FPG <160 mg/dl) at baseline and at day 1 and 4 weeks after treatment 
with SGLT2i (empagliflozin) 
Specific Aim 2: Measure autonomic balance (with heart rate variability) and 
sympathetic nervous system activity (3H-norepinephrine turnover) in IFG, NGT and 
T2DM subjects (drug naïve with FPG <160 mg/dl) at baseline and at day 1 and 4 weeks 
of treatment with SGLT2i (empagliflozin).  
 
SIGNIFICANCE 
The present study will add new insight about the mechanism(s) underlying the multiple 
metabolic actions of SGLT2 inhibitors in T2DM patients (1). Our preliminary results 
demonstrate that, this class of drugs, in addition to the inhibition of renal glucose 
transport, exerts a direct effect on glucose oxidation. We and others previously have 
shown that SGLT2 inhibitors exert multiple metabolic actions on glucose and fat 
metabolism with significant clinical implication in T2DM patients: (1) stimulate the basal 
rate of HGP which offset approximately 50% of urinary glucose loss produced by the 
drug and ameliorate the drug efficacy in lowering the plasma glucose concentration; 
(2) increase fat oxidation and ketone production, thus, increasing the susceptibility to 
diabetic ketoacidosis; and (3) increase plasma glucagon concentration which could 
contribute to the increase in HGP and ketone production. The results of the present 
study will define the mechanisms responsible for these actions of SGLT2 inhibitors and 
pave the rode for the development of therapeutic strategies to mitigate their adverse 
events, e.g. risk of ketoacidosis, and increase the efficacy of this class of drugs to lower 
the plasma glucose concentration in T2DM patients. 
 
 
 
Item 5 
Study Population(s) Being 
Recruited 
 
In your recruitment plan, how 
many different populations of 
prospective subjects do you plan to 
target? Provide number: 2  
 
 
 
Identify the criteria for inclusion: 
 
 
Identify the criteria for exclusion: 

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
11 
e.g., a population can be individuals 
with type 2 diabetes controlled with 
diet and/or a population of healthy 
controls.  Or a population can be 
individuals attending an education 
program, etc. 
 
List each different population on a 
separate row and provide a short 
descriptive label:   
(e.g., normal-healthy, diabetics, 
parents, children, etc.) 
 
To add rows use copy & paste 
Type 2 diabetic subjects 
1) FPG< 240 mg/dl 
2) Age 18-70 years 
3) eGFR>60 ml/min 
4) weight stable (±4Lb) in the 
preceding 3 months 
5) Good general health 
1) Use of an insulin or SGLT2 inhibitor 
medications affects glucose 
tolerance 
2) Major organ disease 
3) eGFR <60 ml/min 
4) type 1 diabetes 
 
 
Subjects with FPG<126 mg/dl 
1) FPG<126 and 2-h PG <200 mg/dl 
2) Age 18-70 years 
3) eGFR>60 ml/min 
4) weight stable (±4Lb) in the 
preceding 3 months 
5) Good general health 
1) Use of medications that affect 
glucose tolerance 
2) Major organ disease 
3) eGFR <60 ml/min 
Sub-Study: Type 2 diabetic 
1) current diagnosis of Type 2 
Diabetes 
2) fasting plasma glucose 
concentration <300 mg/dl 
3) Subjects must be drug naïve or on 
stable dose of metformin and/or 
sulfonylurea in the preceding 3 
months 
4) body weight has been stable (± 3 
lbs) over the preceding three 
months 
5) does not participate in an 
excessively heavy exercise 
6) good general health as determined 
by physical exam, medical history, 
blood chemistries, CBC, TSH, 
urinalysis, and EKG 
1) eGFR > 60 ml/min 
2) hematocrit > 34%, 
3) allergy to iodine 
4) Major organ disease 
Sub-Study: non-diabetic 
1) No diagnosis of diabetes (FPG <126 
mg/dl) 
2) body weight has been stable (± 3 
lbs) over the preceding three 
months 
3) does not participate in an 
excessively heavy exercise 
4) good general health as determined 
by physical exam, medical history, 
blood chemistries, CBC, TSH, 
urinalysis, and EKG 
1) eGFR > 60 ml/min 
2) hematocrit > 34%, 
3) allergy to iodine 
4) Major organ disease 
 

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
12 
 
Item 6 
Research Plan / Description of the Research Methods a.  Provide a comprehensive narrative describing the research methods.   
Provide the plan for data analysis (include as applicable the sample size calculation). 
 
Step-by-Step Methods: Study Design:  
After screening, subjects will receive the following tests: (1) Measurement of hepatic glucose 
production, substrate oxidation and plasma ketone/lactate/insulin/glucagon concentrations; (2) 
(Measurement of whole body norepinephrine turnover with 
3H-norepinephrine infusion; (3) 
Measurement of heart rate variability; (4) Measurement of liver fat content with 1H-MRS; (5) 75-grams 
oral glucose tolerance test (OGTT) 
 
The studies will be performed in a random order. 
 
After the completion of the above studies, subjects in each group will be randomized (in 2:1 ratio) to 
receive for 12 weeks, empagliflozin 25 mg per day (n=24) or placebo (n=12), in a double blind fashion. 
The drug treatment will be started (day 1) on the day of the second hepatic glucose measurement 
(see specific details below). On day 2, the measurement of heart rate variability and norepinephrine 
turnover will be repeated. Subjects will continue treatment with the drug (empagliflozin or placebo) 
that they received during the second hepatic glucose measurement for 12 weeks and at the end of 
12 weeks, all baseline tests will be repeated. 
 
The treatment period could be extended based upon the circumstances until the repeat studies are 
completed. 
 
Tests 
Baseline Day 1-3 
12 
Weeks 
OGTT 
OGTT 
OGTT 
HGP 
Short 
Long 
Short 
NE  
NE 
NE 
NE 
MRI 
MRI 
MRI 
DEXA 
DEXA 
HRV 
HRV 
HRV 
HRV 
 
The metabolic effects of empagliflozin lasted for 2 weeks in our previous studies (7). However, 
Ferrannini and colleagues (40) reported that at 4 weeks, the glucose effects of empagliflozin (HGP) 
are attenuated while the fat effects (fat oxidation and ketone production) are enhanced. Therefore, to 
evaluate the long term metabolic impact of SGLT2 inhibition, we decided to extend the treatment 
period in the current study to 12 weeks.  
 
Specific Methods: 
 
Screening: Medical history and physical examination will be performed. Blood samples will be drawn 
for CBC, blood chemistries, lipid profile, thyroid function and HbA1c. EKG will be performed. 
 
Measurement of Hepatic Glucose Production with 3H-glucose infusion (HGP – short): The rate 
of endogenous (primarily reflecting liver) glucose production will be measured with 3H-glucose 
infusion as previously described (7). After a 10 hour overnight fast, a prime (25 µCiXFPG/100) -
continuous (0.25 µCi) infusion of 3H-glucose will be started and continued for 3 (4 in T2DM patients) 
hours. After 2 hours (3 hours in T2DM patients) of tracer equilibration, blood samples will be drawn 
from the catheter on the dorsum of the hand at 120, 130, 140, 150, 160, 170 and 180 minutes (from 
180-240 minutes in T2DM patients). Plasma glucose, insulin, glucagon, GLP-1, cortisol, growth 

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
13 
hormone, ketones, lactate, bicarbonate and catecholamine concentrations, and 3H-glucose specific 
activity will be measured. Indirect calorimetry to measure energy expenditure and substrate oxidation 
rates will be performed between 60-90 minutes. 
 
Measurement of Hepatic Glucose Production With Drug Administration (HGP – Long): On the 
day of starting the treatment, the rate of endogenous (hepatic) glucose production will be measured 
with 3H-glucose as described above with one difference. The 3H-glucose infusion will be continued 
for 7 hours (8 in T2DM patients). After 2 hours of tracer equilibration (3 hours in T2DM patients), 
blood samples will be drawn from the catheter on the dorsum of the hand at -30, -20,-15,-10,-5 and 
0 (time zero is drug ingestion time) minutes. At time zero, subjects will receive the first dose of the 
drug (placebo or empagliflozin 25 mg) and blood samples will be obtained from the retrogradely-
placed catheter on the dorsum of the hand every 20-30 minutes for 5 hours. Plasma glucose, 
insulin, C-peptide, glucagon, GLP-1, cortisol, growth hormone, ketone, lactate, bicarbonate, and 
catecholamine concentrations, and 3H-glucose  specific activity will be measured. Urine will be 
collected during the equilibration period and after drug administration. Urinary volume and glucose 
concentration will be measured and urinary glucose excretion rate will be calculated. Indirect 
calorimetry to measure energy expenditure and glucose and fat oxidation will be performed 3 times 
through the study, from -60 to -30, from 150 to 180 and from 270 to 300 minutes. Blood will also be 
drawn to monitor renal function, electrolytes, glucosoria, and monitor and mitigate urinary tract 
infections.   
 
Norepinephrine turnover rate: The rate of total body norepinephrine spillover (release), which 
reflects total body sympathetic nervous system activity, will be measured with 3H-norepinephrine 
(3H-NE) infusion. Subjects will report to the CRC at 7 AM after a 10 hour overnight fast, and a 
catheter will be placed into an anticubital vein for tracer (3H-NE) infusion. A second catheter will be 
placed retrogradely into a vein on the dorsum of the hand, which is placed in a heated box (60°C) 
for sampling of arterialized blood. The arterialized blood will be ascertained by measuring O2 
saturation (>95%). After drawing 3 baseline blood samples, a prime (3.6 µCi) -continuous (0.36 µCi) 
infusion of 3H-NE will be started and continued for 2 hours. 4-6 arterialized blood samples will be 
drawn from the catheter on the dorsum of the hand at every  10-30 minutes. Plasma NE and 3,4-
dihydroxyphenylglycol (DHPG) concentrations and 3H-NE and 3H-DHPG radioactivity will be 
measured. Previous studies (47,48) have demonstrated that this 3H-NE infusion rate produces a 
steady state of plasma 3H-NE radioactivity within 12 minutes without significant increase (<3%) in 
plasma 3H-DHPG (the primary intraneuronal metabolite of norepinephrine) radioactivity. Blood will 
also be drawn to monitor renal function, electrolytes, glucosoria, and monitor and mitigate urinary 
tract infections.   
 
Heart Rate Variability: Heart rate variability (HRV) will be measured in the morning after a 10-12 
hour overnight fast. The measurement will be performed using the SphygmoCor device (AtCor 
Medical, Sydney, Australia) with the participant lying in the resting supine position for 10 min. This 
device takes into account only the normal heart beats, ignoring ectopic beats, and derives the time 
(SDNN and rMSSD) and frequency (LF and HF) domain parameters of the normal R-R intervals of 
the electrocardiogram (49). Parasympatheric nervous system (primarily is represented by 
decreased high frequency domain [0.15-0.4 HZ] and rMSSD), and sympathetic nervous (primarily is 
represented by decreased low frequency domain [0.04-0.15] and SDNN) system activity will be 
assessed before and at day 2 and week 12 of treatment with empagliflozin. Blood will also be drawn 
to monitor renal function, electrolytes, glucosoria, and monitor and mitigate urinary tract infections.  
 
The following additional measurements will be performed:  1) Central aortic pulse pressure, 2) Pulse 
wave velocity (PWV), 3) Ankle-Brachial Index (ABI), 4) Toe-Brachial Index (TBI), and 5) Below knee 
blood flow.  All measurements will be done on the same day of the HRV. Central aortic pulse 
pressure and pulse wave velocity (PWV) are performed by the same instrument (Sphygmacore) 
with which heart rate variability is measured. They will be measured together with HRV with the 
patient lying supine in bed. For the central aortic pulse pressure, blood pressure is measured while 
patient is lying supine on bed, and a probe is placed on the radial pulse to detect radial pulse wave. 

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
14 
The measurement takes approximately 10 minutes.   The PWV will be measured as carotid-femoral 
and carotid-radial PWV. The patient will be connected to ECG electrodes, and the probe will be 
placed on the carotid pulse, femoral pulse and radial pulse and the pulse wave at each site is 
recorded together with the ECG. This measurement will take approximately 20 minutes.  Ankle-
Brachial Blood Pressure Index (ABI) and Toe-Brachial Blood Pressure Index (TBI) are measured 
with an US machine with the patient lying supine on bed. During the measurement, a blood 
pressure cuff is placed around the arm and a second cuff around the leg, and a probe is connected 
to the toe. The measurement takes 10-15 minutes.  
 
Blood flow in the leg is measured with the same machine with a special U/S probe which is place on 
the anterior tibial region below the knee. It measured blood flow in the anterior tibial artery. This 
measurement takes about 10-15 minutes.  All vascular measurements will be performed on the 
same visit and will take one and a half to two hours to complete.       
 
Hepatic Fat Content: Hepatic triglyceride content will be measured by magnetic resonance and 
proton spectroscopy (1H-MRS) with 3-T magnetic resonance imaging scanner (Siemens, Germany) 
as previously described (50). Measurements will be performed in the morning after 10 h overnight 
fast. Blood will also be drawn to monitor renal function, electrolytes, glucosoria, and monitor and 
mitigate urinary tract infections.   
 
Follow Up Visits: at weeks 1, 2, 4, 6, 8 and 10 after the start of treatments, subjects will come to the 
CRC for follow-up visits. At week 4 and 8 visits, medical history and physical exam will be performed. 
Body weight, blood pressure and pulse will be measured. Blood sample will be drawn for the 
measurement of fasting plasma glucose, insulin, glucagon, FFA, HbA1c, Pro BNP and ketones. Blood 
will also be drawn to monitor renal function with ketones, electrolytes, glucosoria, and monitor and 
mitigate urinary tract infections. 
 
If the treatment period is extended, patients will continue to be followed up every 2-4 weeks and 
provided with the study medications until the repeat tests are completed. If the circumstances prohibit 
clinic visits for follow-up, the study team will perform a phone follow-up visit to ensure patient 
adherence, information collection, and adverse events monitoring.         
 
Sub-Study 1: 
36 T2DM patients will participate in this study, The T2DM subjects enrolled in the main study will also 
be subjects enrolled into the sub-study. Each subject will receive a measurements of hepatic glucose 
production with stable isotope (6,6,-D2-glucose) plus indirect calorimetry with empagliflozin (n=24) or 
placebo (n=12) administration. Norepinephrine turnover measurement will be performed at baseline 
(before the empagliflozin or placebo administration) and 5 hours after the drug administration.  
On a separate day subjects will return to the CRC and the measurement of hepatic glucose production 
and NE turnover studies will be repeated with a glucose clamp. 
 
Specific Methods: 
Measurement of Hepatic Glucose Production With 6,6,D2-glucose, 3H-NE  infusion and Drug 
Administration: In this study, HGP measurement will be done with 6,6,D2-glucose infusion and NE 
turnover measurement with 3H-NE infusion. A prime (4 mg/kg) continuous (0.04 mg/kg.min) infusion 
of 6,6-D2-glucose (Cambridge Isotopes, Boston, MA) will be started and continued to the end of the 
study (8 hours). After a 3-hour basal tracer equilibration period, patients will ingest empagliflozin 25 
mg (n=24) or placebo (n=12). The 6,6-D2-glucose will be continued for additional 5 hours after drug 
ingestion. Blood samples will be drawn from the catheter on the dorsum of the hand at -30, -20,-10, 
and 0 (time zero is drug ingestion time) minutes. At time zero, subjects will receive the drug (placebo 
or empagliflozin 25 mg) and blood samples will be obtained from the retrograde-placed catheter on 
the dorsum of the hand every 20 minutes for 6 hours. Plasma glucose, insulin, glucagon, GLP-1, 
cortisol, growth hormone, ketone, lactate, and bicarbonate, concentrations, and 6,6-D2-glucose 
enrichment will be measured.  
 

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15 
At -60 minutes, 3H-NE infusion will be started for 60 minutes (from -60 to 0 minutes) for the 
measurement of NE turnover as described above (4 blood samples are collected every 10-30 
minutes between -60 and time zero). The 3H-NE infusion will be repeated at 240 minutes and 4 
blood samples are collected before and after 3H NE infusion. Urine will be collected during the 
equilibration period and after drug administration (0 to 5 hours). Urinary volume and glucose 
concentration will be measured and urinary glucose excretion rate will be calculated. Indirect 
calorimetry to measure energy expenditure and glucose and fat oxidation will be performed 2 times 
through the study, from -90 to -60, from and from 210 to 240 minutes. Blood will also be drawn to 
monitor renal function, electrolytes, glucosoria, and monitor and mitigate urinary tract infections.   
 
 
Measurement of Hepatic Glucose Production With 6,6,D2-glucose, 3H-NE  infusion and glucose 
clamp: 
HGP will be measured with 6,6,D2-glucose and NE turnover will be measured as described above 
with one difference. After the drug ingestion, the plasma glucose concentration will be measured 
every 5 minutes and variable 20% dextrose solution will be adjusted to maintain the plasma glucose 
concentration at the fasting level. Blood will also be drawn to monitor renal function, electrolytes, 
glucosoria, and monitor and mitigate urinary tract infections.   
 
Effect of Empagliflozin on EGP and NE turnover with Pancreatic Clamp:  
In this study, HGP measurement will be done with 6,6,D2-glucose infusion and NE turnover 
measurement with 3H-NE infusion as described in Study above, and plasma insulin and glucagon 
concentrations will be clamped at the basal level using the pancreatic clamp technique (17,62), 
while the plasma glucose concentration will be allowed to decrease spontaneously after 
empagliflozin and placebo administration. Somatostatin (750 µg/h) infusion will be started 15 
minutes before the start of 6,6-D2-glucose infusion along with basal infusions of glucagon (0.3 
ng/kg.min) and insulin (0.1 mU/kg.min) to replace basal plasma glucagon and insulin 
concentrations. The somatostatin, glucagon and insulin infusions will be continued for the entire 
duration of the study (until 2 PM). A prime (4 mg/kg) continuous (0.04 mg/kg.min) infusion of 6,6-
D2-glucose (Cambridge Isotopes, Boston, MA) will be started and continued to the end of the study 
(9 hours). After a 3-hour basal tracer equilibration period (time zero), patients will ingest 
empagliflozin 25 mg (n=24) or placebo (n=12). The 6,6-D2-glucose will be continued for additional 5 
hours after drug ingestion. Blood samples will be drawn from the catheter on the dorsum of the 
hand at -30, -20,-10, -5, and 0 (time zero is drug ingestion time) minutes. At time zero, subjects will 
receive the drug (placebo or empagliflozin 25 mg) and blood samples will be obtained from the 
retrograde-placed catheter on the dorsum of the hand every 20 minutes for 5 hours. Plasma 
glucose, insulin, glucagon, GLP-1, cortisol, growth hormone, ketone, lactate, and bicarbonate, 
concentrations, and 6,6-D2-glucose enrichment will be measured.  
 
At -60 minutes, 3H-NE infusion will be started for 60 minutes (from -60 to -0 minutes) for the 
measurement of NE turnover as described above ( 4 blood samples are collected every 10-30 
minutes between -60 and time zero). The 3H-NE infusion will be repeated at 240 minutes and 4 
blood samples are collected before and after 3H NE infusion. Urine will be collected during the 
equilibration period and after drug administration (0 to 5 hours). Urinary volume and glucose 
concentration will be measured and urinary glucose excretion rate will be calculated. Indirect 
calorimetry to measure energy expenditure and glucose and fat oxidation will be performed 2 times 
through the study, from -90 to -60, and from 210 to 240 minutes. 
 
Sub-Study 2 (only conducted at TDI): 
In initial studies, we observed that the increase in EGP after empagliflozin administration is 
influenced by the amount of fluids (saline infused intravenously plus amount of water drank by 
patient) taken by the patient during the study. Because empagliflozin causes glucosuria, and 
increase in urinary sodium and fluid excretion, this observation raises the possibility that plasma 
volume is an important parameter in the action of SGLT2 inhibitors. Further, since decreased 
plasma volume is strong stimulus of sympathetic activity, the decrease in plasma volume could 

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
16 
provide the link between empagliflozin and sympathetic activation (which is the primary focus of the 
present study).     
 
To explore possible role of change in plasma volume in the link between empagliflozin 
administration and increase in EGP, we request to add a pilot study (Sub-study 2) in which the 
effect of empagliflozin (or placebo) on EGP production is measured with and without replacement of 
fluids lost in the urine during the study.   
 
Research Design and Methods: 
Subjects: Two groups of subjects will participate in this sub-study: (1) 36 T2DM patients, and (2) 18 
non-diabetic healthy subjects . T2DM subjects must have fasting plasma glucose concentration 
<300 mg/dl. There is no limit on the upper level of HbA1c. Subjects must be drug naïve or on stable 
dose of metformin and/or sulfonylurea in the preceding 3 months. Other than diabetes, subjects 
must be in good general health as determined by physical exam, medical history, blood chemistries, 
CBC, TSH, urinalysis, and EKG. Only subjects whose body weight has been stable (± 3 lbs) over 
the preceding three months and who do not participate in an excessively heavy exercise program 
will be included. Individuals with eGFR < 60 ml/min, hematocrit <34%, and subjects with allergy to 
iodine will be excluded.  
 
Pregnant women and women planning to become pregnant will be excluded from the study. We will 
perform a pregnancy test at the time of screening. Women of child baring age will be requested to 
use at least one barrier method before being enrolled in the study. 
 
Study Design: 
After screening, eligible subjects will receive DEXA scan and 4 measurements of EGP as described 
below.  
 
Each EGP measurement will be performed on a different day 1-3 weeks apart, after 10-12 hour 
overnight fast. Subjects will be instructed to ingest a weight maintaining diet containing 45% CHO, 
25% fat, and 30% protein throughout the entire study period. 
 
EGP Measurement 
Subjects will fast after 8 PM on the preceding day and report to the Clinical Research Center at 6 
AM on the following morning. Subjects will not take their metformin or sulfonylurea on the day of 
study. At 6 AM a catheter will be inserted into an antecubital vein for the infusion of all test 
substances. A second catheter will be placed retrogradely into a vein on the dorsum of the hand, 
which will be placed in to a heated box (60°C) for arterialized blood withdrawal. The retrograde 
catheter will be continuously infused with half normal saline solution at rate 2 ml/hour to maintain it 
patency. At 6 AM (-180 minute), a prime-(25 X FPG/100 µCi) continuous (0.25 µCi/min)  infusions of 
3H-3-glucose for determination of rates of total body glucose appearance and disappearance will be 
started and continued for 10 hours (420 minute).   
 
Blood sample will be withdrawn at -180 minute to measure plasma glucose concentration and 
background 3H-3-glucose specific activity. Between -40 to -10 minutes, 5 additional blood samples 
will be withdrawn every 5-20 minutes to measure 3H-3-glucose specific activity, plasma glucose, 
insulin, glucagon, FFA, ketone, norepinephrine, ACTH, cortisol, renin, angiotensin-II, NT-proBNP, 
and electrolyte (Na, K, Cl, HCO3) concentrations. 
 
At time -10 minute, a bolus of (0.25 mg/kg) ICG will be injected and 8 blood samples (from -8 to 
zero minutes) will be withdrawn every 20 seconds to one minute to measure plasma volume.  
 
At time zero each subject will ingest the treatment drug (empagliflozin 25 mg or placebo). Plasma 
volume measurements will be repeated with a bolus injection of ICG at 380 minute. 7 blood samples 
will be drawn after time zero for the measurement 3H-3-glucose specific activity, plasma glucose, 

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
17 
insulin, glucagon, FFA, ketone, norepinephrine, ACTH, cortisol, renin, angiotensin-II, NT-proBNP, 
and electrolyte (Na, K, Cl, HCO3) concentrations. 
 
  30-45 minutes of continuous indirect calorimetry will be performed at -60, 180, and 390 minutes to 
determine rates of glucose/lipid oxidation and energy expenditure. The total blood draw in this sub 
study will be 165 ml. 
At 6 AM subjects will void (and discard) and urine will be collected from -180 to zero, and from time 
zero to 420 minute. Urine volume and concentrations of glucose, -OH-B, sodium, and chloride will 
be measured.  
 
Figure 1: Scheme of EGP Measurement in Sub-study 2 
 
Measurement I.  Subjects will ingest placebo at time zero, and blood and urine samples will 
be collected as described above. The retrograde line will be kept patent by infusing 0.5 N 
saline at 2 ml per hour using high precision infusion pumps (2ml/hour x 7 hours = 14 ml total 
over 7 hours). 
Measurement II. Subjects will ingest empagliflozin (25 mg) at time zero, and blood and urine 
samples will be collected as described above.  Each intravenous line will be kept patent as 
described in Measurement I.  Using this protocol we previously have shown that SGLT2i 
(dapagliflozin, empagliflozin, and canagliflozin) stimulate endogenous glucose production 
(EGP) compared to placebo and increase plasma ketone concentration by 0.3-0.6 mmol/L, 
in association with a 4-7 uU/ml decrement in plasma insulin concentration that was 
paralleled by a similar decrease in plasma C-peptide concentration and increase in plasma 
glucagon concentration (3).  
Measurement III.  Subjects will ingest empagliflozin (25 mg) at time zero and blood and urine 
samples will be collected as described above.  At time zero, an intravenous infusion of saline 
will be started to replace the amount of fluid and electrolites excreted in the urine during the 
EGP measurement without volume replacement. Approximately 70 meq of Na+ and 70 meq 
of Cl- are excreted in the urine by SGLT2i treatment within 6 hours. Thus, 2.2 ml/min (of half 
normal saline), or 1.1 ml/min of normal saline (0.9%) will be infused until the study end at 
420 minute. In previous studies with empalgilfozin, as well as with dapagliflozin and 
canagliflozin (3,5), we demonstrated that 8-hour urine volume and 8-hour urine sodium 
chloride excretion increased by ~600 ml and ~70 meq, respectively.  Thus, administration of 
462 ml (or 924 ml if normal saline is infused) of fluid and 70 meq of Na+Cl- over a 7-hour 
period should be more than sufficient to prevent any decrease in plasma (represents 5% of 
body weight), and extracellular (represents 20% of body weight) fluid volume. The 
measurement of plasma volume with ICG will document the absence of any change 
(decrease) in fluid space. In addition we will measure NT proBNP throughout the study.   
Time (min) 

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18 
Measurement IV: This measurement will be performed similar to Measurement III with one 
difference. At time zero, subjects will ingest placebo instead of empagliflozin.  
Data Analysis and Statistical Methods.  Under steady-state postabsorptive conditions, the 
basal rate of endogenous glucose appearance (Ra = bEGP) equals 3-3H-glucsoe infusion 
rate divided by steady state plasma tritiated glucose specific activity.  After drug 
administration, non-steady conditions for 3-3H-glucose specific activity prevail and the rate 
of whole body of glucose appearance (Ra) is calculated from the Steele equation.  The 
difference in EGP during the last half hour of the study (390 to 420 minutes) between 
subjects receiving empagliflozin (Measurement II) vs placebo (Measurement I) represents 
the increase in EGP caused by empagliflozin. The difference in EGP between subjects 
receiving empagliflozin without volume repletion (Measurement II) versus with volume 
repletion (Measurement III) represents the role of intravascular volume depletion in the 
stimulation of EGP. The difference in the change in EPG between Measurement II and 
Measurement III will be compared to the difference between Measurement I and 
Measurement IV will be compared with ANOVA.  Post hoc testing will be performed with 
Bonferroni correction for multiple comparisons. 
The same statistical/analytical approach will be employed for the measurement of plasma 
FFA, and beta-OH-butyrate concentrations.  
Sample Size Calculation.  In previous studies, we demonstrated that the mean difference in 
EGP during the last hour of EGP measurement in empagliflozin-treated (Measurement II) 
and placebo-treated (Measurement I) subjects was 0.70±0.34 mg/kg.min (mean±SD) (3).  To 
detect a 50% decrease in this difference at alpha <0.05 during the volume repletion study 
(Measurement III) with 90% power and alpha = 0.0125 (=0.05/4), we computed that 32 
subjects are required.  Therefore, to ensure 32 completers, we have set the sample size at 
36. 
 
 
Risks in Sub-Study 2: 
Blood loss: blood loss in sub-study 2 will be 165 ml at each EGP measurement. Thus, the total 
blood loss will be 660 ml 
 
Radiation: total amount of radiation in sub-study 2 will be 59.6 mrem (13.4 at each EGP 
measurement and 6 mrem at DEXA scan) 
 
Indocyanine Green (ICG): is approved by the FDA for the measurement of cardiac output and 
hepatic function. It is cleared from the body by excretion to the biliary system. The only known 
adverse event is allergic reaction to iodine. Subjects with allergy to iodine will be excluded from the 
study. Although ICG is rapidly cleared from the body, it is recommended that subjects will not 
perform radioiodine uptake studies until one week after the use of ICG.   
 
Empagliflozin: approved by the FDA for the treatment of T2DM. All patients in this sub-study have 
T2DM. The drug increases the risk of genital and urinary infections by ~5%. Rare adverse events 
(<1%) include volume depletion, decreased blood pressure, ketoacidosis, necrotizing fasciitis, and 
acute kidney injury. These adverse events were observed with long treatment (6 months) with the 
drug. However, subjects will receive only two tablets in the present study.  
 
 
Assessment of adverse events 
Definitions of adverse events: 
Adverse event - An AE is defined as any untoward medical occurrence in a patient or clinical 
investigation subject administered a medicinal product and which does not necessarily have to have 
a causal relationship with this treatment.  
 

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19 
An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of a medicinal product, whether or 
not considered related to the medicinal product. 
 
The following should also be recorded as an AE in the CRF and SAE form (if applicable): 
• 
Worsening of the underlying disease or of other pre-existing conditions  
• 
Changes in vital signs, ECG, physical examination and laboratory test results, if they are 
judged clinically relevant by the investigator 
 
Serious adverse event: 
A serious adverse event (SAE) is defined as any AE, which fulfils at least one of the following 
criteria:  
• 
results in death,  
• 
is life-threatening, which refers to an event in which the patient was at risk of death at the 
time of the event; it does not refer to an event that hypothetically might have caused death if 
more severe,  
• 
requires inpatient hospitalization or prolongation of existing hospitalization  
• 
results in persistent or significant disability or incapacity,  
• 
is a congenital anomaly / birth defect,  
• 
is deemed serious for any other reason if it is an important medical event when based on 
appropriate medical judgement which may jeopardise the patient and may require medical or 
surgical intervention to prevent one of the other outcomes listed in the above definitions. 
Examples of such events are intensive treatment in an emergency room or at home for 
allergic bronchospasm, blood dyscrasias or convulsions that do not result in hospitalisation 
or development of dependency or abuse.  
• 
Cancers of new histology and exacerbations of existing cancer must be classified as a 
serious event regardless of the time since discontinuation of the drug and must be reported. 
 
Adverse events of Special Interest (AESIs): 
 
The term AESI relates to any specific AE that has been identified at the project level as being of 
particular concern for prospective safety monitoring and safety assessment within this trial, e.g. the 
potential for AEs based on knowledge from other compounds in the same class.  AESIs need to be 
reported to the Pharmacovigilance Department of Boehringer Ingelheim within the same timeframe 
that applies to SAEs. 
 
Patients with AESIs need to be followed up appropriately, regardless of the origin of the laboratory 
data (e.g. central, local etc.). The Investigator should consider which, if any, concomitant therapies 
should not be taken during evaluation.  Discontinued treatments can be reintroduced per 
Investigator discretion. 
 
The following are considered as AESIs: 
Hepatic injury - A hepatic injury is defined by the following alterations of hepatic laboratory 
parameters after randomisation:  
• 
an elevation of AST and/or ALT ≥ 3 fold ULN combined with an elevation of total bilirubin ≥ 2 
fold ULN measured in the same blood sample 
• 
an isolated elevation of ALT and/or AST ≥ 5 fold ULN 
 
These laboratory findings constitute a hepatic injury alert and the patients showing these 
abnormalities need to be followed up according to medical judgement. . 
 
In case of clinical symptoms of hepatic injury (icterus, unexplained encephalopathy, unex-plained 
coagulopathy, right upper quadrant abdominal pain, etc.) without laboratory results (ALT, AST, total 

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
20 
bilirubin) available, the Investigator should make sure these parameters are analysed, if necessary 
in an unscheduled blood test. 
 
Decreased renal function 
 
Decreased renal function is defined by a creatinine value showing a ≥ 2 fold increase from baseline 
and is above the ULN. 
 
For the AESI “decreased renal function” the Investigator shall collect an unscheduled laboratory 
sample for creatinine as soon as possible and initiate follow-up laboratory tests of creatinine 
according to medical judgement. 
 
Metabolic acidosis, ketoacidosis and diabetic ketoacidosis (DKA) 
In case of metabolic acidosis, ketoacidosis and DKA further investigations should be done 
according to the medical judgment and the clinical course until a diagnosis is made and/or the 
patient is recovered. 
DKA is defined by the diagnostic criteria in the table below, and as defined by the American 
Diabetes Association (ADA). 
 
Investigators should note that not all criteria in the table below need to apply for the diagnosis of 
DKA, and clinical judgment should also be taken into consideration. Due to its mechanism of action, 
empagliflozin may potentially modify the clinical presentation of DKA which may occur at lower 
plasma glucose levels than stated in the table below. 
 
 
Table 1 Diagnostic criteria for DKA 
 
DKA 
 
Mild 
Moderate 
Severe 
Plasma glucose (mg/dL) 
>250 
>250 
>250 
Arterial pH 
7.25-7.30 
7.00-7.24 
<7.00 
Serum bicarbonate (mEq/L) 
15-18 
10 to <15 
<10 
Urine ketones* 
Positive 
Positive 
Positive 
Serum ketones* 
Positive 
Positive 
Positive 
Effective serum osmolality (mOsm/kg)** 
Variable 
Variable 
Variable 
Anion gap*** 
>10 
>12 
>12 
Alteration in sensoria or mental 
obtundation 
Alert 
Alert/drowsy 
Stupor/coma 
* 
Nitroprusside reaction method 
** 
Calculation: 2[measured Na (mEq/L) + glucose (mg/dL)/18 
*** 
Calculation: (Na+) – (Cl- + HCO3-) (mEq/L) 
 
Intensity of adverse event 
The intensity of the AE should be judged based on the following: 
• 
Mild: Awareness of sign(s) or symptom(s) which is/are easily tolerated 
• 
Moderate: Enough discomfort to cause interference with usual activity 
• 
Severe: Incapacitating or causing inability to work or to perform usual activities 
 
Causal relationship of adverse event 
Medical judgment should be used to determine the relationship, considering all relevant factors, 
including pattern of reaction, temporal relationship, de-challenge or re-challenge, confounding 
factors such as concomitant medication, concomitant diseases and relevant history. Assessment of 
causal relationship should be recorded in the case report forms.  
 

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21 
Yes: There is a reasonable causal relationship between the investigational product administered 
and the AE.  
 
Arguments that may suggest that there is a reasonable possibility of a causal relationship could be:  
• 
The event is consistent with the known pharmacology of the drug.  
• 
The event is known to be caused by or attributed to the drug class.  
• 
A plausible time to onset of the event relative to the time of drug exposure.  
• 
Evidence that the event is reproducible when the drug is re-introduced.  
• 
No medically sound alternative aetiologies that could explain the event (e.g. pre-existing or 
concomitant diseases, or co-medications).  
• 
The event is typically drug-related and infrequent in the general population not exposed to 
drugs (e.g. Stevens-Johnson syndrome).  
• 
An indication of dose-response (i.e. greater effect size if the dose is increased, smaller effect 
size if dose is reduced).  
 
No: There is no reasonable causal relationship between the investigational product administered 
and the AE.  
 
Arguments that may suggest that there is no reasonable possibility of a causal relationship could 
be:  
• 
No plausible time to onset of the event relative to the time of drug exposure is evident (e.g. 
pre-treatment cases, diagnosis of cancer or chronic disease within days / weeks of drug 
administration; an allergic reaction weeks after discontinuation of the drug concerned).  
• 
Continuation of the event despite the withdrawal of the medication, taking into account the 
pharmacological properties of the compound (e.g. after 5 half-lives).  
 
Of note, this criterion may not be applicable to events whose time course is prolonged despite 
removing the original trigger.  
• 
Additional arguments amongst those stated before, like alternative explanation (e.g. 
situations where other drugs or underlying diseases appear to provide a more likely 
explanation for the observed event than the drug concerned).  
• 
Disappearance of the event even though the trial drug treatment continues or remains 
unchanged. 
 
 
Responsibilities for SAE reporting to Boehringer Ingelheim 
The Sponsor shall report (i.e., from signing the informed consent onwards through the trial defined 
12-week follow-up period)  all SAEs and non-serious AEs which are relevant for a reported SAE and 
Adverse Events of Special Interest (AESI) by fax or other secure method using BI IIS SAE form to 
the BI Unique Entry Point in accordance with timeline specified below  
 
• 
within five (5) calendar days upon receipt of initial and follow-up SAEs containing at least 
one fatal or immediately life-threatening event;  
• 
within ten (10) calendar days upon receipt of any other initial and follow-up SAEs.  
 
BIPI Unique Entry Point:  
Boehringer Ingelheim Pharmaceuticals, Inc.  
900 Ridgebury Road, Ridgefield, CT  
Fax: 1-203-837-4329  
 
For each adverse event, the investigator will provide the onset date, end date, intensity,treatment 
required, outcome, seriousness, and action taken with the investigational drug. The investigator will 
determine the expectedness of the investigational drug to the AEs as defined in the Listed Adverse 
Events section of the Boehringer Ingelheim’s (BI’s) Investigator Brochure for the Productor BI Drug 

17-214H, Abdul-Ghani, Form BC, 05-23-20, AMD.docx 
 
22 
Information e.g. Summary of Product Characteristics (SmPC) or Product Information (PI) for the 
authorised Study Drug provided by BI, 
 
After the individual patient’s end of trial: the investigator does not need to actively monitor the 
patient for new AEs but should only report any occurrence of cancer and trial treatment related 
SAEs which the investigator may become aware of by any means of communication. 
Data Analysis Plan: Data Analysis and Statistical Plan: Glucosuria Induced Increase in Hepatic 
Glucose Production: Under steady-state postabsorptive conditions, the basal rate of endogenous 
(primarily reflecting hepatic) glucose appearance (Ra = bHGP) equals the 3H-glucose infusion rate 
divided by steady state plasma 3H-glucose specific activity. After drug (empagliflozin) administration, 
non-steady state conditions for 3H-glucose specific activity prevail and the of rate whole body glucose 
appearance (Ra) is calculated from Steele’s equation (52). The difference between HGP in 
empagliflozin-treated and placebo-treated subjects, in each group (i.e. NGT, IFG and T2DM), 
represents the increase in HGP stimulated by glucosuria.  
 
During the Oral-IV double tracer infusion study, only the 14C-glucose which escapes the liver and non-
hepatic splanchnic tissues appears in the systemic circulation, while all of the infused 3H-glucose 
appears in the systemic circulation. Therefore, the difference between Ra calculated with 3H-glucose 
and Ra calculated with 14C-glucose represents the basal rate of hepatic plus non-hepatic splanchnic 
tissue glucose uptake. Thus, the basal rate of EGP will be calculated for 3H-glucose as the 3H-glucose 
infusion (dpm/min) divided by the steady-state plasma 3H-glucose specific activity (dpm/mg).  The 
rate of 14C-glucose appearance will be calculated as the 14C-glucose infusion rate (dpm/min) divided 
by the steady-state plasma 14C-glucose specific activity (dpm/mg). The hepatic and non-hepatic 
splanchnic glucose uptake equals Ra3H-glucose minus Ra14C-glucose Norepinephrine clearance rate will be 
calculated as 3H-NE infusion rate (dpm/min) divided by the steady state plasma 3H-NE radioactivity 
(dpm/ml) (between 12 and 20 min). Norepinephrine spill over (release) rate (pg/min), which reflects 
the sympathetic nervous system activity (53), will be calculated as the product of plasma 
norepinephrine concentration (pg/ml) and norepinephrine clearance rate (ml/min). The difference 
between norepinephrine spill over in empagliflozin-treated and placebo-treated subjects, in each 
group (i.e. NGT, IFG and T2DM), represents the effect of glucosuria on the sympathetic nervous 
system activity. The ratio between plasma 3H-DHPG to 3H-NE (between 20 to 120 minutes) will be 
used as an index of neuronal uptake of norepinephrine (54). 
 
Values will be presented as mean±SD. Difference between means will be compared by ANOVA. Post 
hoc testing will be performed with the Bonferroni test.  
 
The increase in HGP caused by glucosuria in each treatment group will be related to the change in 
norepinephrine spill over rate with Pearson’s correlation in each treatment group. We anticipate a 
strong correlation between the two variables.  
 
Anticipated Results and Potential Problems: Based upon our preliminary results, we anticipate 
that empagliflozin will produce a large increase in HGP in NGT subjects which will maintain the FPG 
concentration at the fasting level. Further, because the FPG concentration does not change following 
empagliflozin administration, we anticipate that neither the fasting plasma insulin nor glucagon 
concentrations will be affected by empagliflozin. Likewise, we anticipate a large increase HGP in IFG 
subjects with no significant change in the fasting glucagon to insulin ratio. We also anticipate a shift 
in substrate oxidation (i.e. decrease in glucose oxidation and increase in fat oxidation) in NGT and 
IFG subjects with no significant increase in plasma ketone concentration. These results will provide 
strong evidence against a significant role of the increase in plasma glucagon to insulin ratio in the 
increase in HGP and shift in fuel consumption from glucose to fat oxidation following SGLT2 
administration. Further, we anticipate that SGLT2 inhibitors will alter autonomic balance and increase 
sympathetic nervous system activity in NGT and IFG subjects and that the increase in sympathetic 
nervous system activity will be, at least in part, responsible for the increase in HGP. Thus, we 
anticipate a strong correlation between the increase in sympathetic nervous system activity and the 
magnitude of increase in HGP in IFG and NGT subjects. 

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23 
 
It will be of great interest to examine the effect of empagliflozin on plasma lactate concentration in 
NGT subjects. If, as anticipated, empagliflozin causes a robust increase in fat oxidation, decrease in 
glucose oxidation and increase in lactate concentration in NGT subjects, without a change in ketones, 
FPG, FFA, insulin, and glucagon concentrations, this will provide a strong evidence in support of a 
direct effect of the drug on mitochondrial substrate oxidation, independent of SGLT2 inhibition. In 
such a scenario, future studies with MRI spectroscopy and 13C-acetate infusion to quantitate hepatic 
TCA cycle activity (55,56) before and immediately after the administration of SGLT2 inhibitor in NGT 
subjects will provide direct evidence in support of an inhibitory effect of SGLT2 inhibitor on 
mitochondrial function independent of SGLT2 inhibition.  
 
Consistent with previous studies (57), we anticipate that IFG subjects will manifest increased hepatic 
fat content and reduced HGU. Further, we anticipate that SGLT2 inhibitors will cause a significant 
decrease in hepatic fat content, increase in basal HGU and reduction in FPG concentration back/close 
to the normal level. Because MRI spectroscopy quantitates hepatic triglyceride content, while toxic 
fat metabolites (e.g. fatty acyl Co-A, DAG and ceramide), are responsible for the inhibition of HGU, it 
is possible to observe an improvement in HGU and decrease in the FPG concentration  in IFG and 
T2DM patients on day 3 without a significant reduction in hepatic fat content measured with MRI. 
Nonetheless, in such a scenario, the reduction in hepatic fat content should be detected after 12 
weeks of treatment.  
 
Lastly, we anticipate that, in addition to the increase in HGP, shift in fuel consumption, and change in 
autonomic nervous system activity, SGLT2 inhibitors will produce a significant decrease in FPG 
concentration in T2DM patients and the decrease in the FPG concentration will be accompanied with 
a small decrease in the fasting plasma insulin concentration and large increase in plasma glucagon 
concentration in association with an increase in plasma ketone concentration in T2DM subjects 
following empagliflozin administration. These results will provide strong evidence in support of an 
important role of the changes in plasma insulin and glucagon concentrations in the increase in plasma 
ketone concentration following SGLT2i administration. It follows, therefore, that combination therapy 
with antidiabetic agents which inhibit glucagon secretion and increase insulin secretion, e.g. GLP-1 
RA, plus SGLT2i will decrease the risk of ketoacidosis associated with SGLT2i therapy in T2DM 
patients (14,15). 
We anticipate that empagliflozin will increase norepinephrine spill over to the plasma in T2DM patients 
as well, and that the increase in norepinephrine spillover will strongly correlate with the increase in 
HGP. Because the methodology utilized in the present study measures the rate of total body 
norepinephrine spill over, it is possible that the increase in epinephrine spill over from specific organs, 
e.g. kidney and splanchnic (primarily hepatic) tissue, will be diluted in the systemic circulation. Thus, 
it is possible that only a modest effect of empagliflozin on sympathetic activity will be detected. In 
such a scenario, measurement of organ specific (58), e.g. renal norepinephrine spill over (with renal 
vein catheterization) and splanchnic (with intradoudenal 3H-NE infusion and arterial or hepatic vein 
sampling) can be employed to quantitate the change in sympathetic activity in the kidney and liver. 
Sample Size Calculation We powered the study to detect a significant increase in the basal rate of 
HGP following empagliflozin administration versus placebo. In previous studies, the increase in the 
rate of HGP versus placebo following empagliflozin administration was 0.34±0.26 mg/kg.min. 
Therefore, we computed that 30 subjects in 2:1 randomization (20 empagliflozin and n=10 placebo) 
provide >90% power to detect a significant difference in HGP between empagliflozin-treated and 
placebo-treated subjects (in 3 groups comparison) at alpha <0.05. Therefore, to ensure 30 
completers, we have set the sample size at 36 in each group.  
 
 

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24 
Item 7 Risks Section: 
Complete the following table to describe the risks of all research procedures listed in Step 2, Institutional Form (items 28-34).  Do not list risks of Routine care 
procedures here.  
 X N/A, Risks are described in the informed consent document – do not complete this table. 
Research procedures 
 
example: 
• 
History and physical 
• 
Questionnaire 
• 
Laboratory tests 
 
Add or delete rows as needed 
Risks 
 
List the reasonably expected risks 
under the following categories as appropriate: 
 
o  
 
o  
 
o  
 
o  
 
 
 
o  
 
o