Am J Physiol Endocrinol Metab 304: E747–E756, 2013.
First published February 12, 2013; doi:10.1152/ajpendo.00639.2012.
Effects of 11␤-hydroxysteroid dehydrogenase-1 inhibition on hepatic
glycogenolysis and gluconeogenesis
J. J. Winnick,1 C. J. Ramnanan,2 V. Saraswathi,3 J. Roop,1 M. Scott,1 P. Jacobson,4 P. Jung,4 R. Basu,5
A. D. Cherrington,1 and D. S. Edgerton1
1
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee;
Department of Cellular and Molecular Medicine, University of Ottawa School of Medicine, Ottawa, Ontario, Canada;
3
Department of Medicine, University of Nebraska Medical Center, Omaha, Nebraska; 4Abbott Laboratories, Chicago, Illinois;
and 5Department of Endocrinology, Diabetes, Metabolism, and Nutrition, Mayo Clinic College of Medicine, Rochester, Minnesota
2
Submitted 19 December 2012; accepted in final form 7 February 2013
11␤-hydroxysteroid dehydrogenase-1; cortisol; hepatic glucose production; glycogenolysis; gluconeogenesis
with many physiological effects,
including the regulation of carbohydrate, protein, and fat metabolism. At pathological concentrations, cortisol produces metabolic
abnormalities similar to the metabolic syndrome, including obesity, insulin resistance, fasting hyperglycemia, hypertension, and
dyslipidemia (2, 42, 56, 58). In vivo cortisol action can be
modified by 11␤-hydroxysteroid dehydrogenase-1 (11␤-HSD1),
an enzyme that converts intracellular cortisone into active cortisol
without necessarily modifying plasma cortisol concentrations (4,
6, 59). Liver (55, 57) and adipose (30, 33) 11␤-HSD1 activity has
been shown to be elevated in obese humans with type 2 diabetes
mellitus and the metabolic syndrome, and an intracellular Cushings-like state may contribute to these abnormalities (28, 32). In
mice, hepatic 11␤-HSD1 overexpression can increase hepatic
CORTISOL IS A STEROID HORMONE
Address for reprint requests and other correspondence: D. S. Edgerton,
Molecular Physiology and Biophysics, Vanderbilt University Medical Center,
710 Robinson Research Bldg., Nashville, TN 37232-0615 (e-mail: dale.
[email protected]).
http://www.ajpendo.org
lipid flux, thereby causing dyslipidemia and insulin resistance
(46), whereas overexpression in adipose tissue causes obesity,
impaired glucose tolerance, and hypertension (35, 36). Conversely, when the enzyme is knocked out or acutely inhibited,
mouse models of diabetes are protected against the manifestations
of the metabolic syndrome (1, 31, 43). Together, these data
demonstrate an overarching ability of dysregulated cortisol metabolism to generate a phenotype similar to that of type 2 diabetes
and illustrate the potential therapeutic value of 11␤-HSD1 inhibition in the treatment of insulin resistance.
Cortisol has potent effects on the glucoregulatory actions of
hormones that regulate hepatic glucose production (HGP). For
example, cortisol has been shown to impair insulin action and
potentiate glucagon- and epinephrine-mediated increases in
HGP (21, 22, 53). In addition, we recently showed that acute
administration of an 11␤-HSD1 inhibitor lowered HGP during
combined insulin and glucagon deficiency in healthy dogs (16),
suggesting that cortisol can also regulate glucose production
independent of its interactions with these hormones. In that
study, the reduction in HGP seen during 11␤-HSD1 inhibition
was accounted for by a reduction in the rate of glycogenolysis,
whereas gluconeogenesis remained unchanged. Interestingly,
however, both phosphoenolpyruvate carboxykinase (PEPCK)
and glucose-6-phosphatase (G6Pase) mRNA levels decreased,
raising the possibility that gluconeogenesis would have been
reduced had enough time for meaningful changes in the levels
of those enzymes been allowed. Therefore, the purpose of this
study was to examine the effect of 7 days of 11␤-HSD1
inhibition on basal glucose metabolism and on the metabolic
responses to a hormonal challenge known to augment HGP by
increasing gluconeogenesis and glycogenolysis (23, 25).
RESEARCH DESIGN AND METHODS
Animals and surgical procedures. Studies were carried out in
conscious 42-h-fasted dogs of either sex (20 –23 kg). This length of
fast in the canine does not induce hypoglycemia, raise the plasma
levels of stress hormones, or exhaust liver glycogen (38, 39). In
addition, after a 42-h fast approximately two-thirds of hepatic glucose
production is accounted for by gluconeogenic flux (48), comparable
with overnight-fasted humans. The surgical and animal care facilities
met the standards published by the American Association for the
Accreditation of Laboratory Animal Care, and diet and housing were
provided as described previously (17). The protocol was approved by
the Vanderbilt University Institutional Animal Care and Use Committee.
Approximately 16 days before the study, each dog underwent
surgery for placement of ultrasonic flow probes (Transonic Systems,
Ithaca, NY) around the hepatic portal vein and the hepatic artery as
0193-1849/13 Copyright © 2013 the American Physiological Society
E747
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on June 15, 2017
Winnick JJ, Ramnanan CJ, Saraswathi V, Roop J, Scott M,
Jacobson P, Jung P, Basu R, Cherrington AD, Edgerton DS.
Effects of 11␤-hydroxysteroid dehydrogenase-1 inhibition on hepatic glycogenolysis and gluconeogenesis. Am J Physiol Endocrinol
Metab 304: E747–E756, 2013. First published February 12, 2013;
doi:10.1152/ajpendo.00639.2012.—The aim of this study was to determine the effect of prolonged 11␤-hydroxysteroid dehydrogenase-1
(11␤-HSD1) inhibition on basal and hormone-stimulated glucose
metabolism in fasted conscious dogs. For 7 days prior to study, either
an 11␤-HSD1 inhibitor (HSD1-I; n ⫽ 6) or placebo (PBO; n ⫽ 6) was
administered. After the basal period, a 4-h metabolic challenge followed, where glucagon (3⫻-basal), epinephrine (5⫻-basal), and insulin (2⫻-basal) concentrations were increased. Hepatic glucose
fluxes did not differ between groups during the basal period. In
response to the metabolic challenge, hepatic glucose production was
stimulated in PBO, resulting in hyperglycemia such that exogenous
glucose was required in HSD-I (P ⬍ 0.05) to match the glycemia
between groups. Net hepatic glucose output and endogenous glucose
production were decreased by 11␤-HSD1 inhibition (P ⬍ 0.05) due to
a reduction in net hepatic glycogenolysis (P ⬍ 0.05), with no effect on
gluconeogenic flux compared with PBO. In addition, glucose utilization (P ⬍ 0.05) and the suppression of lipolysis were increased (P ⬍
0.05) in HSD-I compared with PBO. These data suggest that inhibition of 11␤-HSD1 may be of therapeutic value in the treatment of
diseases characterized by insulin resistance and excessive hepatic
glucose production.
E748
EFFECT OF 11␤-HSD1 INHIBITION ON HEPATIC GLUCOSE METABOLISM
A
B
Experimental timeline on day 8
-140
Fig. 1. Twelve dogs were dosed daily with either
an 11␤-hydroxysteroid dehydrogenase-1 (11␤HSD1) inhibitor (compound 392) or placebo for
8 days, after which they were studied during the
basal fasted state and during hormone infusion.
-40
0
240 min
Control
Period
Equilibration
Period
Experimental
Period
Peripheral [3-3H]Glucose & d4-Cortisol
Portal Glucagon (3xB) & Insulin (2xB)
Peripheral Glucose infusion to match arterial
glucose levels between groups
*
*
*
*
*
*
20
10
0
Hepatic d3-Cortisol Production
(ng/kg/min)
B
40
30
the animals were to be studied, hepatic catheters and flow probe leads
were exteriorized from their subcutaneous pockets under local anesthesia. Intravenous (iv) catheters were also inserted into peripheral leg
veins for infusion of hormones and substrates as necessary.
Treatment administration. Each animal was randomly assigned to receive
either 75 mg of a specific 11␤-HSD1 inhibitor (HSD1-I), E-4-{2-methyl-2[4-(trifluoromethyl)benzyloxy]propionylamino}adamantane-1-carboxylic
acid amide (compound 392, n ⫽ 6; Abbott Laboratories, Abbott Park, IL)
D
30
PBO
HSD1-Inh
20
10
0
-10
40
30
*
*
*
*
*
*
20
10
0
6
Arterial Plasma Cortisol
(µg/dl)
C
Visceral d3-Cortisol Production
(ng/kg/min)
A
Whole Body d3-Cortisol Production
(ng/kg/min)
well as insertion of silicone rubber catheters for sampling in the
hepatic vein, hepatic portal vein, and a femoral artery, with portal vein
infusion catheters inserted into splenic and jejunal veins, as described
in detail elsewhere (17). The proximal ends of the flow probes and
catheters were tucked into subcutaneous pockets. All dogs were
determined to be healthy prior to experimentation, as indicated by
1) leukocyte count ⬍18,000/mm3, 2) hematocrit ⬎35%, and 3) good
appetite (consuming ⱖ75% of the daily ration). On the morning when
4
2
0
-40
0
30
60
90
120 150 180 210 240
Time (min)
-30
0
30
60
90
120 150 180 210 240
Time (min)
Fig. 2. Whole body, liver, and visceral (gut) d3-cortisol production rates and arterial plasma cortisol levels in conscious dogs during the basal (⫺40 to 0 min)
and experimental (0 –240 min) periods treated with a placebo (PBO; Œ) or an 11␤-HSD1 inhibitor (HSD1-Inh; ). Data are means ⫾ SE; n ⫽ 6/group. *P ⬍
0.05, HSD1-Inh vs. PBO.
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Peripheral Somatostatin & Epinephrine (5xB)
E749
EFFECT OF 11␤-HSD1 INHIBITION ON HEPATIC GLUCOSE METABOLISM
Glucose turnover, used to estimate endogenous glucose production
and whole body glucose uptake, was measured using [3-3H]glucose
based on the circulatory model described by Mari et al. (34). The use
of isotopically labeled cortisol in the calculation of hepatic cortisol
production has been described previously in detail (3, 8, 16). This
approach takes advantage of the fact that d4-cortisol loses a deuterium
when it is converted into d3-cortisone by 11␤-HSD2. This, in turn, is
converted into d3-cortisol by 11␤-HSD1, thereby providing a measure
of 11␤-HSD1 activity. Hepatic and visceral d3-cortisol uptake were
calculated as hepatic or visceral d3-cortisol Loadin, respectively,
multiplied by hepatic or visceral d4-cortisol fractional extraction, as
described previously. Hepatic d3-cortisol production was calculated as
the difference between hepatic d3-cortisol uptake and net hepatic
d3-cortisol balance. Visceral d3-cortisol production is the difference
between visceral d3-cortisol uptake and net visceral d3-cortisol balance. Whole body d3-cortisol production was determined by dividing
the d4-cortisol infusion rate by the ratio of arterial d4-cortisol to
arterial d3-cortisol. Whole body total cortisol production was calculated by dividing the d4-cortisol infusion rate by the arterial plasma
d4-cortisol enrichment (i.e., arterial d4-cortisol divided by unlabeled
cortisol plus d3-cortisol plus d4-cortisol).
Net hepatic uptake rates of the gluconeogenic precursors alanine,
lactate, glycerol, glycine, threonine, and serine were measured using the
A
Hepatic Sinusoidal
Glucagon (pg/mL)
200
PBO
HSD1-Inh
160
120
80
40
0
B
Arterial Plasma
Epinephrine (pg/mL)
1500
1200
900
600
300
0
C
Hepatic Sinusoidal
Insulin (µU/mL)
60
45
30
15
0
-40
0
30
60
90 120 150 180 210 240
Time (min)
Fig. 3. Hepatic sinusoidal glucagon and insulin and arterial epinephrine levels
in conscious dogs during the basal (⫺40 to 0 min) and experimental (0 –240
min) periods treated with a PBO (Œ) or an HSD1-Inh (). Data are means ⫾
SE; n ⫽ 6/group.
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(Fig. 1A), or placebo (PBO; n ⫽ 6) for each of 7 days prior to being
studied. On the day of each study (day 8), animals received a final dose
of the respective treatment prior to the start of the experiment.
Experimental design. Each experiment consisted of a 100-min tracer
equilibration period (⫺140 to ⫺40 min), a 40-min period for basal
sample collection (⫺40 to 0 min), and a 240-min experimental period
(0 –240 min; Fig. 1B). At ⫺140 min, primed, continuous iv infusions of
both [9,11,12,12-2H4]cortisol (d4-cortisol; 12 ␮g/kg prime and 0.147
␮g·kg⫺1·min⫺1 continuous rate; Isotope Laboratories, Andover, MA)
and [3-3H]glucose (42 ␮Ci prime and 0.35 ␮Ci/min continuous rate;
Isotope Laboratories) were started. At 0 min, a constant iv infusion of
somatostatin (0.8 ␮g·kg⫺1·min⫺1; Bachem, Torrance, CA) was begun to
suppress pancreatic insulin and glucagon secretion, and infusion of
three-times basal intraportal glucagon (1.5 ng·kg⫺1·min⫺1; Eli Lilly,
Indianapolis, IN) and five-times basal peripheral iv epinephrine (91
ng/kg/min; Sigma-Aldrich) was initiated. Also at 0 min, insulin (450
␮U·kg⫺1·min⫺1; Eli Lilly) was infused intraportally to replicate the
hyperinsulinemia that is associated with the hyperglycemia that occurs in
insulin-resistant individuals or when glucagon and epinephrine levels are
elevated (9, 60). Glucose was infused iv as needed to maintain glycemia
at a similar level in each group.
Hematocrit, plasma glucose, [3-3H]glucose, insulin, glucagon, catecholamines, cortisol, d4-cortisol, d3-cortisol, adrenocorticotropic hormone and nonesterified fatty acids (NEFA), and blood alanine, glycine,
serine, threonine, lactate, glutamine, glutamate, glycerol, and ␤-hydroxybutyrate concentrations were determined as described previously (16, 17).
RNA extraction, cDNA synthesis, real-time PCR, SDS-PAGE, and
Western blotting procedures were performed by standard methods (20).
11␤-HSD1-Inhibitor. 11␤-HSD1 is primarily a reductase, whereas
11␤-HSD type 2 (11␤-HSD2) generally catalyzes the reverse dehydrogenase reaction (56, 58). Compound 392 is a very selective and
potent competitive inhibitor of 11␤-HSD1 compared with 11␤-HSD2
across several species, including mouse (0.7 vs. 13,000 nM), rat (5.9
vs. 2,480 nM), dog (2.8 vs. 12,550 nM), monkey (1.8 vs. 32,840 nM),
and human (1.6 vs. 24,120 nM) (IC50 for 11␤-HSD1 vs. 11␤-HSD2,
respectively). Furthermore, it is not metabolized to any compounds
with appreciable 11␤-HSD1 or 11␤-HSD2 inhibitory activity. Oral
bioavailability in the dog following administration of a capsule
containing 75 mg of compound 392 was 100%, with a Cmax of 3.75
␮g/ml, a plasma half-life of 6.4 h, and an area under the curve of 44
␮g·h⫺1·ml⫺1. Near-complete suppression of hepatic d3-cortisol production in the present study indicates near-complete inhibition of
11␤-HSD1 activity in the liver (Fig. 2B). This is consistent with
previous results in the monkey, where a single oral 10 mg/kg dose
reduced hepatic 11␤-HSD1 enzymatic activity to 2%.
Calculations and data analysis. Net hepatic substrate balance (NHB)
was calculated as LOADout ⫺ LOADin. The LOADout ⫽ FH ⫻ [H], and
LOADin ⫽ (FA ⫻ [A]) ⫹ (FP ⫻ [P]), where A, P, and H refer to the
arterial, portal vein, and hepatic vein substrate concentrations, respectively, and FA, FP, and FH refer to the arterial, portal vein, and hepatic
vein (total liver) blood or plasma flow, respectively, as appropriate for
the particular substrate. Net hepatic fractional substrate extraction was
calculated as NHB/LOADin. Nonhepatic glucose uptake equaled the
glucose infusion rate minus net hepatic glucose ouput, where the rate
was corrected for changes in the size of the glucose pool, using a pool
fraction of 0.65 ml/kg (12) and assuming that the volume of distribution for glucose equaled the volume of the extracellular fluid, or
⬃22% of the dog’s weight (54). For all glucose balance calculations,
glucose concentrations were converted from plasma to blood values
using correction factors (ratio of the blood to the plasma concentration) established previously in our laboratory (26, 45). Net visceral
(gut) substrate balance (NVB) was calculated using the formula NVB ⫽
Loadout ⫺ Loadin, where Loadout ⫽ FP ⫻ [P] and Loadin ⫽ FP ⫻ [A],
abbreviated as indicated above. Net hepatic and visceral fractional
extraction were calculated as NHB ⫼ hepatic Loadin and NVB ⫼
visceral Loadin, respectively.
112 ⫾ 16
155 ⫾ 33
353 ⫾ 49
392 ⫾ 66
4.9 ⫾ 0.7
5.3 ⫾ 0.8
1.5 ⫾ 0.2
2.2 ⫾ 0.2
262 ⫾ 15
294 ⫾ 27
2.2 ⫾ 0.2
2.7 ⫾ 0.6
55 ⫾ 10
44 ⫾ 10
1.1 ⫾ 0.4
1.0 ⫾ 0.3
388 ⫾ 71
365 ⫾ 28
5.5 ⫾ 0.6
6.1 ⫾ 0.5
1.6 ⫾ 0.3
1.5 ⫾ 0.6
270 ⫾ 22
300 ⫾ 25
2.5 ⫾ 0.4
2.9 ⫾ 0.3
52 ⫾ 7
46 ⫾ 9
1.3 ⫾ 0.4
1.5 ⫾ 0.3
⫺20
0
84 ⫾ 16
78 ⫾ 15
15
1.2 ⫾ 0.2
1.3 ⫾ 0.4
47 ⫾ 6
47 ⫾ 12
2.0 ⫾ 0.2
2.7 ⫾ 0.4
273 ⫾ 16
335 ⫾ 41
1.5 ⫾ 0.2
2.3 ⫾ 0.3
4.5 ⫾ 0.4
5.9 ⫾ 1.1
1.4 ⫾ 0.2
2.0 ⫾ 0.6
58 ⫾ 8
53 ⫾ 14
2.7 ⫾ 0.2
3.0 ⫾ 0.3
270 ⫾ 11
344 ⫾ 39
2.1 ⫾ 0.3
2.5 ⫾ 0.3
5.5 ⫾ 1.2
7.5 ⫾ 1.0
325 ⫾ 17 404 ⫾ 32
511 ⫾ 175 554 ⫾ 68
113 ⫾ 14
125 ⫾ 11
Control Period, min
125 ⫾ 22
115 ⫾ 13
⫺40
0.7 ⫾ 0.2
1.2 ⫾ 0.4
43 ⫾ 7
46 ⫾ 11
2.5 ⫾ 0.3
3.2 ⫾ 0.2
259 ⫾ 13
331 ⫾ 39
1.4 ⫾ 0.2
2.2 ⫾ 0.3
5.0 ⫾ 1.1
8.3 ⫾ 1.4
635 ⫾ 85
769 ⫾ 150
87 ⫾ 15
109 ⫾ 31
30
0.4 ⫾ 0.1
0.7 ⫾ 0.4
36 ⫾ 7
40 ⫾ 12
2.9 ⫾ 0.3
3.5 ⫾ 0.3
290 ⫾ 21
369 ⫾ 56
1.6 ⫾ 0.2
1.9 ⫾ 0.3
5.6 ⫾ 0.8
6.5 ⫾ 2.4
1,019 ⫾ 158
1,253 ⫾ 372
78 ⫾ 17
97 ⫾ 20
60
0.5 ⫾ 0.2
0.5 ⫾ 0.2
33 ⫾ 8
28 ⫾ 8
2.9 ⫾ 0.1
3.6 ⫾ 0.3
318 ⫾ 25
400 ⫾ 66
1.7 ⫾ 0.3
1.5 ⫾ 0.2
5.8 ⫾ 1.1
6.7 ⫾ 2.1
1,371 ⫾ 195
1,568 ⫾ 322
0.6 ⫾ 0.3
0.4 ⫾ 0.1
29 ⫾ 7
23 ⫾ 5
3.5 ⫾ 0.3
4.1 ⫾ 0.3
344 ⫾ 25
417 ⫾ 63
1.9 ⫾ 0.3
1.4 ⫾ 0.2
8.2 ⫾ 1.2
8.3 ⫾ 2.5
1,752 ⫾ 215
1,761 ⫾ 360
111 ⫾ 25
96 ⫾ 17
120
108 ⫾ 21
120 ⫾ 22
1.8 ⫾ 0.3
1.4 ⫾ 0.2
399 ⫾ 34
430 ⫾ 61
3.9 ⫾ 0.2
4.6 ⫾ 0.4
25 ⫾ 2
23 ⫾ 6
102 ⫾ 15
114 ⫾ 24
1,839 ⫾ 356
1,777 ⫾ 361
9.2 ⫾ 1.2
5.1 ⫾ 1.7
1.8 ⫾ 0.3
1.2 ⫾ 0.2
381 ⫾ 30
433 ⫾ 61
3.5 ⫾ 0.2
4.1 ⫾ 0.2
33 ⫾ 9
25 ⫾ 8
0.3 ⫾ 0.1
0.3 ⫾ 0.2
86 ⫾ 16
122 ⫾ 31
1,900 ⫾ 280
1,792 ⫾ 355
8.1 ⫾ 1.0
7.6 ⫾ 2.9
1.5 ⫾ 0.2
1.2 ⫾ 0.2
375 ⫾ 24
431 ⫾ 62
3.4 ⫾ 0.2
4.4 ⫾ 0.3
28 ⫾ 4
25 ⫾ 8
0.3 ⫾ 0.0
0.4 ⫾ 0.2
0.3 ⫾ 0.1
0.4 ⫾ 0.2
12.4 ⫾ 1.1
8.6 ⫾ 1.7
2,153 ⫾ 312
1,726 ⫾ 359
210
180
150
Hormone Infusion Period, min
91 ⫾ 11
86 ⫾ 17
90
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Data are means ⫾ SE. PBO, placebo; HSD1-Inh, 11␤-hydroxysteroid dehydrogenase-1 inhibitor.
Arterial norepinephrine levels,
pg/ml
PBO
HSD1-Inh
Arterial blood lactate, ␮mol/l
PBO
HSD1-Inh
Net hepatic lactate uptake,
␮mol 䡠 kg⫺1 䡠 min⫺1
PBO
HSD1-Inh
Net hepatic glycerol uptake,
␮mol 䡠 kg⫺1 䡠 min⫺1
PBO
HSD1-Inh
Arterial blood alanine, ␮mol/l
PBO
HSD1-Inh
Net hepatic alanine uptake,
␮mol 䡠 kg⫺1 䡠 min⫺1
PBO
HSD1-Inh
Arterial blood ␤-hydroxybutyrate,
␮mol/l
PBO
HSD1-Inh
Net hepatic ␤-hydroxybutyrate
output, ␮mol 䡠 kg⫺1 䡠 min⫺1
PBO
HSD1-Inh
Group
Table 1. Hormone and substrate concentrations and net hepatic balances
0.3 ⫾ 0.1
0.5 ⫾ 0.3
25 ⫾ 3
25 ⫾ 1
3.7 ⫾ 0.3
4.1 ⫾ 0.3
402 ⫾ 37
419 ⫾ 59
1.9 ⫾ 0.3
1.4 ⫾ 0.2
11.4 ⫾ 1.2
9.4 ⫾ 1.4
2,022 ⫾ 315
1,656 ⫾ 326
122 ⫾ 21
140 ⫾ 36
240
E750
EFFECT OF 11␤-HSD1 INHIBITION ON HEPATIC GLUCOSE METABOLISM
E751
11␤-HSD1 activity. Whole body d3-cortisol production (via
11␤-HSD1 activity) was reduced by two-thirds in HSD1-I compared with PBO (P ⬍ 0.05 between groups; Fig. 2A), whereas
hepatic d3-cortisol production was completely eliminated between
⫺40 and 240 min (23 ⫾ 2 vs. 0 ⫾ 0 ng·kg⫺1·min⫺1 in PBO vs.
HSD1-I, P ⬍ 0.05; Fig. 2B). Visceral d3-cortisol production was
undetectable in both groups (Fig. 2C), consistent with previous
studies (4, 6, 16). Whole body total cortisol production rates were
233 ⫾ 23 and 225 ⫾ 8 ng·kg⫺1·min⫺1 in PBO and HSD1-I,
respectively (between ⫺40 and 240 min). Since circulating cortisol levels are tightly regulated by the hypothalamic-pituitaryadrenal axis and net hepatic cortisol balance is relatively low, it is
not surprising that the inhibitor had no effect upon arterial cortisol
levels (Fig. 2D).
Basal period. Despite prolonged suppression of intracellular
cortisol production (Fig. 2), hepatic sinusoidal glucagon and
insulin levels and arterial plasma epinephrine (Fig. 3) did not
differ during the basal period. However, there was a tendency for
basal insulin levels to be lower and glucagon and epinephrine
levels to be increased with 11␤-HSD1 inhibition. Subtle (statistically insignificant but biologically important) differences in the
concentrations of these hormones may explain partially why
plasma glucose levels, liver glucose production, net hepatic glycogenolysis, hepatic gluconeogenic flux, and glucose utilization
did not differ between groups during the basal period (Figs. 4 – 6).
In addition, arterial glycerol, NEFA, lactate, and alanine levels
and net hepatic ␤-hydroxybutyrate output, an index of fatty acid
oxidation in the liver, did not differ between groups during the
basal period (Fig. 7 and Table 1).
Hormone challenge period. During the metabolic challenge
(0 –240 min), the hepatic sinusoidal glucagon and arterial epinephrine levels were clamped at three- and fivefold basal, respectively, and were similar between groups (Fig. 3). As expected,
arterial norepinephrine levels remained basal throughout the study
in both groups (Table 1). In response to the metabolic hormone
challenge, the arterial glucose levels rose from 109 to 165 mg/dl
in PBO (Fig. 4A). Glucose was infused to match the plasma glucose
levels between groups, and this rate was greater in HSD1-I than in
PBO (2.07 ⫾ 0.97 vs. 0.21 ⫾ 0.14 mg·kg⫺1·min⫺1, respectively,
during the last hour, P ⬍ 0.05; Fig. 4B). Hepatic insulin levels
were clamped at twofold basal to replicate the hyperinsulinemia
200
160
120
80
PBO
HSD1-Inh
40
0
B
4
Glucose Infusion Rate
(mg/kg/min)
RESULTS
A
*
* *
3
*
*
2
1
0
-40
0
30
60
90 120 150 180 210 240
Time (min)
Fig. 4. Arterial plasma glucose level and glucose infusion rates in conscious
dogs during the basal (⫺40 to 0 min) and experimental (0 –240 min) periods
treated with a PBO (Œ) or an HSD1-Inh (). Data are means ⫾ SE; n ⫽
6/group. *P ⬍ 0.05, HSD1-Inh vs. PBO.
that is associated with the hyperglycemia that occurs with insulin
resistance and stress (9, 60).
The hormone infusion protocol was associated with transient
increases in net hepatic glucose output (Fig. 5A) and endogenous glucose production (Fig. 5B) in PBO. In contrast, 11␤HSD1 inhibition diminished these effects (P ⬍ 0.05) by
reducing net hepatic glycogenolysis (P ⬍ 0.05; Fig. 5C).
Hepatic gluconeogenic flux increased modestly in both groups
(Fig. 5D) but was not affected by 11␤-HSD1 inhibition.
11␤-HSD1 inhibition also affected the regulation of nonhepatic glucose metabolism. Although the hormone infusion
challenge only transiently increased nonhepatic glucose uptake
in PBO and had little to no effect on whole body glucose
utilization, both parameters were increased in HSD1-I compared with PBO (P ⬍ 0.05; Fig. 6, A and B). The area under the
curve for nonhepatic glucose uptake over the final hour was
141 ⫾ 30 vs. 81 ⫾ 14 mg·kg⫺1·60 min⫺1, in HSD1-I and PBO,
respectively (P ⬍ 0.05). Arterial glucose clearance was reduced in PBO, probably due to the effects of epinephrine on
muscle glucose uptake, whereas 11␤-HSD1 inhibition prevented that reduction (P ⬍ 0.05; Fig. 6C). The effect of the
inhibitor on glucose utilization may have also been due in part
to greater suppression of lipolysis, since arterial glycerol and
NEFA levels (Fig. 7) were reduced by ⬃40% in HSD1-I compared with PBO (P ⬍ 0.05). Epinephrine is a potent simulator of
skeletal muscle glycogen degradation and glycolysis. Thus, arterial lactate levels and net hepatic lactate uptake increased in both
groups. Arterial ␤-hydroxybutyrate, alanine, and lactate levels and
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arteriovenous difference method. It was assumed that all of the gluconeogenic precursors taken up by the liver were completely converted into
glucose 6-phosphate (G6P) (17, 18). Gluconeogenic flux to G6P was
estimated by summing net hepatic gluconeogenic precursor uptake and
dividing by two (to convert the data into glucose equivalents). Net hepatic
gluconeogenic flux was calculated by subtracting glycolysis from gluconeogenic flux. Glycolysis was estimated by summing net hepatic lactate
output (when it occurred) and glucose oxidation over the course of each
experiment. In earlier studies, glucose oxidation was 0.2 ⫾ 0.1
mg·kg⫺1·min⫺1 even when the concentrations of circulating insulin,
glucose, and NEFA varied widely (40, 51). Because glucose oxidation
did not change appreciably under conditions similar to the present study,
it was assumed to be constant (0.2 mg·kg⫺1·min⫺1). Net hepatic glycogenolysis was determined by subtracting net hepatic gluconeogenic flux
from net hepatic glucose output (18).
Statistical analysis. Statistical comparisons were carried out with
SigmaStat Software (San Jose, CA), using ANOVA for repeated
measures with post hoc analysis. Statistical significance was accepted
when P ⬍ 0.05. Data are expressed as means ⫾ SE.
Arterial Plasma Glucose
(mg/kg/min)
EFFECT OF 11␤-HSD1 INHIBITION ON HEPATIC GLUCOSE METABOLISM
E752
3
**
2
B
PBO
HSD1-Inh
* *
*
*
*
1
Endogenous Glucose
Production (mg/kg/min)
Net Hepatic Glucose
Output (mg/kg/min)
A
EFFECT OF 11␤-HSD1 INHIBITION ON HEPATIC GLUCOSE METABOLISM
2
*
1
0
0
D
1
3
Net Glycogen Breakdown
* *
*
*
*
0
-1
GNG Fluxto G6P
(mg/kg/min)
**
2
2
1
Net Glycogen Synthesis
0
-40
0
30
60
90 120 150 180 210 240
Time (min)
-40
0
30
60
90
120 150 180 210 240
Time (min)
Fig. 5. Net hepatic glucose output, endogenous glucose production, net hepatic glycogenolysis, and gluconeogenic flux rates in conscious dogs during the basal
(⫺40 to 0 min) and experimental (0 –240 min) periods treated with a PBO (Œ) or an HSD1-Inh (). Data are means ⫾ SE; n ⫽ 6/group. *P ⬍ 0.05, HSD1-Inh
vs. PBO.
net hepatic balance of these substrates were not affected significantly by 11␤-HSD1 inhibition (Table 1).
Previously, we showed that acute 11␤-HSD1 inhibition decreased the gluconeogenic mRNA levels of both PEPCK and
G6Pase as well as the PEPCK protein level (16). However, in the
current study, neither PEPCK (1.0 ⫾ 0.1 and 0.9 ⫾ 0.2 in PBO
and HSD1-I, respectively, arbitrary units), G6Pase (1.0 ⫾ 0.2 and
1.2 ⫾ 0.1, respectively), nor pyruvate carboxylase (1.0 ⫾ 0.2 and
0.8 ⫾ 0.2, respectively) liver mRNA levels differed between
groups at the end of the study. Likewise, PEPCK (1.0 ⫾ 0.2 and
1.2 ⫾ 0.1 in PBO and HSD1-I, respectively) and pyruvate
carboxylase (1.0 ⫾ 0.1 and 1.0 ⫾ 0.2, respectively) protein levels
did not differ.
DISCUSSION
11␤-HSD1 increases intracellular cortisol concentrations,
which can have deleterious effects on substrate metabolism in
tissues where it is expressed, such as the liver and adipose tissues
(2, 42, 56, 58). Although most individuals with diabetes do not
exhibit elevated circulating cortisol concentrations, an intracellular Cushings-like state may contribute to glucose dysregulation
(28, 32). In the present study, we show that inhibition of 11␤HSD1 during a metabolic hormone challenge inhibits hepatic
glucose production (by reducing glycogenolysis) and increases
whole body glucose utilization and the suppression of lipolysis.
Splanchnic (gut and liver) and whole body rates of 11␤HSD1-mediated cortisol production were determined to verify
the effect of the 11␤-HSD1 inhibitor. In agreement with previous findings (4, 6, 16), release of cortisol by the gut was
undetectable in both groups, suggesting that 11␤-HSD1 activity in visceral adipose tissue is negligible. Whereas the inhibitor eliminated hepatic cortisol production, cortisol produced
by the liver in the control group was almost as great as whole
body 11␤-HSD1 cortisol production, indicating that the liver is
the primary site of nonadrenal cortisol production. Consistent
with our previous report (16), arterial cortisol levels were
similar between groups. This is reflective of low (⬃10%) liver
cortisol production relative to the rest of the body and the fact
that splanchnic cortisol uptake accounts for the majority of
splanchnic production (6). Since intracellular cortisol concentrations and action are determined by the balance between
adrenally derived and 11␤-HSD1-facilitated cortisol production, tissues with high 11␤-HSD1 activity, like the liver, could
be expected to display more pronounced effects of 11␤-HSD1
inhibition, whereas glucocorticoid-sensitive tissues lacking
11␤-HSD1 expression would not.
Hyperglycemia in type 2 diabetes is characterized by reduced glucose utilization and elevated HGP due to increased
gluconeogenesis and/or glycogenolysis (5, 7, 44). To determine whether the effects of 11␤-HSD1 inhibition would be
apparent during conditions in which these processes were
elevated, glucagon and epinephrine were infused. These hormones cause insulin resistance; glycogenolysis is stimulated,
gluconeogenesis is amplified due to increased substrate supply
and lipolysis, and glucose clearance is reduced (9, 25, 60).
Consequent hyperglycemia leads to hyperinsulinemia, but the
anti-insulin effects of cortisol, glucagon, and catecholamines
mitigate insulin’s effects. Since cortisol both antagonizes the
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C
Net Hepatic Glycogenolysis
(mg/kg/min)
3
E753
EFFECT OF 11␤-HSD1 INHIBITION ON HEPATIC GLUCOSE METABOLISM
4
PBO
HSD1-Inh
3
* *
2
1
#
0
4
3
*
2
1
0
3
2
*
1
A
0
-40
0
30
60 90 120 150 180 210 240
Time (min)
Fig. 6. Nonhepatic and whole body glucose uptake and arterial glucose
clearance rates in conscious dogs during the basal (⫺40 to 0 min) and
experimental (0 –240 min) periods treated with a PBO (Œ) or an HSD1-Inh ().
Data are means ⫾ SE; n ⫽ 6/group. *P ⬍ 0.05, HSD1-Inh vs. PBO; #P ⬍
0.05, HSD1-Inh vs. PBO area under the curve.
effects of insulin and augments the actions of glucagon and
epinephrine (21, 22, 53), we hypothesized that intrahepatic
reduction of cortisol would improve liver glucose metabolism
when circulating glucagon, epinephrine, and insulin levels
were elevated.
In the absence of 11␤-HSD1 inhibition, the hormonal challenge led to predictable increases in HGP and arterial plasma
glucose levels, which were accounted for by increases in
glycogenolysis and gluconeogenesis. Meanwhile, peripheral
glucose utilization did not increase in the control group despite
hyperglycemic and hyperinsulinemic conditions, an effect that
was most likely due to the inhibitory effect of epinephrine on
insulin-stimulated muscle glucose uptake (10). In contrast,
whole body and hepatic responses to the hormonal challenge
were altered significantly during 11␤-HSD1 inhibition. In
particular, stimulation of hepatic glucose production was prevented and glucose utilization increased, thereby making exogenous glucose infusion necessary to match glycemic levels
between groups.
B
Change from Basal Arterial
Blood Glycerol (µmol/L)
Glucose Clearance
(ml/kg/min)
C
PBO
HSD1-Inh
80
60
*
40
* * *
20
0
-20
-40
400
200
* * *
0
-200
-400
-40
0
30
60
90 120 150 180 210 240
Time (min)
Fig. 7. Change from basal arterial blood glycerol and plasma nonesterified free
fatty acid (NEFA) levels in conscious dogs during the basal (⫺40 to 0 min) and
experimental (0 –240 min) periods treated with a PBO (Œ) or an HSD1-Inh ().
Data are means ⫾ SE; n ⫽ 6/group. *P ⬍ 0.05, HSD1-Inh vs. PBO. Average basal
glycerol levels were 77 ⫾ 5 and 97 ⫾ 9 ␮mol/l, and basal NEFA levels were
695 ⫾ 65 and 779 ⫾ 87 ␮mol/l in PBO and HSD1-Inh, respectively.
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Whole Body Glucose
Uptake (mg/kg/min)
B
Although cortisol can increase hepatic gluconeogenesis,
acute inhibition of 11␤-HSD1 (16) or intrahepatic glucocorticoid receptor signaling (19) was shown previously to regulate
HGP through effects on glycogen metabolism. In addition,
chronic hypercortisolemia had a more pronounced effect on
glycogenolysis than gluconeogenesis during acute insulin deficiency (24). Consistent with those results, our data show that
even after prolonged 11␤-HSD1 inhibition it was hepatic
glycogen metabolism, not gluconeogenic flux, that was affected by 11␤-HSD1 inhibition. However, it remains possible
that gluconeogenesis might be reduced under other circumstances, such as when the gluconeogenic precursor supply is
elevated (23, 37). Nevertheless, the initial glycogenolytic surge
that occurred in the control group was completely absent in the
presence of the inhibitor such that net hepatic glycogenolysis
remained suppressed throughout the study period despite elevated glucagon and epinephrine levels. This effect may have
been mediated by increased insulin sensitization and/or decreased glucagon and epinephrine action in response to the
reduction in cortisol during 11␤-HSD1 inhibition. Cortisol can
decrease insulin receptor binding (13) and affect postreceptor
insulin action (50) in part by decreasing PI3K activity (11).
Cortisol has also been shown to maintain physiological glycogen phosphorylase levels (52) and increase glucagon binding to
hepatocytes (15). In addition, the potent effects of epinephrine
and glucagon on glycogenolysis (25) are synergistically increased by cortisol (21, 53), probably via sensitization of the
glycogenolytic process to cAMP (22). Thus, a reduction in
Change from Basal Arterial
Plasma NEFA (µmol/L)
Non-Hepatic Glucose
Uptake (mg/kg/min)
A
E754
EFFECT OF 11␤-HSD1 INHIBITION ON HEPATIC GLUCOSE METABOLISM
GRANTS
This research was supported in part by National Institutes of Health
Diabetes Research and Training Center Grant SP-60-AM-20593. C. J. Ramnanan was supported by an American Diabetes Association Mentor-based
Fellowship. A. D. Cherrington was supported by the Jacquelyn A. Turner and
Dr. Dorothy J. Turner Chair in Diabetes Research.
DISCLOSURES
P. Jacobson and P. Jung are employees of Abbott Laboratories, which
provided financial support and compound 392 to complete the reported studies.
A. D. Cherrington is a consultant for Abbott Laboratories. There are no other
conflicts of interest to declare. D. S. Edgerton is the guarantor of this work, had
full access to all of the data, and takes full responsibility for the integrity of the
data and the accuracy of the data analysis.
AUTHOR CONTRIBUTIONS
J.J.W., C.J.R., V.S., J.R., M.S., P. Jung, R.B., and D.S.E. performed the
experiments; J.J.W., C.J.R., V.S., M.S., and D.S.E. analyzed the data; J.J.W.,
C.J.R., V.S., R.B., A.D.C., and D.S.E. interpreted the results of the experiments; J.J.W., A.D.C., and D.S.E. prepared the figures; J.J.W., A.D.C., and
D.S.E. drafted the manuscript; J.J.W., C.J.R., V.S., J.R., P. Jacobson, P. Jung,
R.B., A.D.C., and D.S.E. edited and revised the manuscript; J.J.W., C.J.R.,
V.S., J.R., M.S., P. Jacobson, P. Jung, R.B., A.D.C., and D.S.E. approved the
final version of the manuscript; P. Jacobson, A.D.C., and D.S.E. contributed to
the conception and design of the research.
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