An 11-Beta Hydroxysteroid Dehydrogenase Type 1 Inhibitor, JTT-654 Ameliorates Insulin Resistance and Non-obese Type 2 Diabetes

Abstract

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is the only enzyme that converts inactive glucocorticoids to active forms and plays an important role in the regulation of glucocorticoid action in target tissues. JTT-654 is a selective 11β-HSD1 inhibitor and we investigated its pharmacological properties in cortisone-treated rats and non-obese type 2 diabetic Goto-Kakizaki (GK) rats because Asians, including Japanese, are more likely to have non-obese type 2 diabetics. Systemic cortisone treatment increased fasting plasma glucose and insulin levels and impaired insulin action on glucose disposal rate and hepatic glucose production assessed by hyperinsulinemic-euglycemic clamp, but all these effects were attenuated by JTT-654 administration. Cortisone treatment also reduced basal and insulin-stimulated glucose oxidation in adipose tissue, increased plasma glucose levels after administration of the pyruvate, the substrate of gluconeogenesis, and increased liver glycogen content. Administration of JTT-654 also inhibited all of these effects. Cortisone treatment decreased basal and insulin-stimulated 2-deoxy-D-[1-³H]-glucose uptake in 3T3-L1 adipocytes and increased the release of free fatty acids and glycerol, a gluconeogenic substrate, from 3T3-L1 adipocytes, and JTT-654 significantly attenuated these effects. In GK rats, JTT-654 treatment significantly reduced fasting plasma glucose and insulin levels, enhanced insulin-stimulated glucose oxidation in adipose tissue, and suppressed hepatic gluconeogenesis as assessed by pyruvate administration. These results demonstrated that glucocorticoid was involved in the pathology of diabetes in GK rats, as in cortisone-treated rats, and that JTT-654 ameliorated the diabetic conditions. Our results suggest that JTT-654 ameliorates insulin resistance and non-obese type 2 diabetes by inhibiting adipose tissue and liver 11β-HSD1.

INTRODUCTION

In recent years, the number of patients with type 2 diabetes has been steadily increasing worldwide.¹⁾ Type 2 diabetes is characterized by an initial phase of progressive insulin resistance and a subsequent phase of β-cell exhaustion.²⁾ The main cause of this disease is considered to be decreased insulin efficiency, i.e., the development of insulin resistance due to factors such as overeating, insufficient exercise and aging, in addition to several genetic factors that are thought to predispose individuals to diabetes. Glucocorticoids (mainly cortisol, which accounts for almost all glucocorticoid activity) secreted by the adrenal cortex, have long been implicated as one of the causes of insulin resistance. Glucocorticoids are known to reduce the action of insulin in blood glucose homeostasis, as does glucagon, and to further disrupt glucose metabolism disorders. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is widely expressed in liver, adipose tissue, etc., and is the only enzyme that converts inactive glucocorticoid intracellularly to its active form, and it plays an important role in regulating the expression of glucocorticoid actions in the target tissues.³⁾ Various metabolic disorders have been observed in transgenic mice overexpressing 11β-HSD1 specifically in the liver or adipose tissue.^4–6)

A drug that inhibits 11β-HSD1 is expected to be a promising antidiabetic agent to improve insulin sensitivity in type 2 diabetes by suppressing the intracellular actions of glucocorticoids. Several 11β-HSD1 inhibitors have been investigated for the treatment of type 2 diabetes, and studies have reported that they could improve insulin sensitivity or glucose intolerance in some animal models.^7–11) However, the mechanisms of action of these compounds in adipose and liver tissue have not been reported in detail. In addition, these reports refer to the obese diabetes model, and pharmacological evaluation in non-obese diabetes is lacking. To date, 11β-HSD1 inhibitors have never been reported to exert antidiabetic effects in Goto-Kakizaki (GK) rats, a non-obese type 2 diabetic model rat. Asian populations tend to have more intra-abdominal fat accumulation and low muscle mass. Asian Indians, in particular, have the aforementioned abnormalities, which account for the high prevalence of insulin resistance and diabetes at low levels of body mass index (BMI).¹²⁾ In this study, we evaluated the effects of JTT-654 on adipose tissue and liver glucose metabolism using cortisone-treated rats and GK rats.

MATERIALS AND METHODS

Chemicals and Reagents

JTT-654 ([1-({4-[5-cyclopropyl-4-({(3S)-3-[2-(trifluoromethyl)phenyl]pyrrolidin-1-yl}carbonyl)-1H-pyrazol-1-yl]piperidin-1-yl}carbonyl)cyclopropyl]methanol) was synthesized at the Central Pharmaceutical Research Institute of Japan Tobacco Inc. (Osaka, Japan). The purity of JTT-654 was 99.0%. [1,2-³H]-Cortisone was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO, U.S.A.). 2-Deoxy-D-[1-³H]-glucose (2-DG), D-[U-¹⁴C]-glucose, and polyvinyl toluene (PVT) scintillation proximity assay (SPA) antibody-binding beads (anti-mouse reagent) were purchased from PerkinElmer, Inc. Japan Co., Ltd. (Kanagawa, Japan). Mouse anti-cortisol monoclonal antibody was purchased from EastCoast Bio, Inc. (North Berwick, ME, U.S.A.). Other chemicals were purchased from Sigma-Aldrich Japan (Tokyo, Japan).

Animals and Diets

For the evaluation of adipose tissue and liver 11β-HSD1 activity, male Sprague-Dawley (SD) rats were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan). For the cortisone-treated rat study, male Wistar rats were also purchased from Charles River Laboratories Japan, Inc. For the GK rat study, male GK rats and Wistar rats, the latter as normal control, were purchased from CLEA Japan, Inc. (Tokyo, Japan). All animals were maintained on CRF-1 (Charles River Laboratories Japan, Inc.), standard laboratory chow, and water ad libitum. Animals were housed under specific pathogen-free conditions in a room with temperature controlled at 23 ± 3 °C and humidity at 55 ± 15%, with lighting provided on a 12-h light/dark cycle (lights on from 8:00 AM to 8:00 PM). All procedures were performed in accordance with the guidelines of the Animal Care Committee of Japan Tobacco Inc.

Enzyme Assays

Recombinant human, rat, and mouse 11β-HSD1 with a FLAG epitope (DYKDDDDK) at the C-terminus was cloned into pFastBac1 vectors and expressed in Express SF+ insect cells using a baculovirus expression system. Virus-infected stable Express SF+ cells were lysed with HN buffer (25 mmol/L Tris–HCl (pH 7.4), 137 mmol/L NaCl, 2.68 mmol/L KCl, 10% glycerol, 1% Triton X-100), and the lysates were passed through affinity columns loaded with anti-FLAG antibody (anti-FLAG M2-agarose gel, SIGMA Corporation, Tokyo, Japan). Recombinant 11β-HSD1 proteins were eluted with FLAG peptide (SIGMA Corporation), followed by overnight dialysis with dialysis buffer (50 mmol/L N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES)-NaOH (pH 7.5), 150 mmol/L NaCl, 0.1% Triton X-100). Human (120 ng/mL), rat (80 ng/mL), or mouse (80 ng/mL) 11β-HSD1, substrate [1,2-³H]-cortisone (10 nmol/L), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) (500 µmol/L) were added to an assay buffer (phosphate buffered saline (PBS) (−) containing 0.1% bovine serum albumin (BSA)) with or without JTT-654 and incubated for 2 h at room temperature. To measure the inhibitory activity of JTT-654, the amount of [1,2-³H]-cortisol in the reaction mixture was measured using the SPA system (PerkinElmer, Inc. Japan Co., Ltd.).

To analyze the inhibition type of JTT-654, lipids were extracted by adding chloroform : methanol (2 : 1) solvent to the reaction mixture. After mixing, the reaction mixture was centrifuged (2500 × g, 5 min), and the lipids extracted in the organic phase were separated by TLC using a dichloromethane: methanol (10 : 1) solvent system. The radioactivity of [1,2-³H]-cortisol was measured using an FLA-7000 imaging system (FUJIFILM Corporation, Tokyo, Japan).

To measure the inhibitory activity of JTT-654 on 11β-HSD2, human kidney microsomes as a source of 11β-HSD2 (40 µg/mL), substrate [1,2-³H]-cortisol (35 nmol/L), and β-nicotinamide adenine dinucleotide (NAD) (500 µmol/L) were added to an assay buffer with or without JTT-654 or carbenoxolone (CBX, non-selective 11β-HSD1/2 dual inhibitor) and incubated for 1 h at room temperature. After the reaction, lipid extraction and [1,2-³H]-cortisone detection were performed in the same manner as for the analysis of the inhibition type of JTT-654. The percentage inhibition was calculated and plotted against the concentration of the test compound to obtain the IC₅₀.

Evaluation of Adipose Tissue and Liver 11β-HSD-1 Activity

These experiments were performed to determine the dose of JTT-654 for in vivo experiments. JTT-654 (1, 3, or 10 mg/kg) or vehicle (0.5% methylcellulose) was administered orally to SD rats (8 weeks old). Epididymal adipose tissue and liver were harvested from individual rats at 2, 4, 8, and 24 h after administration of JTT-654 or vehicle. Collected portions of adipose tissue (approximately 50 mg) or liver tissue (approximately 25 mg) were used as tissue pieces for incubation. The adipose and liver tissue pieces were incubated in Krebs–Ringer phosphate buffer (KRP) consisting of 122 mmol/L NaCl, 5.0 mmol/L KCl, 1.16 mmol/L MgSO₄, and 16.7 mmol/L Na₂HPO₄ (pH 7.4). The 11β-HSD1 substrate [1,2-³H]-cortisone (10 nmol/L) was then added to the assay buffer and incubated for 1 h at 37 °C (fat pad) or 10 min at room temperature (liver). The amount of [1,2-³H]-cortisol secreted in the reaction solution was measured by the SPA system.

Cortisone-Treated Rat

Repeated doses of JTT-654 and cortisone were administered once daily for 4 d to 7-week-old male Wistar rats. Cortisone was administered 1 h after JTT-654 administration on each day of dosing. Blood samples were collected prior to administration of JTT-654 on day 4 to assess its effect under fed conditions. After the administration of cortisone on day 4, the rats were fasted to assess the effect of the test article on fasted animals. Blood samples were collected from the tail vein. To clarify the effect of JTT-654 on adipose tissue and liver insulin resistance, pyruvate tolerance test (PTT), glucose oxidation assay, or hyperinsulinemic-euglycemic clamp test were performed on day 5 under overnight-fasted conditions. On day 5, the effects of JTT-654 on liver glycogen content were investigated under non-fasted, 4-h fasted, and 16-h fasted (overnight-fasted) conditions. Plasma glucose levels were measured using commercially available kits (Roche Diagnostics, Basel, Switzerland) and a biochemistry automatic analyzer (Hitachi 7180, Hitachi, Ltd., Tokyo, Japan). Plasma insulin levels were measured using a rat-insulin enzyme-linked immunosorbent assay (ELISA) kit (Morinaga Institute of Biological Science, Inc., Yokohama, Japan).

Evaluation of Hepatic Gluconeogenesis

To clarify the effect of JTT-654 on hepatic gluconeogenesis, PTT was performed in the cortisone-treated rats under overnight-fasted conditions. Sodium pyruvate (1 g/kg, dissolved in saline) was administered intraperitoneally. Blood samples were taken to measure glucose levels before and at 10, 20, and 30 min after sodium pyruvate administration. Changes in plasma glucose concentrations were calculated as the differences between the plasma glucose concentrations before sodium pyruvate administration and those at 10, 20, and 30 min after sodium pyruvate administration.

Evaluation of Adipose Tissue Insulin Resistance

To clarify the effect of JTT-654 on adipose tissue insulin resistance, a glucose oxidation assay was performed on epididymal fat from overnight-fasted cortisone-treated rats. Small portions (approximately 200 mg) of epididymal adipose tissue were incubated in Hank’s balanced salt solution (pH 7.4) containing D-[U-¹⁴C]-glucose in the absence or presence of insulin (1, 10, or 100 nmol/L) at 37 °C for 2 h. After the reaction was stopped by the addition of 0.05 mol/L H₂SO₄, the generated ¹⁴CO₂ was trapped in wet filter paper. The radioactivity in the filter paper was measured using a liquid scintillation counter (TRI-CARB 2500TR, Packard BioScience Co., Whatman, MA, U.S.A.).

Hyperinsulinemic-Euglycemic Clamp Test

Hyperinsulinemic-euglycemic clamp tests were performed in cortisone-treated rats under overnight fasting conditions. After the onset of fasting on the evening of day 4, a first cannula was implanted through the jugular vein and a second cannula was placed in the carotid artery under isoflurane anesthesia. Prior to clamping, a blood sample (approximately 250 µL) was obtained through the carotid cannula. A bolus dose (740 kBq/rat) of [3-³H]-glucose was administered via the jugular vein cannula, followed by a continuous infusion at a rate of 7.4 kBq/min. Blood samples were taken at 110, 115, and 120 min after the start of the [3-³H]-glucose infusion to determine basal parameters. After blood sampling, a bolus dose (0.15 U/kg) of insulin was administered via the jugular vein cannula, followed by continuous infusion of [3-³H]-glucose and insulin at rates of 7.4 kBq/min and 5 mU/kg/min, respectively. Thereafter, blood samples were taken every 5 min for blood glucose monitoring, and the infusion rate (mL/min) of the 25% glucose solution through the jugular vein cannula was adjusted to maintain blood glucose levels in the range of 110 ± 10 mg/dL (steady state). Once the steady state was observed at three consecutive points (for 10 min), blood samples were taken at the following three points (for 10 min) to determine the clamped state parameters: Clamped blood samples were collected at least 90 min after the start of insulin infusion. Glucose infusion rate (GIR), rate of glucose disposal (Rd), and hepatic glucose production (HGP), measures of whole-body, peripheral, and hepatic insulin sensitivity, respectively, were calculated using the following formulas:

  

Measurement of Liver Glycogen Content

To clarify the effect of JTT-654 on hepatic glucose production, liver glycogen content was measured in cortisone-treated rats. Rats were fasted over time on day 4 after administration, and dissection was performed under non-fasted, 4-h fasted, and 16-h fasted (overnight-fasted) conditions. Rats were laparotomized and exsanguinated from the abdominal inferior vena cava under isoflurane anesthesia to rapidly isolate the liver, which was frozen and stored in liquid nitrogen. The frozen liver was mixed with a 2-fold volume (v/w) of purified water and, after thawing, homogenized in an ice bath using a homogenizer. The homogenate was diluted 200-fold with purified water to prepare a sample. A volume of 100 µL of sample was mixed with 100 µL of 0.4 mol/L acetic acid buffer (pH 4.8) containing 10 U/mL amyloglucosidase (AMG) and incubated at 40 °C for 2 h with shaking (170 r.p.m.) to degrade glycogen to glucose. A blank was not treated with AMG. The incubated mixture was centrifuged at 1000 × g for 10 min at 4 °C, and 40 µL of the supernatant was transferred to a 96-well plate and neutralized with 10 µL/well of 0.25 mol/L NaOH. The glucose concentration in this neutralized sample was measured using a commercially available kit (Glucose CII Test Wako, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The protein content in the above-diluted sample was measured by the Lowry method (BIORAD Protein Assay Kit, Bio-Rad Laboratories, Inc., Osaka, Japan). Glycogen content was calculated as the concentration of glucose equivalents per mg of protein.

Cell Culture and Evaluation of Adipocyte Glucose Uptake, Glycerol and FFA Release

The 3T3-L1 fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Life Technologies Japan Ltd., Tokyo, Japan) in an atmosphere of 5% CO₂ at 37 °C. Two days after the fibroblasts reached confluence, the medium was changed to DMEM containing 10 µg/mL insulin, 0.5 mmol/L 3-isobutyl-1-methylxanthine, 1 µmol/L dexamethasone, and 10% fetal bovine serum, and the cells were further cultured for 48 h to induce differentiation. The cells were fed with DMEM supplemented with 5 µg/mL insulin and 10% fetal bovine serum every other day for the next 4–8 d. More than 90% of the cells expressed the adipocyte phenotype. The glucose uptake assay was performed according to the method reported by Sakoda et al.¹³⁾ with some modifications. 10 µmol/L cortisone was added to differentiated 3T3-L1 adipocytes in the presence of JTT-654. After incubation for 24 h, the medium was collected and changed, and 2-DG was added in the presence of JTT-654, cortisone, and insulin. After incubation for 10 min, the medium was removed and the cells were lysed with a surfactant to measure the radioactivity of the intracellular 2-DG. The amount of intracellular 2-DG in each sample was calculated using the formula of the standard curve. Based on this value, the inhibitory effect of JTT-654 on impaired glucose uptake in cortisone treated adipocytes was evaluated. Glycerol or free fatty acid (FFA) release assays were performed using medium obtained from 24-h culture of 3T3-L1 adipocytes as described above. Released glycerol was measured as triglyceride using the Triglyceride E-test kit (FUJIFILM Wako Pure Chemical Corporation). Released FFA was measured using NEFA-C test kit (FUJIFILM Wako Pure Chemical Corporation).

GK Rat

JTT-654 (1.5, 5, 15 mg/kg) was administered orally twice daily to 8-week-old male GK rats for 19 d (Days 0–18). On day 15, blood glucose and insulin concentrations were determined, and PTT was performed under overnight-fasted conditions. On day 18, glucose oxidation assay was performed using epididymal adipose tissue under overnight-fasted conditions.

Statistical Analysis

Statistical analyses were performed with SAS version 8.2 (SAS Institute Inc., CA, U.S.A.). Data are expressed as the mean ± standard deviation (S.D.) Bartlett’s homoscedasticity test was performed in the dose-response study. Dunnett’s multiple comparison test was performed when homoscedasticity was confirmed, and Steel’s multiple comparison test was used when homoscedasticity was not confirmed. An F-test was performed to test for homoscedasticity in a single comparison. If homoscedasticity was confirmed, Student’s t-test was performed; if homoscedasticity was not confirmed, Welch’s test was used. Differences were considered significant at p < .05 (2-sided).

RESULTS

Inhibitory Effects of JTT-654 on Human, Rat, Mouse 11β-HSD1 and Human 11β-HSD2

The structure of JTT-654 is shown in Fig. 1A. The IC₅₀ of JTT-654 for 11β-HSD1 was 4.65 ± 0.28, 0.97 ± 0.019, and 0.74 ± 0.050 nmol/L in human, rat, and mouse recombinant enzymes, respectively (Table 1). JTT-654 showed competitive inhibition against human recombinant enzyme (Fig. 1B). The IC₅₀ value for human 11β-HSD2, which is responsible for the reverse reaction against human 11β-HSD1, was greater than 30 µmol/L (Table 1).

Fig. 1. Chemical Structure (A) and Inhibition Mode (B) of JTT-654

Table 1. Inhibitory Effects of JTT-654 on Human, Rat, Mouse 11β-HSD1 and Human 11β-HSD2

Species	11β-HSD1			11β-HSD2
	Human	Rat	Mouse	Human
IC50 (nmol/L)	4.65 ± 0.28	0.97 ± 0.019	0.74 ± 0.050	>30000

11β-HSD1 enzyme activities were determined using [1,2-³H]-cortisone and recombinant enzymes. 11β-HSD2 enzyme activities were determined using [1,2-³H]-cortisol and human kidney microsomes. IC₅₀ values were calculated in a semilogarithmic proportional manner from the two points flanking 50% inhibition. Each value represents the mean ± S.D. of three independent experiments.

Effect of JTT-654 on Liver and Adipose Tissue 11β-HSD1 Activity

To evaluate the inhibitory efficacy and duration of JTT-654 on liver and adipose tissue 11β-HSD1 activity, we examined the cortisone-cortisol conversion activity in these tissues. The cortisone-cortisol conversion inhibition rates (% inhibition) of the isolated rat liver or fat after a single administration of JTT-654 are shown in Fig. 2. The inhibitory effect of JTT-654 in liver and fat was dose dependent. In the 10 mg/kg JTT-654 group, the % inhibition in both tissues was almost 100% up to 8 h post-dose, and approximately 70% inhibition was still observed even at 24 h post-dose.

Fig. 2. Effect of JTT-654 on 11β-HSD1 Activity in Liver and Adipose Tissue

After a single dose of JTT-654 (1, 3, 10 mg/kg) to rats, liver and fat were isolated at 2, 4, 8, and 24 h post-dose. The cortisone-cortisol conversion activity was measured at each time point to calculate the conversion inhibition rate in the test compound group relative to the vehicle group. Inhibitory effects of JTT-654 on liver (A) and adipose tissue (B) are shown. The basal 11β-HSD1 activities (cortisone to cortisol conversion rate of the vehicle group) in liver and adipose tissue were approximately 45% in liver and about 35% in adipose tissue, respectively. Data represent the mean ± S.D. (n = 5).

Effect of JTT-654 on Insulin Resistance in Cortisone-Treated Rats

To evaluate the effect of JTT-654 on insulin resistance, JTT-654 was administered to cortisone-treated rats for 4 d. Fasted plasma glucose and insulin levels are shown in Figs. 3A and B, respectively. Plasma glucose and insulin levels were significantly higher in the control group (cortisone treated) than in the normal control group (cortisone untreated). JTT-654 (1, 3, 10 mg/kg) administration significantly attenuated the increase in fasted plasma glucose and insulin levels in a dose-dependent manner. The plasma glucose and insulin levels in the 10 mg/kg group were comparable to those in the normal control group. To elucidate the mechanism of JTT-654 responsible for the amelioration of insulin resistance, a hyperinsulinemic-euglycemic clamp test was performed. The hyperinsulinemic-euglycemic clamp test is used to assess whole-body insulin resistance because the amount of glucose injected correlates with systemic insulin sensitivity. As shown in Table 2, there were no significant differences in the basal Rd and HGP between the groups. The GIR was decreased in the control group as compared to the normal control group in the clamped state. Moreover, Rd was decreased and the HGP tended to be increased in the control group compared to the normal control group in the clamped state. Administration of JTT-654 inhibited the decrease in GIR induced by cortisone treatment to the level of the normal control group. In addition, it also inhibited the decrease in Rd and increase in HGP induced by cortisone treatment under the clamped condition.

Fig. 3. Effects of JTT-654 on Plasma Glucose and Insulin Levels, Adipose Tissue Insulin Sensitivity, and Liver Glycogen Content in Cortisone-Treated Rats

JTT-654 and cortisone were administered for 4 d. After the last administration, the rats were fasted overnight and blood samples were collected for measurement of plasma glucose and insulin. To assess adipose tissue insulin sensitivity and hepatic glucose production, glucose oxidation test, PTT and measurement of liver glycogen content were performed. Fasting plasma glucose (A), insulin (B), adipose tissue glucose oxidation assay (C), PTT (D, E), and liver glycogen content (F) are shown. Data represent the mean ± S.D. (n = 5). ^†† p < .01 vs. control (Dunnett’s test). ^‡ p < 0.05 vs. control (Steel’s test). ^# p < .05, ^## p < .01 vs. normal control. * p < .05, ** p < .01 vs. control (Student’s t test or Welch’s test).

Table 2. Effect of JTT-654 on Blood Glucose and Plasma Insulin Levels, GIR, Rd, and HGP in the Hyperinsulinemic-euglycemic Clamp Study

	Normal control	Control	JTT-654 (10 mg/kg)
Plasma glucose (mg/dL)
Basal	99 ± 12	110 ± 8#	106 ± 14
Clamp	111 ± 4	110 ± 2	112 ± 5
Plasma insulin (ng/mL)
Basal	0.89 ± 0.20	2.39 ± 1.14##	1.06 ± 0.41†
Clamp	4.94 ± 0.51	6.68 ± 2.28	5.15 ± 0.55
GIR (mg/kg/min)	18.3 ± 3.5	12.9 ± 4.8#	20.9 ± 3.6††
Rd (mg/kg/min)
Basal	6.6 ± 0.5	8.3 ± 2.3	7.3 ± 1.3
Clamp	18.6 ± 2.9	14.2 ± 3.9#	19.7 ± 4.0††
HGP (mg/kg/min)
Basal	6.6 ± 0.5	8.3 ± 2.3	7.3 ± 1.3
Clamp	0.3 ± 2.9	1.3 ± 2.4	−1.3 ± 1.4†

JTT-654 and cortisone were administered for 4 d. Hyperinsulinemic-euglycemic clamp tests were performed under overnight fasting conditions. Data represent the mean ± S.D. (n = 8–10). ^# p < .05, ^## p < .01 vs. normal control (Student’s t-test or Welch’s test). ^† p < .05, ^†† p < .01 vs. JTT-654 (10 mg/kg) group (Student’s t-test or Welch’s test).

Effect of JTT-654 on Adipose Tissue Glucose Oxidation and Hepatic Glucose Production

Next, we evaluated the effect of JTT-654 on basal and insulin-stimulated adipose tissue glucose oxidation in cortisone-treated rats. As shown in Fig. 3C, epididymal fat from the control group showed a marked decrease in basal and insulin-stimulated glucose oxidation compared to that from the normal control group. JTT-654 administration improved the glucose oxidation reduced by cortisone treatment. This improvement was more pronounced at higher insulin concentrations.

As shown in Figs. 3D and E, the increase in plasma glucose concentration after administration of the gluconeogenesis substrate was significantly greater in the control (cortisone treated) group than in the normal control (cortisone untreated) group. JTT-654 attenuated this increase to approximately the same level as in the normal control group. To investigate the effect of JTT-654 on cortisone-induced hepatic glucose production, we measured hepatic glycogen content (Fig. 3F). Under the non-fasted, 4-h fasted, and 16-h fasted conditions, the amount of liver glycogen in the control group increased significantly compared with the normal control group. In the control group, the amount of liver glycogen did not decrease even after 16 h of fasting. In contrast, the amount of liver glycogen in the JTT-654 group was decreased according to the fasting time as in the non-cortisone group.

Effect of JTT-654 on Glucose Uptake and Gluconeogenic Substrate Release in Cortisone-Treated 3T3-L1 Adipocytes

To elucidate a mechanism of action of JTT-654 in adipose tissue, glucose uptake, glycerol and FFA release assays were performed in cortisone-treated 3T3-L1 adipocytes. The 2-DG uptake in cortisone-treated adipocytes (control group) was significantly decreased compared to that of the normal control group. JTT-654 significantly improved the decreased 2-DG uptake induced by cortisone to a level comparable to that of the normal control group in a dose-dependent manner. This effect of JTT-654 was observed both under basal conditions (without insulin) and at various insulin concentrations. The basal levels of 2-DG uptake were as follows: Normal control; 0.170 ± 0.05 pmol/10 min, control; 0.059 ± 0.04 pmol/10 min, JTT-654 (0.1 µmol/L); 0.074 ± 0.06 pmol/10 min, JTT-654 (1 µmol/L); 0.140 ± 0.013 pmol/10 min, and JTT-654 (10 µmol/L); 0.154 ± 0.02 pmol/10 min. The changes in 2-DG uptake in all groups increased with higher insulin concentrations (Fig. 4A). It was assumed that insulin sensitivity was decreased in the control group because the insulin-sensitive 2-DG uptake changes in the control group were significantly smaller than those in the normal control group. JTT-654 attenuated the reduction in insulin sensitivity induced by cortisone treatment in a dose-dependent manner (Fig. 4A). Secreted glycerol and FFA levels in the control group were significantly increased compared to those in the normal control group (Figs. 4B, C). JTT-654 significantly improved the increase in secretion of these molecules by cortisone treatment to the same level as the normal control group in a dose-dependent manner (Figs. 4B, C).

Fig. 4. Effects of JTT-654 on 2-DG Uptake and Gluconeogenic Substrate Release in Cortisone-Treated 3T3-L1 Adipocytes

In the presence or absence of JTT-654, 10 µmol/L cortisone was added to differentiated 3T3-L1 adipocytes and the cells were incubated for 24 h. The culture medium was collected and the secreted glycerol and FFA were measured. After the medium was changed, the 2-DG uptake assay was performed. Changes in 2-DG uptake (A), released FFA (B), and glycerol (C) are shown. Data represent the mean ± S.D. (n = 4). ^†† p < .01 vs. control (Dunnett’s test). ^# p < .05; ^## p < .01 vs. normal control (Student’s t-test or Welch’s test).

Effect of JTT-654 on Insulin Resistance in Type 2 Diabetic GK Rats

To verify whether JTT-654 has the same effect of ameliorating insulin resistance in a spontaneous non-obese type 2 diabetic model as in the cortisone-treated model, we conducted a repeated dose study using the GK rat. JTT-654 (1.5, 5, 15 mg/kg, twice daily) was administered to GK rats for 19 d. There were no differences in food consumption or body weight after JTT-654 treatment (Figs. 5A, B). A significant decrease in plasma insulin levels was observed in GK rats treated with 30 mg/kg JTT-654 on day 7 under fed conditions (Fig. 5D). On the other hand, there were no significant differences in plasma glucose levels in JTT-654-treated GK rats on the same day (Fig. 5C). Fasting plasma glucose and insulin levels were significantly decreased in JTT-654-treated GK rats (except for plasma glucose level treated with 3 mg/kg JTT-654) (Figs. 5E, F). Furthermore, we performed PTT and adipose tissue glucose oxidation assay to evaluate the mechanism of action of JTT-654 in liver and adipose tissue. During PTT, plasma glucose levels of JTT-654-treated GK rats were significantly decreased compared to vehicle-treated GK rats (Fig. 5G). Moreover, JTT-654 significantly improved insulin-stimulated glucose oxidation in isolated adipose tissue from GK rats (Fig. 5H).

Fig. 5. Effects of JTT-654 on Food Intake, Body Weight, Diabetic Parameters, Hepatic Glucose Production, and Adipose Tissue Insulin Sensitivity in the GK Rat, a Model of Type 2 Diabetes

Repeated doses of JTT-654 (1.5, 5, and 15 mg/kg) were administered orally twice daily (BID) to GK rats for 19 d. Blood samples were collected 7 d (fed conditions) and 15 d (fasted conditions) after the start of treatment to measure plasma glucose and insulin. PTT was then performed on day 15 under fasted conditions to evaluate hepatic glucose production. Furthermore, a glucose oxidation test was performed on isolated adipose tissue to evaluate insulin sensitivity in adipose tissue (day 19). Food consumption was calculated as the sum of the food intake per cage divided by 6 rats (three rats were kept in one cage). Food intake (A), body weight (B), fed plasma glucose (C) and insulin (D), fasted plasma glucose (E) and insulin (F), PTT (G), and adipose tissue glucose oxidation assay (H) are shown. Data represent the mean ± S.D. (n = 6). ^† p < .05, ^†† p < .05 vs. control (Dunnett’s test), * p < .05, ** p < .01 vs. control (Student’s t-test).

DISCUSSION

For more than a dozen years, many 11β-HSD1 inhibitors have been investigated for the treatment of type 2 diabetes because glucocorticoids cause insulin resistance. It has been reported that 11β-HSD1 inhibitors could improve insulin sensitivity or glucose intolerance in animal models.^7–11) However, the mechanisms of action of these compounds in the liver and adipose tissue have not been reported in detail. In this study, we demonstrated whether JTT-654, an 11β-HSD1 inhibitor that exhibits potent and long-lasting inhibition of 11β-HSD1 enzyme activity in liver and adipose tissue, has therapeutic efficacy. In addition, we elucidated the mechanism of action of JTT-654 to improve liver and adipose tissue insulin resistance and glucose metabolism using cortisone-treated rats and non-obese type 2 diabetic GK rats.

First, we investigated the inhibitory effect and pattern of JTT-654 against 11β-HSD1. The inhibitory effect of JTT-654 on 11β-HSD1 enzyme activity is comparable in humans, rats and mice. JTT-654 demonstrated a competitive pattern of inhibition against cortisone on 11β-HSD1. The potency of JTT-654 in inhibiting human 11β-HSD1 is quite potent, with an IC₅₀ as low as 4.65 nmol/L in vitro, comparable to other potent 11β-HSD1 inhibitors that have been reported.^7–11) Single administration of JTT-654 inhibited cortisone-cortisol conversion activity in isolated rat fat and liver tissues in a dose-dependent manner, and this effect was shown at 24 h post-dose. In particular, the activity was completely inhibited at the dose of 10 mg/kg 8 h after administration in normal rats. These data suggest that JTT-654 has a potent and long-lasting inhibitory effect on 11β-HSD1 enzyme activity in vivo.

We used cortisone-treated rats to evaluate the mechanism of action of JTT-654 in ameliorating liver and adipose tissue insulin resistance. Fasting plasma glucose and insulin levels in the cortisone-treated rats were significantly increased compared to normal control rats. In general, excess glucocorticoid in the liver induces gluconeogenesis and glycogen synthesis.^14,15) Pyruvate-induced gluconeogenesis was enhanced and liver glycogen content was increased in cortisone-treated rats, and liver glycogen content was maintained in cortisone-treated rats even under overnight-fasted conditions. Moreover, HGP tended to increase in cortisone-treated rats in the hyperinsulinemic-euglycemic clamp test. These results indicate that our data are consistent with the results of previous reports.^16,17) JTT-654 (10 mg/kg) significantly ameliorated these effects of excessive glucocorticoids to normal levels.

JTT-654 improved GIR in the hyperinsulinemic-euglycemic test. This means JTT-654 could improve whole body insulin resistance. In this evaluation system, which doesn’t use overeating or obesity model, we think Rd indicate insulin sensitivity primarily in skeletal muscle. Therefore, we think JTT-654 could improve skeletal muscle insulin resistance.

We wanted to evaluate insulin sensitivity not only in skeletal muscle but also in adipose tissue. In adipose tissue, glucocorticoids play a pleiotropic role in insulin sensitivity. Human visceral adipocytes treated with glucocorticoids have been found to have impaired insulin-stimulated glucose uptake.¹⁸⁾ JTT-654 improved glucose uptake and insulin sensitivity, both of which were impaired by cortisone treatment, in 3T3-L1 adipocytes. Moreover, JTT-654 also improved adipose tissue insulin sensitivity in cortisone-treated rats. It was thought that the decrease in glucose oxidation capacity by cortisone treatment was due to the decrease in glucose uptake capacity in adipocytes. In general, glucocorticoids have catabolic functions that promote lipid mobilization and release FFA. JTT-654 significantly ameliorated the increase in FFA and glycerol secretion caused by cortisone treatment. This released glycerol was thought to be used as a substrate for gluconeogenesis in the liver.

Next, we evaluated the effect of JTT-654 in a diabetic model. Currently, the number of diabetic patients is increasing in Asian countries, including Japan. The prevalence of obesity and overweight is relatively lower in Asia compared to Western populations.¹²⁾ However, most reports evaluating 11β-HSD1 inhibitors use obese diabetic animal models. In a preliminary study, we also confirmed that JTT-654 ameliorated the development of diabetes in obese type 2 Zucker Diabetic Fatty (ZDF) rats. In this preliminary study, we observed that JTT-654 decreased plasma glucose and mRNA levels of PEPCK and G6Pase in the liver of ZDF rats, suggesting that JTT-654 improves gluconeogenesis (data not shown). It has been reported that the expression and activity of 11β-HSD1 in the liver and adipose tissue differ between obese and non-obese diabetic animals.¹⁹⁾ In the current study, we used non-obese diabetic GK rats to clarify whether JTT-654 also ameliorates insulin resistance in non-obese type 2 diabetes. Because we required long-acting and maximal inhibition of liver and adipose tissue 11β-HSD1 within 24 h, JTT-654 (1.5, 5, and 15 mg/kg) was administered orally twice daily. At the dose of 30 mg/kg of JTT-654, both fasting and fed plasma glucose and insulin levels were decreased without altering food intake or body weight compared to the control group. In normal rats, a single dose of 10 mg/kg JTT-654 provided over 90% 11β-HSD1 inhibition in both adipose tissue and liver for 8 h. However, the potency of 11β-HSD1 inhibition decreased to approximately 70% from 8 to 24 h after dosing. Therefore, it was concluded that the administration of 15 mg/kg JTT-654 twice daily was capable of completely inhibiting 11β-HSD1 activity in both adipose tissue and liver for 24 h. These results indicate that for JTT-654 to exert its maximum efficacy, liver and adipose tissue 11β-HSD1 activity must be maintained at a level of at least 90% inhibition for 24 h. In contrast, only fasting plasma insulin levels were reduced at the 3 mg/kg dose of JTT-654. JTT-654 improved adipose tissue glucose oxidation activity in GK rats. Comparing Fig. 5H with Fig. 3C, glucose oxidation in adipose tissue of GK rats is similar to that of cortisone-treated rats. As shown in Fig. 3C, JTT-654 improved adipose tissue glucose oxidation to normal control level. It was suggested that JTT-654 has the effect of reducing insulin demand in peripheral tissues, including adipose tissue. JTT-654 also improved gluconeogenesis in the liver of GK rats. The reason why even the lowest dose of JTT-654 reduced fasting plasma insulin was thought to be a synergistic effect of ameliorating insulin resistance in peripheral tissues and inhibiting hepatic gluconeogenesis. These results indicate that JTT-654 ameliorated insulin resistance in the non-obese type 2 diabetic GK rats by the same mechanism as in the cortisone-treated rats. Considering that JTT-654 ameliorated insulin resistance in type 2 diabetes regardless of the presence of obesity, 11β-HSD1 inhibitors are expected to be effective against both types of type 2 diabetes. Therefore, JTT-654 can be expected to adapt to Cushing’s syndrome caused by excessive glucocorticoid action.

In conclusion, we identified an 11β-HSD1 inhibitor, JTT-654, which exhibited potent and long-acting 11β-HSD1 enzymatic inhibition in liver and fat pad as target tissues. JTT-654 ameliorated impaired glucose oxidation activity and release of FFA and glycerol, which is a substrate for gluconeogenesis release in adipose tissue. JTT-654 also improved gluconeogenesis and glycogen storage in the liver. The information gained from the detailed analysis of the mechanism of action of JTT-654 may represent a new therapeutic approach for the treatment of non-obese type 2 diabetic patients.

Conflict of Interest

All authors are employed by Japan Tobacco Inc. The authors declare that there is no conflict of interest regarding this article. No specific grant was received for this research from public, commercial, or non-profit funding agencies.

Data Availability

The data supporting the results of this study are available from the corresponding author upon reasonable request.

REFERENCES

• 1) Khan MAB, Hashim MJ, King JK, Govender RD, Mustafa H, Al Kaabi J. Epidemiology of type 2 diabetes-global burden of disease and forecasted trends. J. Epidemiol. Glob. Health, 10, 107–111 (2020).
• 2) Fonseca VA. Defining and characterizing the progression of type 2 diabetes. Diabetes Care, 32 (Suppl. 2), S151–S156 (2009).
• 3) Seckl JR, Walker BR. Minireview: 11beta-hydroxysteroid dehydrogenase type 1—a tissue-specific amplifier of glucocorticoid action. Endocrinology, 142, 1371–1376 (2001).
• 4) Masuzaki H, Paterson J, Shinyama H, Morton NM, Mullins JJ, Seckl JR, Flier JS. A transgenic model of visceral obesity and the metabolic syndrome. Science, 294, 2166–2170 (2001).
• 5) Masuzaki H, Yamamoto H, Kenyon CJ, Elmquist JK, Morton NM, Paterson JM, Shinyama H, Sharp MG, Fleming S, Mullins JJ, Seckl JR, Flier JS. Transgenic amplification of glucocorticoid action in adipose tissue causes high blood pressure in mice. J. Clin. Invest., 112, 83–90 (2003).
• 6) Paterson JM, Morton NM, Fievet C, Kenyon CJ, Holmes MC, Staels B, Seckl JR, Mullins JJ. Metabolic syndrome without obesity: hepatic overexpression of 11beta-hydroxysteroid dehydrogenase type 1 in transgenic mice. Proc. Natl. Acad. Sci. U.S.A., 101, 7088–7093 (2004).
• 7) Gathercole LL, Lavery GG, Morgan SA, Cooper MS, Sinclair AJ, Tomlinson JW, Stewart PM. 11beta-Hydroxysteroid dehydrogenase 1: translational and therapeutic aspects. Endocr. Rev., 34, 525–555 (2013).
• 8) Hong SP, Han D, Chang KH, Ahn SK. A novel highly potent and selective 11beta-hydroxysteroid dehydrogenase type 1 inhibitor, INU-101. Eur. J. Pharmacol., 835, 169–178 (2018).
• 9) Li J, Kennedy LJ, Walker SJ, et al. Discovery of clinical candidate BMS-823778 as an inhibitor of human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1). ACS Med. Chem. Lett., 9, 1170–1174 (2018).
• 10) Oh H, Jeong KH, Han HY, Son HJ, Kim SS, Lee HJ, Kim S, Sa JH, Jun HS, Ryu JH, Choi CS. A potent and selective 11beta-hydroxysteroid dehydrogenase type 1 inhibitor, SKI2852, ameliorates metabolic syndrome in diabetic mice models. Eur. J. Pharmacol., 768, 139–148 (2015).
• 11) Okazaki S, Takahashi T, Iwamura T, Nakaki J, Sekiya Y, Yagi M, Kumagai H, Sato M, Sakami S, Nitta A, Kawai K, Kainoh M. HIS-388, a novel orally active and long-acting 11beta-hydroxysteroid dehydrogenase type 1 inhibitor, ameliorates insulin sensitivity and glucose intolerance in diet-induced obesity and nongenetic type 2 diabetic murine models. J. Pharmacol. Exp. Ther., 351, 181–189 (2014).
• 12) Ramachandran A, Snehalatha C, Shetty AS, Nanditha A. Trends in prevalence of diabetes in Asian countries. World J. Diabetes, 3, 110–117 (2012).
• 13) Sakoda H, Ogihara T, Anai M, Funaki M, Inukai K, Katagiri H, Fukushima Y, Onishi Y, Ono H, Fujishiro M, Kikuchi M, Oka Y, Asano T. Dexamethasone-induced insulin resistance in 3T3-L1 adipocytes is due to inhibition of glucose transport rather than insulin signal transduction. Diabetes, 49, 1700–1708 (2000).
• 14) Jitrapakdee S. Transcription factors and coactivators controlling nutrient and hormonal regulation of hepatic gluconeogenesis. Int. J. Biochem. Cell Biol., 44, 33–45 (2012).
• 15) Margolis RN, Curnow RT. Effects of dexamethasone administration on hepatic glycogen synthesis and accumulation in adrenalectomized fasted rats. Endocrinology, 115, 625–629 (1984).
• 16) Kimura M, Daimon M, Tominaga M, Manaka H, Sasaki H, Kato T. Thiazolidinediones exert different effects on insulin resistance between dexamethasone-treated rats and wistar fatty rats. Endocr. J., 47, 21–28 (2000).
• 17) Mokuda O, Sakamoto Y, Ikeda T, Mashiba H. Sensitivity and responsiveness of glucose output to insulin in isolated perfused liver from dexamethasone-treated rats. Horm. Metab. Res., 23, 53–55 (1991).
• 18) Lundgren M, Buren J, Ruge T, Myrnas T, Eriksson JW. Glucocorticoids down-regulate glucose uptake capacity and insulin-signaling proteins in omental but not subcutaneous human adipocytes. J. Clin. Endocrinol. Metab., 89, 2989–2997 (2004).
• 19) Czegle I, Margittai E, Senesi S, Benedetti A, Banhegyi G. Different expression and distribution of 11beta-hydroxysteroid dehydrogenase type 1 in obese and lean animal models of type 2 diabetes. Acta Physiol. Hung., 95, 419–424 (2008).