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A novel adrenaline derivative, AZ002, and its hypoglycemic action in yellow KK mice.

AZ002 (L-threo-(3,4-dihydroxy phenyl)-N-methyl serine methyl ester) is a newly synthesized adrenaline derivative. AZ002 caused relaxation of rat jejunum (beta 3-receptors) (ED50 = 18 microM), but did not affect the atrial rate (beta 1) or tracheal relaxation (beta 2) at a concentration of 0.3 mM. The pA2 values for propranolol in inhibiting the isoproterenol- and AZ002-stimulated relaxation of rat jejunum were 6.27 and 6.33, respectively. Thus, AZ002 is a selective agonist for beta 3-adrenoceptor. AZ002 stimulated lipolysis (ED50 = 10 microM) and glucose uptake (ED50 = 1 microM) in rat adipocytes. In both cases, stimulation was antagonized by high concentrations of the beta-adrenoceptor antagonist propranolol, but not by the alpha-adrenoceptor antagonist phentolamine. The effect of AZ002 on glucose uptake was synergistic with that of insulin. AZ002 was also assessed in vivo by using genetically obese mice (KK/Ay strain) with hyperglycemia. Administration of AZ002 in the diet for a week decreased blood glucose and non-esterified fatty acids.

present in the majority of patients with impaired glucose tolerance and non-insulin dependent diabetes mellitus (NIDDM) (1,2). However, current oral therapy for NIDDM relies on the sulfonyl ureas and predominantly addresses the insulin-secretory defects rather than the insulin-sensitivity defects of this disease (3,4). Therefore, research effort has been directed toward the discovery of drugs that will improve glucose uptake and hyperglycemia.
At the cellular level, impaired glucose uptake has been demonstrated in isolated adipocytes (5) and muscle strips (6,7) from obese and diabetic patients. Therefore, we have screened compounds that stimulate glucose uptake in isolated rat adipocytes, because such compounds may represent an improved therapeutic approach to the treatment of NIDDM. Among the compounds synthesized in our laboratory, we found that AZO02 showed potent stimulation of glucose uptake and lipolysis.
Recently, it was demonstrated that the lipolytic adrenoceptor associated with thermogenesis in brown adipose tissue was of neither the 31-nor the j32-subtype (8). In addition, the existence of adrenoceptors distinct from classical a-and /3-subtypes has been demonstrated in rat and guinea pig intestine (9). These were referred to as atypical /3-adrenoceptors and termed the 83-receptor. 13-Adrenoceptor agonists stimulate lipolysis and thermogenesis by selectively increasing the energy expenditure within brown adipose tissue, thereby providing a potential pharmacological treatment for obesity and NIDDM (10).
In this report, we describe the agonistic activities of AZO02 on j3-adrenoceptors and on the stimulation of glucose uptake and lipolysis in rat adipocytes. We also examined whether AZO02 improved hyperglycemia in a mouse model of NIDDM which is genetically obese and hyperglycemic (10,11).

Chemicals
Insulin (porcine pancreas), collagenase (type II), (-)isoproterenol hydrochloride, (±)adrenaline hydrochloride, DL-propranolol and bovine serum albumin (Fraction V) were purchased from Sigma (St. Louis, MO, USA). D-[3-3H]Glucose (654.9 GBq/mmol) was from NEN (DuPont, Wilmington, DE, USA), and toluene scintillation cocktail was from Packard Instrument Company (Meriden, CT, USA). AZO02 (L-threo-(3,4dihydroxy phenyl)-N-methyl serine methyl ester) and NB108 (L-threo-(3,4-dihydroxy phenyl)-N-methyl serine) ( Fig. 1)  Measurement of glucose uptake stimulation The adipose cell suspension (about 105 cells/ml) was stirred to maintain homogeneity and 800-u1 aliquots were transferred to polyethylene tubes. After the addition of the test compounds, the tubes were preincubated for 5 min in the presence or absence of insulin. Reactions were started by adding 0.3 nCi of D-[3-3HJglucose to a final volume of 1 ml and incubated for a further 120 min. Reactions were terminated by the addition of 3 ml of toluene scintillation cocktail and vortexing to extract lipids into the organic scintillation phase. This method was originally developed for adipocytes by Moody et al. (13). In the presence of glucose at the low concentration used here, glucose uptake is rate-limiting for the conversion of glucose into lipid (14,15). In this assay, there is a good linear relationship between the glucose uptake and incubation time (0-6 hr). Adenosine deaminase did not affect the glucose uptake activities.

Lipolysis
The lipolytic response was determined by measuring the amount of non-esterified fatty acids according to the method of Duncombe (16). A control response in the absence of agonists and a maximal response in the presence of isoproterenol were obtained for each experiment. The lipolytic responses are expressed as a percentage of the maximal lipolysis obtained with isoproterenol.

Rat jejunum
The mid-portion of the small intestine (jejunum) was removed from male SD rats (400-500 g) (9). After the intraluminal contents were flushed, longitudinal segments (± 1.5 cm) were mounted in 20-ml organ baths under 0.5 g of tension in Krebs-Henseleit solution (composition: 118 mM NaCI, 5.6 mM KCI, 1.2 mM MgSO4i 2.5 mM CaC12, 1.3 mM NaH2PO4, 25 mM NaHCO3 and 5.5 mM glucose) oxygenated with 95 % 02 / 5 % CO2 (pH = 7.4) and kept at 37C. Responses were measured isotonically. After a steady baseline was obtained, the test compound was added cumulatively to the organ baths, until a maximal response was obtained. The response to the test compound is expressed as a percentage of the maximal relaxation response obtained with isoproterenol.

Guinea pig trachea
The trachea was removed from male Hartley guinea pigs (500-600 g). Tracheal transverse preparations were mounted in 5-ml organ baths under 0.6 g of tension in Krebs-Henseleit solution oxygenated with 9507o 02 / 5 % CO2 (pH = 7.4) and kept at 37C. °Responses were measured isometrically. After a steady contraction to 100 pM histamine was obtained, the test compound was added cumulatively to the organ baths, until a maximal response was obtained. The response to the test compound is expressed as a percentage of the maximal relaxation response obtained with isoproterenol.

Guinea pig right atrium
The right atrium was removed from male Hartley guinea pigs (500-600 g). The tissue was mounted in 20-ml organ baths under 1 g of tension in Krebs-Henseleit solution oxygenated with 95 % 02 / 5 % CO2 (pH = 7.4) and kept at 31'C. After reaching a steady baseline rate, the test compound was added cumulatively to the organ baths, until a maximal response was obtained. The response to the test compound is expressed as a percentage of the maximal rate obtained with isoproterenol.
Drug administration and blood sampling Male yellow KK mice were purchased from Clea Japan (Tokyo). They were housed in individual cages and maintained on a moderately high-calorie laboratory diet, Oriental CMF (Oriental Yeast, Tokyo), and water ad libitum. They were used for experiments when their body weights were 40-46 g (4-5 months of age), and they had well-developed hyperglycemia (15 -20 mM) and hyperinsulinemia (10-12 nM) (17). Animals were given either powdered diet alone or diet mixed with AZO02 (30 mg/100 g diet) for 7 days. For blood sampling, animals were fasted for 24 hr; then the tip of the tail vein was cut by a razor at 9-10 a.m., and blood was collected in heparinized hematocrit tubes. The plasma, separated by centrifugation, was stored in a refrigerator and measured for glucose and non-esterified fatty acids (NEFA) with commercial kits (Wako, Osaka). An oral glucose tolerance test (2 mg of glucose/g body weight) was also performed. The data are expressed as means±S.D. or S.E.M. and analyzed by Mann-Whitney's U-test.

Glucose uptake and lipolysis in the absence of insulin
The effects of AZO02, adrenaline, noradrenaline and isoproterenol on glucose uptake and lipolysis were investigated in rat adipocyte suspensions (Fig. 2). AZO02, isoproterenol, noradrenaline and adrenaline, in the absence of insulin, induced dose-dependent increases in glucose uptake ( Fig. 2A) and lipolysis (Fig. 2B). Bell-shaped dose-response curves were observed for stimulation of glucose uptake, but not for lipolysis. The ED50 values of isoproterenol, noradrenaline, adrenaline and AZO02 for glucose uptake were 15, 30 and 60 nM and 1 ,uM and for lipolysis, 50, 110 and 300 nM and 10 pM, respectively. The sensitivity (ED50) for glucose uptake was several times more potent than that for lipolysis with all compounds. AZO02 stimulated lipolysis by 1.4-fold above the maximum response (Vm) with isoproterenol, whereas noradrenaline and adrenaline showed a response similar to that of isoproterenol. NB108 (Fig. 1), which is the free acid of AZO02, did not stimulate either glucose uptake or lipolysis even at 1 mM (not shown). This indicates that a methyl ester moiety at the carboxyl group in AZO02 is essential to elicit adrenoceptor agonistic activity.

Glucose uptake and lipolysis in the presence of insulin
The effects of insulin on AZO02-induced glucose uptake and lipolysis were investigated in rat white adipocytes. The effect of AZO02 on glucose uptake was synergistic with that of insulin (Fig. 3A). In contrast, as expected, insulin significantly inhibited AZOO2-stimulated lipolysis in a dose-dependent manner (Fig. 3B). These results indicated that AZO02 predominantly stimulated glucose uptake rather than lipolysis in the presence of a high concentration of insulin.
Effects of adrenoceptor antagonists on glucose uptake and lipolysis by AZO02 Phentolamine, an a-adrenoceptor antagonist, failed to antagonize glucose uptake or lipolysis. However, high concentrations of propranolol, a (3-adrenoceptor antagonist, antagonized both glucose uptake and lipolysis in a dose-dependent manner (Fig. 4), indicating that AZO02 acts via the p-adrenergic receptor in stimulating both processes. It should be noted that propranolol showed different potencies in the inhibition of AZO02-stimulated glucose uptake (IC50=2.5 eM) and lipolysis (IC50=0.65 PM).

Action mechanism of AZO02
To investigate the mechanism of action of AZO02, the relaxation of rat jejunum (F33) (9) and guinea pig trachea (p2) (8), and the stimulation of guinea pig atrial rate (f3,) (about 80% of the maximal isoproterenol response) with a relatively high potency. Propranolol caused parallel shifts to the right in the dose-response curves of isoproterenol and AZ002. The pA2 values (negative logarithm of the molar concentration of propranolol causing a twofold shift in dose-response curves) for isoproterenoland AZO02-stimulated relaxation were 6.27 and 6.33, respectively. NB 108 showed no effect on relaxation of the jejunum at concentrations as high as 0.3 mM.
Isoproterenol and BRL37344 produced dose-dependent stimulatory effects on atrial rate. The values of ED50 (M) for isoproterenol and BRL37344 were 2.0±0.2 x 10-9 and 1.3±0.1 x 10-6, respectively (Fig. 6B). In contrast, neither AZO02 nor NB 108 affected the atrial rate at concentrations as high as 0.3 mM.   Fig. 7A, AZO02 also produced some improvement in glucose tolerance. No significant differences in body weight or food intake were observed between the control and AZO02-treated groups. However, in accordance with improved hyperglycemia, water intake volume in the AZO02-treated group (127±4 ml/7 days) was moderately decreased compared to that in the control group (148 ± 7 ml/7 days).
It was reported that the biological effects of BRL35134A, the parent methylester of BRL37344, were mediated by its free acid, BRL37344 (8). NB108, the free acid of AZO02, did not display /3-adrenoceptor agonistic activity. Thus, unlike BRL35134A, AZO02 is not a prodrug. It is well-known that adrenaline, the mother compound of AZO02, shows agonistic activity against not only r33-adrenoceptors but also all subtypes of a-and /3adrenoceptors. Therefore, it should be noted that the simple modification of the a-carbon in the chemical structure of adrenaline, as in AZO02, induces selective agonistic activity for the /33 subtype. This finding may be useful for the study of molecular interactions between catecholamines and their receptors.
Only the catecholamines are full agonists for human adipocytes, whereas for rat adipocytes, both catecholamines and non-catecholamine /3-adrenoceptor agonists have full agonistic action (19,20). Therefore, AZO02 is expected to show a higher level of intrinsic activity than non-catecholamine agonists in human adipocytes since it is a catecholamine derivative.
In rat adipocytes, isoproterenol, noradrenaline, adrenaline and AZO02 stimulated glucose uptake and lipolysis with the same order of potency. This suggests that the stimulation of glucose uptake and lipolysis are functionally linked, at least at low agonist concentrations. Indeed, catecholamines were less effective for stimulating glucose uptake when used at high concentrations, resulting in bell-shaped dose-response curves. In contrast, the bell-shaped curves were not seen for stimulation of lipolysis. Such a relationship was also demonstrated for stimulation of glucose uptake (bell-shaped curves) and respiration (non bell-shaped curves) in rat brown adipocytes by noradrenaline, dibutyryl cAMP, 3isobutyl-1-methylxanthine and palmitic acid (21). Transport of glucose into cells is accomplished by a membrane-associated carrier protein, specific glucose transporter. Five functional glucose transporter isoforms have been identified that have distinct tissue distributions and biochemical properties (22)(23)(24). Adipocytes predominantly contain the glucose transporter, GLUT4 isoform, which is responsible for insulin-stimulated uptake of glucose in these tissues. This action of insulin is accomplished via a translocation of GLUT4 transporters from low density microsomes to the plasma membrane.
In this report, we demonstrated the stimulating effect of AZO02 on glucose uptake and found that the action of AZO02 was synergistic with that of insulin (Fig. 3). This would indicate that part of the stimulating effect of AZO02 on glucose uptake could be related to the pathway of insulin action. Indeed, cAMP regulates translocation of insulin-sensitive GLUT4 (25). We administered AZO02 to genetically obese and hyperglycemic mice (10,11,18). The experiments revealed that AZO02 improved hyperglycemia in a mouse model of NIDDM. BRL35135, a selective ~3-adrenoceptor agonist, normalized glucose tolerance in obese Zucker rats (26). The lipolytic and insulin-releasing actions of /33adrenoceptor agonists may be important factors in considering the etiology of their effects on the NIDDM model (26,27). In addition, the anti-diabetic action of /33agonists may be related to their stimulation of glucose uptake into peripheral tissues (adipose or muscle tissue). However, the precise mechanisms for how BRL35135 improves the condition of diabetic rodents have not been identified (26).
In conclusion, we demonstrated here that AZO02, a newly synthesized catecholamine derivative, is a selective R3-agonist, and it stimulates glucose uptake and lipolysis in rat adipocytes. In addition, oral administration of AZO02 to NIDDM mice improved hyperglycemia. Further pharmacological evaluation is necessary for determining the mechanism whereby AZO02 improves hyperglycemia.