Camp. Biochem. Physiol. Vol. 97C, No. 1, pp. 133-138, 1990 Printed in Great Britain 0306~4492/90 163.00 + 0.00 0 1990 Pergamon Press plc zyxwvut EFFECT OF INSULIN AND ADRENERGIC AGONISTS ON GLUCOSE TRANSPORT OF PORCINE ADIPOCYTES K. A. AKANBI, D. C. ENGLAND and C. Y. Hu* Oregon State University, Corvallis, OR 97331-6702, U.S.A. [Telephone: (503) 737-34311 zyxwvutsrqponmlk (Received 11 April 1990) Abstract-l. Insulin increased basal 2-deoxyglucose uptake in isolated swine adipocytes by 75%. In the absence of insulin, isoproterenol did not inhibit basal 2-deoxyglucose transport. 2. Adenosine deaminase plus isoproterenol or theophylline alone reduced insulin effect by 10 and 40%, respectively. Isoproterenol alone or with 2-chloroadenosine did not inhibit insulin effect on glucose transport activity. 3. Insulin effect was inhibited by isoproterenol in the presence of theophylline but not in the presence of adenosine deaminase. 4. These results suggest that catecholamines do not counter-regulate basal and insulin-stimulated glucose transport in s&e adipocytes. INTRODUCTION One of the primary actions of insulin is to enhance glucose uptake and utilization in muscle and adipose tissue (Hilf et al., 1981; Simpson and Cushman, 1986). Insulin stimulates in vitro glucose uptake and lipogenesis in rat adipocytes (May and Mikulecky, 1983; Green, 1983). Walton and Etherton (1986) and Liu et al. (1989) have demonstrated that INS stimulates glucose incorporation into lipids in porcine adipose tissue and adipocytes. Adrenergic agonists stimulate lipolysis and decrease lipogenesis in laboratory animals (Volpe and Marasa, 1975; Fain and Garcia-Sainz, 1983), pigs [Mersmann et al., 1974), cattle (Yang and Baldwin, 1973) and sheep (Hu et al., 1988). The lipolytic action of p-adrenergic agonists was effectively inhibited by insulin in swine (Mersmann, 1986; Mersmann and Hu, 1987). Adrenergic agonists inhibit insulin stimulated glucose uptake in muscle (Chiasson et al. 1981). However, studies of adrenergic agonists in rat adipose tissue and adipocytes generally show t ‘lern to enhance glucose uptake (Cahill et al., 1960; Lynn et al., 1960; Luzio et al., 1974; Smith et al., 1984). Inhihition of insulin stimulated glucose transport by adrenergic agonists in rat adipocytes occurred only in the presence of adenosine deaminase (Green, 1983; Smith et al., 1984; Kuroda et al., 1987). Since there have been no published reports on the effect of insulin and adrenergic agonists on glucose transport of swine adipocytes, our objective was to investigate whether insulin and adrenergic agonists affect swine adipocyte lipogenesis at the glucose transport level. MATERIALS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA AND METHODS Maferials (-)Isoproterenol bitartrate (12760), theophylline (T1633), adenosine deaminase (A6648), (-)epinephrine bitartrate (E4375), m-propranolol HCl (PO884), 2- *To whom correspondence should be addressed. chloroadenosine (C5134), yohimbine (Y3125), prazosin (P7791), phloretin (P4268), N-2-hydroxyethylpiperazine-N- 2-ethanesulfonic acid, HEPES (H3375), 2-deoxy-D-glucose (D8375), 2-deoxy-D-[l-‘4C]glucose (D6784), crystalline porcine insulin (13505) were purchased from Sigma Chemi- cal Co. (St Louis, MO). Bovine Serum Albimin (BSA; Bovuminar Reagent CRG-7) was purchased from Armour Pharmaceutical Co. (Tarrytown, NY), collagenase from Worthington Biochemical (Freehold, NJ), thiamylal-sodium (Biotal) from Boehringer-Inglheim Animal Health Inc. (St Joseph, MO) and osmium tetroxide (0~0,) was from E. M. Corp. (Chestnut Hill, MA). Animal and biopsy procedure Crossbred gilts and barrows (Sus scrofa) weighing 2845 kg were fed ad libitum triticale-soybean-meal contain- ing 16% protein. Pigs were iniected with 5 ml of 10% Biotal (Boehringer-Inglheim, St Josdph, MO) through the ear vein and the dorsal neck region was scrubbed with Prepodyne (AMSCO, Erie, PA) and rinsed twice with 70% ethanol. Adipose tissue was obtained using a biopsy gun made by attaching a sterile S/8” stainless steel hole drill bit to a Sears Craftsman 3/8” cordless drill. The drill was operated on low speed (100 rpm) to cut through the subcutaneous fat in the dorsal neck region and the tissue was excised with sterile surgical blade. Furazolidone (Topazone, Norden Labora- tories Inc., Lincoln, NE) was applied to the biopsy site. Tissue transport and adipocyte isolation Tissue was transported to the laboratory in Krebs-Ringer bicarbonate buffer (KRB, 37°C. PH 7.4) containing 118 mM NaCl, 4.8 mM Kel, 1.3 mM CaCl,; 1.2 mM XH,PO,, 1.3 mM MgSO,, 10 mM NaHCO,, 10 mM HEPES, 5 mM glucose, and 40 mg/l gentamicin sulfate and equilibrated with 95% 0,: 5% CO,. Adipocytes were isolated as de- scribed by Etherton and Chung (1981) with slight modifi- cation. Five grams of minced tissue was incubated in 15 ml KRB buffer described above containing 3% BSA and 32.25 mg collaaenase for 1 hr at 37°C. Digested tissue was filtered through a single layer of polyesier chiffon, and isolated cells were washed three times with the digestion buffer without collagenase or glucose. Measurement of glucose transport Approximately 2 x 10’ cells/ml were pre-incubated for 1 hr in a 37°C gyratory water bath (New Brunswick 133