EFFECT OF PITUITARY LIPID MOBILIZER (ASTWOOD'S PEPTIDE I) UPON GLUCOSE UPTAKE OF THE ISOLATED PERFUSED RABBIT HEART.

A lipid mobilizing peptide of pituitary origin (Astwood's Peptide I) has been tested as a direct inhibitor of glucose transport and intracellular phosphorylation in the perfused rabbit heart. No effects were observed. Accelerated release of unesterified fatty acids from adipose tissue has been confirmed. (Endocrinology 74: 504, 1964) G uptake is inhibited in hearts from alloxan diabetic rats (1). This inhibition is due to depressed rates of both glucose transport and phosphorylation. The phosphorylation inhibition is relieved by hypophysectomy and adrenalectomy (2). Injection of growth hormone into hypophysectomized diabetic rats results in an inhibition of phosphorylation, particularly if combined with injection of cortisol (2). Addition of these hormones in vitro does not cause inhibition (2, 3). These results suggested that the inhibitory effects of pituitary and adrenal hormones were either indirect or involved Received October 21, 1963. Supported in part by grants from Life Insurance Medical Research Fund and National Institutes of Health (Grant No. AM-3722). 1 Fellow of the Life Insurance Medical Research Fund. Present address: II Medizinische Universitats-Klinik, Ziemssenstrasse 1, Munich, Germany. 2 Investigator of the Howard Hughes Institute. hormones other than growth hormone and cortisol. Recently Astwood and co-workers (4, 5) have isolated metabolically active peptides (Peptides I and II) from porcine pituitary glands. These peptides accelerate the release of unesterified fatty acids from adipose tissue in rabbits. The present study was designed to assess the effect of Peptide I upon glucose uptake of the perfused rabbit heart. Materials and Methods Perfusion of Hearts. Hearts were removed as described earlier (6) from young domestic rabbits (14-21 days old) which had been anesthetized with sodium pentobarbital, 2-3 mg/100 g body weight, intraperitoneally. The experimental animals had been taken from the doe 6-8 hr prior to death and, in manj' cases, had stomachs filled with milk. The hearts were perfused with KrebsRinger bicarbonate buffer, gassed with 95% O2-5% CO2, at 37 C, containing glucose (150 mg/100 ml) and sorbitol (50 mg/100 ml). Insulin, when included, was added to a final concentration of 10 mU/ml. After a 5-min "wash-through" period, the heart was perfused March 1964 NOTES AND COMMENTS 505 by recirculating a measured volume of the same buffer for 20 min. Sorbitol-l-C (1 pc) was added to the recirculating perfusate to estimate the actual volume of the perfusate and the extracellular space of the heart. On completion of the perfusion, the heart was dropped into cold saline, the chambers were incised, the surfaces were quickly blotted, and the heart was frozen in a mixture of acetone and Dry Ice. Incubation of Adipose Tissue. At the time the heart was removed for perfusion, portions of mesenteric and perirenal adipose tissue were removed by gentle blunt dissection, rinsed in warm buffer, quickly weighed on a torsion balance, and incubated for 3 hr in Krebs-Ringer bicarbonate buffer containing 5% bovine serum albumin gassed with 95%O2-5% CO2 at 37 C. Approximately equal portions of tissue from each rabbit were incubated in duplicate, one serving as control and the other experimental. Material. Peptide I was the generous gift of Dr. E. B. Astwood. The substance was dissolved in distilled water when administered intravenously, or in the buffers to be used for in vitro studies. Other materials have been previously listed (6). Analytical Methods. Samples of perfusate and the heart were analyzed for glucose and radioactivity as described earlier (6). Free fatty acids (FFA) in samples of plasma and incubation medium were estimated by a modification of Dole's method (7). Sugar and sorbitol spaces and intracellular glucose concentrations were calculated as described earlier (6). Results Glucose uptake of the perfused rabbit heart was determined: 1) 10 min after the intravenous injection of Peptide I; or 2) 5-7 hr after injection; and 3) following the addition of Peptide I in vitro to the perfusate. In the absence of insulin, membrane transport is the major rate-limiting step for glucose uptake of the isolated rat heart (6). As shown in the top line of each panel of Table 1, intracellular free glucose was not detected in control hearts, an indication that glucose was phosphorylated as rapidly as it entered the cell. This is evidence that membrane transport was rate-limiting for glucose uptake. Following the addition of insulin, uptake was TABLE 1. Effect of Peptide I on glucose uptake and intracellular glucose concentration in the perfused heart, on plasma levels of FFA, and on FFA release from adipose tissue. (The experiments were performed as described in the Materials and Methods section. Peptide I was injected into the inferior vena cava of anesthetized rabbits; the comparable control animals received an equal amount of saline. The body temperature of animals injected 5-7 hr before sacrifice was maintained by placing the rabbits in an incubator at 32 C. The number of animals injected, hearts perfused, or samples of adipose tissue incubated are indicated by the figures in parentheses. All measurements are listed as the mean ± standard error of the mean. Rates and spaces are expressed per g of wet tissue. ND =none detected.)