Somatostatin is an endogenous peptide and neurotransmitter

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1 Rapid Publication Somatostatin Impairs Clearance of Exogenous Insulin in Humans ELI IPP, YSEF SINAI, BENJAMIN BAR-Z, RAFAEL NESHER, AND ERL CERASI SUMMARY Somatostatin has been widely used to suppress endogenous pancreatic hormone secretion in research studies. Many of these studies required the simultaneous infusion of a hormone together with somatostatin. A critical assumption for its use in metabolic investigation is that somatostatin has no effect on the action or clearance of a concomitantly infused hormone. To test whether clearance of an exogenously infused hormone is affected, we infused insulin with or without somatostatin in two sets of studies. Insulin (40 mu kg 1 h 1 ) was infused for min (n = 6). Plasma glucose levels fell to 55 ± 4.1 mg/dl with insulin alone and significantly lower, to 44 ± 1.9 mg/dl, when somatostatin (250 ag h) was also infused (P <.01). Plasma immunoreactive insulin (IRI) rose to 57 ± 12.5 xll/ml with insulin alone, which was significantly different from 88 ± 15 fxll/ml when insulin was infused together with somatostatin (P <.01). When a smaller dose of insulin (30 mu kg 1 h 1 ) was infused for min (n = 4), similar results were observed. When somatostatin was infused together with insulin, plasma glucose fell to lower levels (41 ± 4.2 vs. 62 ± 9.5 mg/dl; P <.01) and plasma IRI rose higher (39 ± 8.5 vs. 27 ± 5.9 jxu/ml; P <.01) than when insulin was infused alone. C-peptide was equally suppressed by hypoglycemia regardless of whether somatostatin was administered, indicating suppression of endogenous insulin during these studies. We conclude that somatostatin infusion impairs the clearance of exogenous insulin. The resulting higher plasma IRI levels may contribute to the exaggerated hypoglycemia observed when somatostatin is infused with insulin. Impairment of clearance must be considered in the interpretation of metabolic studies where insulin and perhaps other From the Department of Endocrinology and Metabolism, Hadassah-Hebrew University Medical School, Jerusalem, Israel, Address correspondence and reprint requests to Eli Ipp, M.D., Division of Endocrinology and Metabolism, Harbor-UCLA Medical Center, 0 W. Carson Street, Torrance, CA Received for publication 11 February 1987 and accepted 24 February peptide hormones are infused together with somatostatin. Diabetes 36: , 1987 Somatostatin is an endogenous peptide and neurotransmitter that has been used as a pharmacological tool in endocrine and metabolic studies (1,2). Its powerful inhibitory characteristics have been exploited to create transient deficiency states of one or more hormones. Somatostatin has been used in various clinical investigations, including glucose counterregulation during hypoglycemia (3-5) and glucose-clamping experiments to eliminate endogenous insulin secretion (6,7), and in establishing selective insulin or glucagon deficiency to study the metabolic action of these hormones (8-14). In these studies, somatostatin was often infused together with other peptide hormones, such as insulin, glucagon, or growth hormone, to replace basal physiologic plasma concentrations of hormones where a deficiency state was not desired (3-15). We describe the effect of somatostatin on simultaneously infused exogenous insulin in normal human subjects. These studies were prompted by the unexpected finding in preliminary investigations that plasma immunoreactive insulin concentrations (IRI) were higher when insulin was infused with somatostatin than when insulin was infused alone. MATERIALS AND METHDS Human subjects. Healthy normal-weight volunteers were studied after obtaining informed consent. Ten subjects volunteered for a total of 20 studies. None had a history of disease, and subjects with first-degree family history of diabetes mellitus and women on oral contraceptives were not included. All studies were performed with approval of the institutional human subjects review committee. Protocols. All subjects were admitted after an overnight fast to a clinical-studies unit where they were rested, and catheters were inserted into antecubital veins in each arm. An infusion set was connected to one catheter, and the other was used for drawing blood samples. Another catheter was DIABETES, VL. 36, MAY

2 SMATSTATIN EFFECT N INSULIN CLEARANCE 10CH I+SS MINUTES FIG. 1. Effect of continuous insulin (I) infusion (40 mil kg 1 - h 1 ) on plasma glucose and immunoreactive insulin (IRI) concentration with and without concomitant somatostatin (SS) infusion (250 ^g/h). Mean ± SE. n = 6. *P <.05, "P <.01. placed in retrograde fashion on the dorsum of the hand and heated with a heat lamp to arterialize venous blood for glucose measurements. xygen saturation was maintained >90% by this technique. Subjects rested for 60 min before initiating the studies. During infusion experiments, insulin and somatostatin were infused separately via two infusion pumps (IVAC) set at an infusion rate of 1 ml/min. Regular porcine insulin (Actrapid, Novo, Bagsvaerd, Denmark) was infused for min at the rate of 40 mu kg~ 1 hr 1 in six subjects (protocol A) or 30 mu kg" 1 h~ 1 in four subjects (protocol B) after obtaining baseline samples over 20 min. The subjects were also studied with the same insulin infusion rates with the addition of somatostatin (Stilamin, Serono, Milan, Italy) at a rate of 250 xg/h. Each pair of studies, in which insulin was infused with or without somatostatin over min, was performed in randomized fashion >1 wk apart. Blood samples were drawn at 10- to 20-min intervals throughout the study. Sample processing and analysis. Heparinized blood samples were immediately processed by centrifugation, and plasma glucose levels were determined with a Beckman glucose analyzer (Fullerton, CA). Plasma was frozen at -20 C until subsequent radioimmunoassay. C-peptide was measured with a radioimmunoassay kit distributed by Novo. Plasma insulin immunoreactivity was measured by radioimmunoassay with human insulin standards and polyethylene glycol for separation of bound and free ligand. Somatostatin at a concentration of 1 xg/ml did not cross-react in the insulin or C-peptide assays. Statistics. Data were analyzed with the two-tailed Student's t test for paired samples. Data are presented as means ± SE. RESULTS Infusion of insulin at the rate of 40 mu kg~ 1 hr 1 resulted in a fall of plasma glucose from 86 ± 3.0 to 55 ± 4.1 mg/dl at 60 min, which was maintained through min (Fig. 1). This was associated with a rise in plasma insulin concentration from 16 ± 4.1 to 52 ± 12.9 uxi/ml at 20 min, with a further small rise to 57 ± 12.5 uaj/ml at 60 min. This level remained constant until min. When insulin was infused with somatostatin, plasma glucose fell to considerably lower levels, from 88 ± 2.0 to 44 ± 1.9 mg/dl at 60 min, remaining at this level until the end of the infusion (Fig. 1). Subjects were more symptomatic at this plasma glucose level. Plasma insulin concentrations rose from 16 ± 4.0 to 67 ± 17 fiu/ml at 20 min and still further to 88 ± 15 xu/ml at 50- min. These levels were consistently and significantly higher than those observed with insulin infusion alone. When insulin was infused at the rate of 30 mu/kg~ 1 h~\ plasma glucose fell from 85 ± 3.4 to 62 ± 9.5 mg/dl with 674 DIABETES, VL. 36, MAY 1987

3 E. IPP AND ASSCIATES -1 C7> J LJ C 3 _l 40 -J * * * * 20H FIG. 2. Effect of continuous insulin (I) infusion (30 mil kg* 1 h') on plasma glucose and immunoreactive insulin (IRI) concentration with and without concomitant somatostatin (SS) infusion (250 xg/h). Mean ± SE. n = 4. *P.05, "P < MINUTES insulin alone and from 89 ± 5.2 to 41 ± 4.2 mg/dl with insulin and somatostatin (Fig. 2). Plasma insulin rose from 10 ± 2.2 to 39 ± 8.5 xu/ml when somatostatin was added (Fig. 2), which was significantly different from 27 ± 5.9 xu/ml when insulin alone was infused. Plasma C-peptide concentrations were measured in three pairs of studies (2 from protocol A and 1 from B) to determine whether differences in suppressibility of endogenous insulin secretion had occurred during hypoglycemia with insulin alone or when somatostatin was also present. Table 1 demonstrates, as expected, that plasma C-peptide concentrations are equally well suppressed by insulin-induced hypoglycemia with or without somatostatin. DISCUSSIN These studies demonstrate that somatostatin infused together with insulin into normal human subjects causes higher steady-state concentrations of plasma IRI than when insulin is infused alone. Under these conditions endogenous insulin secretion, as reflected by measurement of C-peptide concentrations, is completely (and equally) suppressed with or without somatostatin. Therefore, the differences in IRI concentration when somatostatin is added are best explained by altered clearance of the exogenously infused insulin. ur data suggest that somatostatin reduces clearance of infused insulin by -25%. It is surprising that this phenomenon was not previously reported, considering the numerous studies performed with somatostatin. However, three of the studies compared the effect of somatostatin plus hormone with that of infusion of the hormone alone. Gerich et al. (3) injected a bolus of insulin and measured its levels 15 and 30 min later. No difference in plasma IRI was observed when insulin and insulin plus somatostatin administration were compared. However, plasma IRI was measured at only two time points in a non-steady-state situation in which rapid changes in the plasma insulin level were taking place. Thus, variability between subjects may have hidden any obvious differences. n the other hand, and in indirect support of our findings, when the same investigators infused glucagon to reach steady-state plasma levels in the presence of somatostatin, they found an unexpected 22% elevation of the plasma glucagon concentrations above the anticipated level (4). This observation may indicate that clearance of glucagon was also impaired during somatostatin infusion. Note that this effect was observed when, like in our studies with insulin, glucagon was infused to produce steady-state plasma levels with multiple plasma measurements. Also, the increase in glucagon concentrations was quantitatively similar to our observations with insulin. Another recent and thoroughly executed study with insulin and somatostatin infusion supports our findings and illustrates how delayed clearance of exogenous insulin may be DIABETES, VL. 36, MAY

4 SMATSTATIN EFFECT N INSULIN CLEARANCE TABLE 1 Effect of continuous insulin infusion with or without somatostatin on plasma immunoreactive C-peptide concentrations Time (min) Insulin + somatostatin Subject 1 Subject 2 Subject 3 Mean ± SE Insulin Subject 1 Subject 2 Subject 3 Mean ± SE ± ± ± ± ± ± ± 0.15 ± ± ± ± ± ± ± 0.01 C-peptide concentrations are expressed as pmol/ml. There were no statistically significant differences between the 2 groups. overlooked (16). The study differed from ours because the investigators maintained euglycemia with the glucose-clamp technique. Euglycemia had the effect of maintaining endogenous insulin secretion during infusion of insulin alone while allowing complete suppression of (3-cell function when somatostatin was added. This effect on endogenous insulin secretion was demonstrated by measuring C-peptide levels throughout their study. As in our study, consistently higher plasma IRI concentrations were observed in the group where somatostatin and insulin were infused than in the group that received insulin alone. This difference was not statistically significant. However, if the contribution of endogenous insulin secretion in the insulin-infused group is corrected by subtracting basal plasma IRI levels, the plasma concentrations of exogenously infused insulin can be approximated. With this rough correction, we estimate the difference in steady-state IRI concentrations between the two groups (insulin alone vs. somatostatin and insulin) to be 18-25%. This estimate is very close to our own findings, where plasma IRI concentrations reflect exogenous insulin only, because hypoglycemia completely suppressed C-peptide secretion in both groups. The reason for the reduced clearance of exogenous insulin is not clear. ne possible explanation relates to the effects of somatostatin to reduce hepatic portal and renal blood flow, thus diminishing access of the infused hormone to its primary sites of degradation (17). ur findings call for greater caution in the interpretation of data derived from studies in which somatostatin is infused together with other peptide hormones. The plasma glucose responses to infused insulin in our studies are a good example. A greater decrease in plasma glucose occurred when insulin infusion was accompanied by somatostatin. The excessive fall in plasma glucose in response to the same insulin dose has been assumed to be the result of the glucagon deficiency induced by somatostatin (3,4,8,9). Furthermore, it would be logical to conclude that other counterregulatory mechanisms, such as epinephrine secretion, were insufficient to compensate fully in the absence of glucagon (3,4). However, our studies indicate that in addition to these explanations, higher IRI levels in the somatostatininfused subjects must also be considered a cause for the greater degree of hypoglycemia. Elevated circulating insulin may also explain why the other counterregulatory processes appear to be ineffective in maintaining glucose homeostasis in the absence of glucagon. These findings may also be of importance in other areas of endocrinological investigation where somatostatin is infused with a hormone. This effect of somatostatin may have therapeutic implications as well. It is unclear if somatostatin analogues have the same effect as native somatostatin on insulin clearance. However, because the possibility exists, clinical situations in which somatostatin analogues and insulin therapy are used together will require careful monitoring for a possible decrease in insulin requirements associated with diminished clearance. This might be of importance in patients with acromegaly who are increasingly being treated with somatostatin analogues (18) and who may also be on insulin therapy for diabetes mellitus secondary to their acromegaly. ACKNWLEDGMENTS We thank Nathan Zinder for expert technical assistance. These studies were supported in part by the Yad Hanadiv Foundation. E.I. was a recipient of a Career Development Award of the Juvenile Diabetes Foundation. REFERENCES 1. Reichlin S: Somatostatin. N Engl J Med 309: , , Gerich J: Somatostatin and diabetes. Am J Med 70:619-26, Gerich J, Davis J, Lorenzi M, Rizza R, Bohannon N, Karam J, Lewis S, Kaplan R, Schultz T, Cryer PE: Hormonal mechanisms of recovery from insulin-induced hypoglycemia in man. Am J Physiol 236:E380-85, Rizza RA, Cryer PE, Gerich JE: Role of glucagon, catecholamines and growth hormone in human glucose counter-regulation. J Clin Invest 64:62-71, Bolli G, DeFeo P, Perriello G, DeCosmo S, Ventura M, Campbell P, Brunetti P, Gerich JE: Role of hepatic autoregulation in defense against hypoglycemia in humans. J Clin Invest 75: , Baron AD, Kolterman G, Bell J, Mandarino LJ, lefsky JM: Rates of noninsulin mediated glucose uptake are elevated in type II diabetic subjects. J Clin Invest 76: , Felber JP, Theibaud D, Malder E, Jequier E, Hendler R, DeFronzo RA: Effect of a somatostatin-induced insulinopenia on glucose oxidation in man. Diabetologia 25:325-30, Cherrington AD, Chiasson JL, Liljenquist JE, Jennings AS, Keller K, Lacy WW: Role of glucagon in maintaining basal glucose production. J Clin Invest 58: , Liljenquist JE, Mueller GL, Cherrington AD, Keller V, Chiasson JL, Pevy JM, Lacy WW, Rabinowitz D: Evidence for an important role of glucagon in the regulation of hepatic glucose production in normal man. 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5 E. IPP AND ASSCIATES glucose production during insulin deficiency in the conscious dog. J Clin Invest 62:664-77, Lickey HLA, Ross GG, Vranic M: Effects of selective insulin or glucagon deficiency on glucose turnover. Am J Physiol 236:E255-62, Ferrannini E, DeFronzo RA, Sherwin RS: Transient hepatic response to glucagon in man: role of insulin and hyperglycemia. Am J Physiol 242:E73-81, Lins PE, Wajngot A, Adamson V, Vranic M, Efendic S: Minimal increases in glucagon levels enhance glucose production in man with partial hy- 18. poinsulinemia. Diabetes 32:633-36, Gerich TE, Lorenzi M, Bier DM, Tsalikian E, Schneider V, Karam JH, Forsham PH: Effects of physiologic levels of glucagon and growth hormone on human carbohydrate and lipid metabolism. J Clin Invest 57:875-84, 1976 Baron AD, Wallace P, Brechtel G, Prager R: Somatostatin does not increase insulin-stimulated glucose uptake in humans. Diabetes 36:33-36, 1987 Jaspan J, Polonsky K, Lewis M, Moossa AR: Reduction in portal vein blood flow by somatostatin. Diabetes 28:888-92, 1979 Lamberts SWJ, Uitterlinden P, Verschoor L, van Dogen KJ, del Pozo E: Long-term treatment of acromegaly with the somatostatin analogue SMS N EnglJ Med 313: , 1985 DIABETES, VL. 36, MAY