Interactions of a-ketoisocaproate, Glucose and Arginine in the Secretion of Giucagon and Insulin from the Perfused Rat Pancreas

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1 Dabetologa 17, (1979) Dabetologa 9 by Sprnger-Verlag 1979 Interactons of a-ketosocaproate, Glucose and Argnne n the Secreton of Gucagon and Insuln from the Perfused Rat Pancreas V. Leclercq-Meyer, J. Marchand, R. Leclercq ~, and W. J. Malasse Laboratory of Expermental Medcne, Free Unversty of Brussels, and 1Laboratory of Bologcal Chemstry, IMC Anderlecht, Brussels, Belgum Summary. The effects of (x-ketosocaproate (KIC, 10retool/) on glucagon and nsuln release were studed n the n vtro perfused rat pancreas, The experments were performed at low glucose concentraton (3.3 mmol/1) n the absence or presence of argnne (10mmol/1). In all the experments KIC nduced a marked and not rapdly reversble nhbton of glucagon release. Ths nhbton was more pronounced n the absence (76 percent) than presence of argnne (61 percent). These nhbtory patterns closely duplcated those whch were seen n parallel experments whch ncluded a rse n the concentraton of glucose (from 3.3 to 11.1 retool/l). KIC was also a potent stmulator of nsuln release. The results are compatble wth the vew that the ntracellular metabolsm of KIC and glucose plays an essental role n the regulaton of glucagon release by exogenous substrates. Key words: a-ketosocaproate, KIC, argnne, glucose, glucagon release, nsuln release, perfuson, rat pancreas. Glucose normally nhbts the secreton of glucagon, both n vvo and n vtro [7, 46, 47]. The ntmate mechansms by whch glucose exerts ts nhbtory effect are not fully elucdated but several hypotheses have been proposed. Accordng to the glucoreceptor hypothess, glucose nteracts wth specfc receptors located at the A2-cell plasma membrane [29, 31, 32]. Another hypothess postulates that the A2-cell s devod of ntrnsc glucose-sensng capacty and that the nhbtory effect of glucose s ndrect and medated by nsuln [41, 47]. Fnally, n the substrate-ste hypothess, t s beleved that the metabolsm of glucose n the A2-cell plays an essental role n the regu- laton of glucagon release. In favour of the latter concept are the observatons that the A2-cells, although havng a low glucose-phosphorylaton capacty [18], possess a hgh enzymatc capacty for degradaton of trose phosphates [19]. Moreover, metabolc studes conducted on A2-cell-rch slets of streptozotocntreated gunea pgs have shown that oxygen consumpton, glucose oxdaton, glucose utlzaton and ATP concentraton were ncreased n the presence of elevated glucose levels [2, 3, 28, 38]. Other nvestgators, however, faled to detect major changes n metabolsm, n partcular n ATP concentraton, n slets from rats treated wth streptozotocn or alloxan [23, 24]. These negatve fndngs may not nvaldate the metabolc hypothess snce t s well establshed that the senstvty of the A2-cell to glucose s markedly mpared n these dabetc models, as n many other forms of chronc dabetes (e. g. 6-8, 23-25, 30, 47). Besdes, ATP may not be the essental messenger medatng the effect of glucose upon glucagon release. The present work was undertaken to gan addtonal nformaton on the role of ntermedary metabolsm n the regulaton of glucagon release from the normal rat pancreas. We have compared the effects of glucose and a-ketosocaproate (KIC) upon glucagon release n the absence or presence of argnne. Both KIC, whch s the frst catabolc product of leucne, and glucose are potent nsulnotropc secretagogues and are actvely metabolzed n rat slets [12, 13, 33, 34, 42, 43]. Materals and Methods Fed female albno rats were used (mean body weght SEM: g; n = 19). The perfuson technque has been descrbed prevously [15]. The anmals were anaesthetzed wth sodum pentobarbtal (42 mg/kg ntrapertoneally) and the pancreas was solated, X/79/0017/0121/$01.20

2 122 V. Leclercq-Meyer et al.: a-ketosocaproate, Glucagon and Insuln Release G "IF... mm oly2...,, ~-C 2 I. n=5 I I"5 I 1.0 GLUCOSE 3.3 mm n=4 (..) O Z c ~E40 5 \ z~ -- I- 0 A - ;-- H/a- Mn 0 25 h t, J t L '5Qo - 0, ]B / 0 1.4b.s-- L J~ 0 25 L0 60, J 90 Fg. 1. The effects of ct-ketosocaproate (KIC, 10 retool/l) upon glucagon and nsuln release from the rat pancreas perfused n the presence of a low concentraton of glucose (3.3 mmol/1, panel to the rght). On the panel to the left are llustrated the effects of a rse n the concentraton of glucose (from 3.3 to 11.1 mmol/1) Table 1. Glucagon secretory rates (ng/mn) from the perfused rat pancreas. The results represent the mean (_+ SEM) of (n) experments and refer to the upper panels of Fg. 1 (parts A and B) and Fg. 2 (parts C and D) Glucagon secretory rates (ng/mn) Part (n) Substrates present n Substrates added n Equlbraton Infuson Post nfuson basc perfusate nfuson perod perod perod perod mn 25 to 42 mn 47 to 66 ran 66 to 87 A (5) Glucose 3.3 mmol/1 Glucose 7.8 mmol/ _ _ (--~ 11.1 mmol/1) B (4) Glucose 3.3 mmol/l KIC 10.0 mmol/ C (6) Glucose 3.3 mmol/1 Glucose 7.8 mmol/l + ARG 10.0retool/1 (--~ 11.1mmol/1) D (4) Glucose 3.3 retool/1 + ARG 10.0retool/1 KIC 10.0mmol/ _+0.13 Non pared t test B vs A NS NS NS D vs C NS NS NS C vs A P < P < 0.01 P < 0.01 D vs B P < 0.02 P < 0.01 P < all adjacent organs, ncludng the duodenum, beng excluded n order to avod nterference n the assay of enterc glucagon-lke mmunoreactvty. The rats were gven ntravenous heparn sodum (125 U) at the end of the solaton procedure. The whole pancreas was perfused n stu wthout recyclng, through the coelac and mesenterc arteres va a cannuta nserted nto the aorta. The perfuson medum comprsed the followng n mmol/l: NaCI, 118.5; KCl, 4.7; KH 2 PO4, 1.2; MgSO4, 1.2, NaHCO3, 25; CaCI2, 2. It was supplemented wth glucose (3.3 retool/l), dextran (40 g/, TT0, Pharmaca, Uppsala, Sweden) and bovne albumn (5 g/l, fracton V, Sgma, St. Lous, {5. S.A.). The medum was equlbrated aganst a mxture of oxygen and carbon doxde (95 : 5, v/v), wth a resultng ph of 7.4, and entered the pancreas wth a temperature of 37.5 ~ The flow rate was set around 2 ml/ mn, wth a resultng (mean + SEM) perfuson pressure of kpa (33.5 _+ 1.5 mmhg). When needed the substrates L(+)- argnne (Merck, Darmstadt, West Germany), glucose (Merck) and KIC (Sgma, St. Lous, U. S. A.) were dssolved n twce dstlled water and admnstered through a sde-arm syrnge at a flow rate of ml/mn (Braun nfuson pump, Melsungen, West Germany). In those experments whch were perfolrned n the presence of argnne (fnal concentraton n the perfusate : 10mmol/1), the nfuson of the amno acd was mantaned throughout the perfusons. In the experments whch ncluded an exposure to glucose (perfusate concentraton : 11.1 retool/l) or the Na salt of KIC (perfusate concentraton : t0 mmol/1), the nfuson of these substrates was started after a 40 ran equlbraton perod and thereafter contnued for 25 mn. In all cases the ncrease n osmolarty nduced by the secretagogues was corrected by a 10 mmol/1 decrease n the amount of NaCI n the perfusate. Samples of the effluent were collected at 1 ran ntervals n

3 V. Leclercq-Meyer et al.: a-ketosocaproate, Glucagon and Insuln Release 123 Table 2. Insuln secretory rates (ng/mn) from the perfused rat pancreas. The results represent the mean (--- SEM) of (n) experments, and refer to the lower panels of Fg. 1 (parts A and B) and Fg. 2 (parts C and D) Insuln secretory rates (ng/mn) Part (n) Substrates present n Substrates added n Equlbraton Infuson perod basc peffusate nfuson perod perod Early phase Late phase ran 25 to 42 ran 42 to 47 ran 47 to 66 A (5) Glucose 3.3 retool/1 Glucose 7.8 mmol/ _ _+ 1.9 (---~ 11.1 retool/l) B (4) Glucose 3.3 retool/1 KIC 10.0 r~gnol/1 0.2_ _ _+11.2 C (6) Glucose 3.3 mmol/l Glucose 7.8 mmol/1 + ARG 10.0mmol/1 (---~ 11.1mmol/1) 22.9_ _ _+13.9 D (4) Glucose 3.3 retool/1 + ARG 10.0mmol/l KIC 10.0mmol/ _+15.2 Non pared t test B vs A NS P < P < D vs C NS NS NS C vs A P < P < P < D vs B P < P < 0.01 NS chlled tubes contanng 2,000 KIU aprotnn (Trasylol; donated by Drs. G. Wald and A. Prard, Bayer, Brussels, Belgum) and frozen at -25 ~ untl assay. At the tmes shown on the fgures, glucagon (0.2ml alquots) and nsuln (0.01 to 0.2ml alquots) were measured wth ndvdual radommunoassays for these hormones [17]. The tracers were 125I-glucagon (4 to 490 mc/mg, CNTS, France) and 125I-nsuln (124 to 132mC/mg, SOIl/N, Italy). The standards were beef-pork glucagon (lot , a gft from Dr. M. A. Root, El Llly and Co., Indanapols, U.S.A.) and rat nsuln (lot R 170, a gft from Dr. J. Schlchtkrull, Novo, Bagsvaerd, Denmark). Glucose was assayed n the pancreatc effluent wth a glucose-oxdase method (Boehrnger-Mannhem) usng an AAI Techncon analyzer. The rates of glucagon and nsuln secreton were expressed as ng/mn. Total glucagon or nsuln release was calculated from the areas under the curves. All results n the text and the fgures are gven as the mean + SEM of n experments. Statstcal analyses were conducted usng two-taled pared or non-pared t tests [44]. Except f otherwse stated, results gven n the text refer to the non-pared t test. Results Effects of KIC on Glucagon and Insuln Release n the Presence of 3.3 mrnol/1 Glucose The results of these experments are shown n Fgure 1 and Tables 1 and 2. The nfuson of KIC frst produced a slght ncrease n glucagon release ( ng/mn at mn 44) whch, however, was not sgnfcantly dfferent from the mean output durng the equlbraton perod (pared t test : P > 0.2). Thereafter, the nfuson of KIC provoked a marked nhbton of glucagon release (pared ttest vs equlbraton perod : P < 0.001). Upon the arrest of KIC nfuson, the glucagon secretory rates showed some osclla- tons but remaned extensvely depressed (pared t test vs equlbraton perod : P < 0.001). These results wth KIC closely duplcated those wth 11.1 mmol/1 glucose, except that the nhbton of glucagon release occurred 1 ran earler n the case of glucose (Fg. 1; Table 1, part A). At no tme was there any sgnfcant dfference between the glucagon secretory rates recorded n the KIC and glucose experments. Concomtantly wth the effects on glucagon, KIC nduced a bphasc stmulaton of nsuln release whch was much greater than that nduced by glucose (Fg. 1, Table 2). Thus, n the presence of KIC the nsuln secretory rate was 3 (early phase) to 5 tmes (late phase) as hgh as that seen n the presence of glucose. The stmulaton of nsuln release ceased abruptly upon termnatng the nfuson of KIC or glucose, Effects of KIC on Glucagon and Insuln Release n the Presence of 3.3 rnmol/l Glucose and 10 rnmol/l Argnne The results of the experments performed wth KIC n the presence of argnne are shown n Fgure 2 and Tables 1 and 2. In agreement wth the well known nverse relatonshp between the effects of glucose and argnne n the alpha cell, the output of glucagon before KIC nfuson was hgher n the presence of argnne than n the absence of the amno acd (Table 1, equlbraton perod, D vs B). The nfuson of KIC markedly reduced these elevated secretory rates (pared t test vs equlbraton perod : P < 0.05). Ths nhbton of

4 124 V. Leclercq-Meyer et al.: a-ketosocaproate, Glucagon and Insuln Release [ ARGININE 10 mn ] [ G 3.3 mn ARI 10 mn ] g= z I (..3 O r- 2/-.(? z c 160 ~E Z ~ _ r'- e 0-/I' ',I Mn 0 25 /-,,0 n=6 J /.0 ~ L n=/. Fg. 2. The effects of a-ketosocaproate (KIC, 10 mmol/1) upon glucagon and nsuln from the rat pancreas perfused n the presence of a low concentraton of glucose (3.3 mmol/1) and argnne (10 mmol/1) (panel to the rght). On the panel to the left are llustrated the effects of a rse n the concentraton of glucose (from,,, 3.3 to 11.1 mmol/1) n the presence of argnne 9() (10 mmol/1) glucagon release was only partally reversed upon the cessaton of KIC nfuson. Thus, despte a slght ncrease, the output of glucagon remaned sgnfcantly lower durng the postnfuson perod than durng the equlbraton perod (pared ttest : P < 0.05). Ths overall pattern of glucagon release mmcked the one obtaned when 11.1 mmol/1 glucose was appled to the pancreas n the presence of argnne (Fg. 2; Table 1, part C). In the glucose experments, the glucagon secretory rate durng the equlbraton perod was slghtly hgher than n the KIC experments. The dfference was not sgnfcant, however (Table 1, equlbraton perod, D vs C). Glucose abruptly nhbted ths argnne-nduced glucagon release to a rate whch was comparable to that seen upon the KIC nfuson (Table 1, nfuson perod, D vs C). Followng the arrest of the glucose nfuson, a lmted bphasc glucagon response was notced. Despte ths slght ncrease the late glucagon output recorded n the glucose experments was not sgnfcantly hgher than the late glucagon output n the KIC experments (Table 1, postnfuson perod, D vs C). It s of nterest to note that both glucose and KIC nhbted the secreton of glucagon to a much messer extent n the presence of argnne (Table 1, nfuson perod, C vs A, and D vs B). As for nsuln release, n the presence of argnne KIC exerted stmulatory effects whch were supermposable on those of 11.1 mmol/1 glucose (Fg. 2). Thus, at no tme throughout the perfusons was there any dfference between the nsuln secretory rates n the two types of experments (Table 2, D vs C). It should be emphassed that argnne markedly augmented nsuln release both at low (3.3 mmol/1) and hgh (ll.lmmol/1) glucose concentratons (Table 2, equlbraton and nfuson perods, C vs A; D vs B). Argnne also facltated the early phase of KIC-nduced nsuln release (Table 2, early phase of nfuson perod, D vs B) but dd not change the late phase (Table 2, late phase of nfuson perod; D vs B). Dscusson The results clearly demonstrate that n the perfused rat pancreas, n both the absence and presence of argnne, KIC (10 mmol/1) nduces an extensve and not rapdly reversble nhbton of glucagon release. These nhbtory effects closely resembled those seen upon the admnstraton of an almost equmolar glucose stmulus (ll.lmmol/1). Concomtantly, KIC nduced a strkng stmulaton of nsuln release, n agreement wth prevous observatons [12, 13, 33, 42, 43]. The fact that, at a nearly equmolar concentraton and n the absence of argnne, KIC was more potent than glucose n enhancng nsuln release (Fg. 1) also corroborates prevous fndngs [12, 42]. The present data are compatble wth the hypothess that changes n ntermedary metabolsm of the A2-cells play a role n the regulaton of glucagon release by exogenous nutrents. Proof of such an assumpton awats metabolc nvestgatons on A2- cell-rch slets, snce a dynamc study of the whole pancreas cannot brng any nformaton as to the specfc metabolc changes n the A2-cells. Nevertheless, the data avalable n the lterature, as already stated n the ntroducton, suggest that there s no major dfference n the metabolc handlng of glucose n A2-cell-rch and whole pancreatc slets, respectvely. It s concevable that the metabolsm of KIC s also smlar n the A2-cells as n ntact slets, n whch

5 V. Leclercq-Meyer et al.: a-ketosocaproate, Glucagon and Insuln Release 125 KIC has been shown to ncrease the rates of oxygen consumpton, to be converted to acetoacetate, CO2 and H20, and to ncrease the concentraton of reduced pyrdne nucleotdes [12, 13]. Moreover, other fndngs from the lterature regardng glucagon release seem to support the metabolc hypothess. Thus, a-d-glucose, whch s preferentally metabolzed n slet cells [20, 21], s more effectve than/3- D-glucose n nhbtng glucagon release [9, 22, 40]. An nhbton of glucagon release s also observed n the presence of metabolc ntermedates such as glyceraldehyde and dhydroxyacetone [ 10, 11, 27]. In contrast, metabolc nhbtors, such as mannoheptulose, 2-deoxyglucose, odoacetamde, odoacetate, 2-4-dntrophenol and malonate, were reported to enhance glucagon secreton or to prevent glucose from suppressng amno acd nduced glucagon release [4, 10, 26, 29, 48]. Whether nsuln partcpated n the KIC-nduced nhbton of glucagon release cannot be decded by our experments. Such a possblty exsts, snce marked elevatons of nsuln were observed durng the KIC nfusons. The least that can be sad s that nsuln s not the key or sole determnant of glucagon release. At the same steady-state level of nsuln release ( and _ 2.5 ng/mn), a tenfold ncrease n glucagon output from (n = 5) to ng/mn (n = 10) occurs when glucose ll.lmmol/1 s substtuted by glucose 3.3mmol/1 plus argnne 10mmol/l. Conversely, glucagon release may occur at the same rate ( and ng/mn) despte a more than four-hundred-fold ncrease n steadystate nsuln output from 0.26 _ (n = 9) to _ 15.2 (n = 4) ng/mn due to the addton of a-ketosocaproate and argnne (both 10 mmol/1) to a medum of low glucose concentraton (3.3 mmol/1). A last queston rased by our experments s whether the nhbtory effect of a-ketosocaproate upon glucagon release s relevant to the effect of leucne upon glucagon output. The effects of leucne on the secreton of glucagon have not been studed n our laboratory. The data n the lterature are conflctng. Thus, the n vvo admnstraton of L-leucne dd not nfluence the plasma levels of glucagon [5, 35, 39], except when nfused ntrapancreatcally [14]. In vtro, n the sole presence of glucose, leucne has been found to enhance glucagon release [1, 36, 37, 45]. In contrast, however, some authors reported nhbton of glucagon release upon the nfuson of leucne when the perfusate was supplemented wth a mxture of fumarate, glutamate and pyruvate [45]. Such an nhbton s thus comparable to that obtaned wth KIC n the presence of argnne, for we have shown prevously that argnne and a mxture of fumarate, glutamate and pyruvate exhbted equvalent glucagonotropc propertes [16]. In concluson, several questons reman to be answered n relaton to the ntmate mechansms nvolved n the nhbtory acton of KIC n the A2- cell. That the nhbtory effect of KIC parallels that of glucose, however, suggests an essental role of the ntracellular metabolsm of these substrates n the regulaton of glucagon release. Acknowledgements. Ths work was supported n part by grants and from the Belgan Foundaton for Scentfc Medcal Research (F. R. S. M., Brussels, Belgum). We are grateful to Mrs C. Demesmaeker for secretaral help. References 1. Assan, R., Attal, J. R., Ballero, G., Bollot, J., Grard, J. R.: Glucagon secreton nduced by natural and artfcal amno acds n perfused rat pancreas. Dabetes 26, (1977) 2. Edwards, J.C.: A-cell metabolsm and glucagon secreton. Postgrad Med. J. (Suppl.) 49, (1973) 3. Edwards, J. C., Hellerstr6m, C., Petersson, B., Taylor, K. W.: Oxdaton of glucose and fatty acds n normal and n A2-cell rch pancreatc slets solated from gunea pgs. Dabetologa 8, (1972) 4. Edwards, J. C., Taylor, K. W.: Fatty acds and the release of glucagon from solated gunea-pg slets of Langerhans ncubated n vtro. Bochm. Bophys. Acta 215, (1970) 5. Fajans, S. S., Qubrera, R,, Pek, S., Floyd, J. C., Chrstensen, H. N., Conn, J. W.: Stmulaton of nsuln release n the dog by a non metabolzable amno acd. Comparson wth leucne and argnne. J. Cn. Endocrnol. Metab. 33, (1971) 6. Frankel, B. J., Gerch, J. E., Hagura, R., Fanska, R. E., Gerrtsen, G. G., Grodsky, G. M.: Abnormal secreton of nsuln and glucagon by the n vtro perfused rat pancreas of the genetcally dabetc hamster. J. Cln. Invest. 53, (1974) 7. Gerch, J.C., Charles, M.A., Grodsky, G.M.: Regulaton of pancreatc nsuln and glucagon secreton. Annu. Rev. Physol. 38, (1976) 8. Gerch, J.E., Langlos, M., Noacco, C., Karam, J.H., Forsham, J.H.: Lack of glucagon response to hypoglycaema n dabetes: evdence for an ntrnsc pancreatc alpha cell defect. Scence 182, (1973) 9. Grodsky, G.M., Fanska, R., Lundqust, I.: Interrelatonshps between a and/3 anomers of glucose affectng both nsuln and glucagon secreton n the perfused rat pancreas. II. Endocrnology 97, (1975) 10. 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6 126 V. Leclercq-Meyer et al.: a-ketosocaproate, Glucagon and Insuln Release 16. Leclercq-Meyer, V., Marchand, J., Malasse, W.J.: An argnne-lke effect of the "fumarate + glutamate + pyruvate" mxture on glucagon relase. Lfe Sc. 20, (1977) 17. Leclercq-Meyer, V., Marchand, J., Rebolledo, O., Malasse, W. J., Leclercq, R.: A combned radommunoassay for glucagun and nsuln. Dabetologa 11, (1975) 18. Lundqvst, G.: Enzymatc studes of glucose phosphorylaton n the glucagon producng cells of duck pancreas. Horm. Metab. Res. 4, (1972) 19. Lundqvst, G.: Enzymatc prerequstes for ATP formaton from trose phosphates n the A 2 and B cells of the endocrne pancreas of the gunea-pg. Horm. Metab. Res. 4, (1972) 20. Malasse, W. J., Sener, A., Koser, M., Herchuelz, A.: Identfcaton of the a stereospecfc glucosensor n the pancreatc B- cell. FEBS Lett. 65, (1976) 21. Malasse, W.J., Sener, A., Koser, M., Herchuelz, A.: Stmulus-secreton couplng of glucose-nduced nsuln release. Metabolsm of a- and fl-d-glucose n solated slets. J. Bol. Chem. 251, (1976) 22. Matschnsky, F.M., Paglara, A. S., Hover, B. A., Haymond, M. W., Stllngs, G. N.: Dfferental effects of alpha- and beta- D-glucose on nsuln and glucagon secreton from the solated perfused rat pancreas. Dabetes 24, (1975) 23. Matschnsky, F. M., Paglara, A. S., Hover, B. A., Pace, C. S,, Ferendell, J.A., Wllams, A.: Hormone secreton and glucose metabolsm n slets of Langerhans of the solated perfused pancreas from normal and streptozotocn dabetc rats. J. Bol. Chem. 251, (1976) 24. Matschnsky, F.M., Paglara, A.S., Stllngs, S.N., Hover, B.A.: Glucose and ATP levels n pancreatc slet tssue of normal and dabetc rats. J. Cln. Invest. 58, (1976) 25. Mfller, W. A., Faloona, G. R., Unger, R. H.: Abnormal alpha cell functon n dabetes. Response to carbohydrate and proten ngeston. N. Engl. J. Med. 283, (1970) 26. MUer, W.A., Faloona, G.R., Unger, R.H.: The effect of expermental nsuln defcency on glucagon secreton. J. Cln. 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(Eds.), pp Oxford: Pergamon Press Unger, R.H., Ruskn, P., Srkant, C.B., Orc, L.: Glucagon and the A cells. Recent Prog. Horm. Res. 33, (1977) 48. Wer, G. C., Knowlton, S. D., Martn, D.B.: Glucagon secreton from the perfused rat pancreas: studes wth glucose and catecholamnes. J. Cn. Invest, 54, (1974) Receved: February 12, 1979, and n revsed form: May 10, 1979 Dr. V. Leclercq-Meyer Laboratory of Expermental Medcne Free Unversty of Brussels Boulevard de Waterloo, 115 B-1000 Bruxelles, Belgum