Springer-Verlag 1991

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1 Diabetlgia (1991) 34: Springer-Verlag 1991 Abnrmal glucagn respnse t arginine and its nrmalizatin in bese hyperinsulinaemic patients with glucse intlerance: imprtance f insulin actin n pancreatic Alpha cells T. Hamaguchi, H. Fukushima, M. Uehara, S. Wada, T. Shirtani, H. Kishikawa, K. Ichinse, K. Yamaguchi and M. Shichiri Department f Metablic Medicine, Kumamt University Medical Schl, Kumamt, Japan Summary. An excessive glucagn secretin t intravenus arginine infusin was fund in bese hyperinsulinaemic patients with glucse intlerance. This study was designed t determine whether the glucagn hyperrespnsiveness t arginine in these patients wuld imprve by insulin infused at a high enugh dse t vercme insulin resistance. By infusing high dse insulin during arginine infusin, the previusly exaggerated glucagn respnse t arginine culd be nrmalized. T nrmalize the abnrmal glucagn respnse, insulin dses f 4.2 _+ 0.7 and IU were required during arginine infusin in bese hyperinsulinaemic patients with impaired glucse tlerance and Type 2 (nn-insulin-dependent) diabetes mellitus, respectively. This achieved plasma peak insulin levels 3 t 4 times higher than thse bserved in nn-bese healthy subjects. Furthermre, we clarified whether r nt the effect f nrmalizing insulin actin and/r glycaemic excursins cntributed t nrmalizing the exaggerated glucagn respnse t arginine in these patients. Bld glucse was clamped while high dse insulin was infused at the same levels as bserved during the arginine infusin test with n insulin infusin. As a result, nrmalizatin f the exaggerated plasma glucagn respnse was achieved, whether hyperglycaemia existed r nt. These results clearly demnstrate that, similar t nn-bese hypinsulinaemic Type 1 (insulin-dependent) and Type2 (nn-insulin-dependent) diabetic patients, the exaggerated Alpha-cell respnse t arginine infusin in bese hyperinsulinaemic patients with glucse intlerance is secndary t the reductin f insulin actin n the pancreatic Alpha cell, and that the expressin f insulin actin plays an imprtant part in nrmalizing these abnrmalities. Key wrds: patient, glucagn, Alpha cell, insulin resistance, arginine infusin, artificial endcrine pancreas. It is well knwn that an exaggerated rise in plasma glucagn during intravenus arginine infusin is ne f several characteristic abnrmalities in bth Type 1 (insulindependent) and Type 2 (nn-insulin-dependent) hypinsulinaemic diabetic patients. Kawamri et al. demnstrated that when the plasma insulin respnse t arginine in hypinsulinaemic diabetic patients simulated thse f healthy subjects with the aid f an artificial endcrine pancreas, the abnrmal plasma glucagn respnses were nrmalized, whether hyperglycaemia existed r nt. Thus, it has been clearly prven that the exaggerated respnse f the pancreatic Alpha celt t arginine in hypinsulinaemic nn-bese diabetic patients is secndary t insulin deficiency [1, 2]. In bese hyperinsulinaemic patients, a similar exaggerated plasma glucagn respnse t arginine has been reprted [3]. Hwever, Raskin et al. [4] reprted that the glucagn hyperrespnsiveness t arginine in Type 2 diabetic patients was nt imprved by supra-physilgical dses f insulin. In their studies, fur ut f the six patients were bese and prbably insulin resistant. Therefre, it is nt yet clear whether this abnrmality in bese hyperinsulinaemic diabetic patients simply represents the cnsequences f deficient insulin actin r reflects an independent and primary disturbance f Alpha-cell functin. The effect f insulin n glucagn secretin shuld be clarified after careful examinatin f bese hyperinsulinaemic Type 2 diabetic patients. In this reprt, therefre, we evaluated the glucagn respnses t arginine in five grups f subjects: nn-bese healthy subjects, bese nrminsulinaemic patients, and bese hyperinsulinaemic patients with nrmal glucse tlerance, impaired glucse tlerance and glucse intlerance (Type 2 diabetes). T clarify the mechanism f abnrmal glucagn respnses in the bese hyperinsulinaemic patients, we als investigated the effect f insulin infused at a high enugh dse t vercme insulin resistance r the exaggerated glucagn respnse. Furthermre, we determined which factrs, physilgical glycaemic excursin and/r insulin actin, cntributed t the

2 802 T. Hamaguchi et al.: Glucagn respnse in hyperinsulinaemic Type 2 diabetes Table 1. Clinical characteristics f the nn-bese healthy subjects, bese nrminsulinaemic patients and bese hyperinsulinaemic patients N. Age Sex BMI Y~IRI (GTT) Glucse metablized (years) (M/F) (kg/m 2) (mu/1) (M) (mg. kg ~-min-~) Nn-bese healthy subjects _ / bese nrminsulinaemic patients _ / " patients NGT / " b 5.4_+ 0.6 b IGT _ / _+ 3.2 ~ _ b b Type 2 diabetes _ / ~ b b BMI, bdy mass index; YlRI, integrated IRI values f five sampling pints frm 0 t 120 min during ral glucse NGT, nrmal glucse tlerance; IGT, impaired glucse tlerance. Results are expressed as mean + SEM. p < 0.05, b p < 0.01 as cmpared with nn-bese healthy subjects tlerance test (GTT). nrmalizatin f the exaggerated glucagn respnses in these grups. Materials and methds Ten nn-bese healthy subjects, six bese nrminsulinaemic patients and 18 bese hyperinsulinaemic patients were investigated. Bdy mass index f the bese patients was equal t r higher than 27 kg/m 2. In this study, hyperinsulinaemic patients have been defined as thse whse plasma immunreactive insulin (IRI) respnses, expressed as the integrated IRI value f five sampling pints frm 0 t 120 rain (ZIRI) during a 75 g ral glucse tlerance _e 15 E 10 ~ 5 " m 0 I I I [ I 200 F. ~. -~..:.:::... k ~100 (..5 w 150 * * ~ ~ ~ E 50 ~ j i 0 I I I I i min Fig. L Bld glucse, plasma immunreactive insulin (IRI) and glucagn (IRG) respnses in bese nrminsulinaemic patients (n = 6, 0--0) and bese hyperinsulinaemic patients with nrmal glucse tlerance (n =6,'- :),withimpairedglucsetlerance(n =6,-- --) and Type 2 (nn-insulin-dependent) diabetes mellitus (n = 6, 9 ~) during a 75 g ral glucse tlerance test (arrw). The mean values in 10 nn-bese healthy subjects are als depicted (shaded area). Mean _+ SEM, * p < 0.05 vs nn-bese healthy subjects test (GTT), were abve 2 SD f the mean value f thse frm nnbese healthy subjects. All subjects gave infrmed cnsent. The bese hyperinsulinaemic patients were divided int three grups accrding t the results f the GTT [5]: bese hyperinsulinaemic with nrmal glucse tlerance (bese hyperinsulinaemic NGT), impaired glucse tlerance (bese hyperinsulinaemic IGT) and Type 2 diabetes meltitus (bese hyperinsulinaemic Type 2 diabetes). All the diabetic patients had been treated with diet alne, and had shwn n islet cell antibdies n testing r had a histry f ketsis. Sex, age, the degree f besity and ZIRI values during GTT in these five grups are shwn in Table 1. The sex distributin and mean age were nt significantly different amng the five grups, and the degrees f besity and hyperinsutinaemia were nt statistically different amng the three bese hyperinsulinaemic grups. Experimental prtcl Euglycaemic clamp test. T estimate the degrees f insulin resistance, ten nn-bese healthy subjects, six bese nrminsulinaemic patients and 18 patients cnsisting f the three bese hyperinsulinaemic grups (six patients in each grup) underwent the euglycaemic clamp test. After vernight fasting, each patient was cnnected t an artificial endcrine pancreas (AER Mdel STG-22, Nikkis, Tky, Japan). A priming infusin f insulin (semisynthetic human shrtacting insulin, Nv, Denmark, 800 mu ver 10 min) was given, fllwed by a cnstant infusin at 40 mu. m 2. rain- 1 fr 110 rain. Glucse was infused t keep bld glucse levels between 4.4 and 5.6 retl/1 fr 120 rain with the amunt f glucse metablized being calculated frm the amunt f glucse infused during the rain perid. During this test, the mean cefficient f variatin f the glucse values was 4.7 _+ 0.6%. A quantitative estimate f insulin resistance was btained by glucse metablized (M) [6]. Intravenus arginine infusin test. Patients were studied n three separate days within a 2-week perid. In all subjects, after vernight fasting, 0.5 g f arginine/kg f bdy weight (10% arginine in distilled water) was infused fr 30 rain. T determine the mechanism f the exaggerated glucagn respnses in bese hyperinsulinaemic patients, the fllwing tw experiments were perfrmed. Experiment (1): Infusin f insulin at a dse high enugh t vercme insulin resistance during intravenus arginine infusin test Each patient frm the bese hyperinsulinaemic IGT and Type 2 diabetes grups was evaluated. After reducing the bld glucse t euglycaemia with the AER euglycaemia was maintained fr at least 1 h befre the arginine infusin was started. In this experiment, the insulin was infused fr 30 rain t increase plasma IRI levels by 3 t 4- fld f thse bserved in nn-bese healthy subjects, by changing the parameters f insulin infusin algrithm f the AEP [1,2, 7, 8].

3 T. Hamaguchi et al.: Glucagn respnse in hyperinsulinaemic Type 2 diabetes 803 Table 2. Bld glucse, plasma IRI, and plasma IRG respnses during and after 30-rain arginine infusin in nn-bese healthy subjects, bese nrminsulinaemic patients and bese hyperinsulinaemic patients Sampling time (min) Bld glucse (mmz/l) Nn-bese healthy subjects _ _ bese nrminsulinaemic patients 4.7_ _ _ _+ 0.5 patients NGT _ _ _ _ _+ 0.4 [GT 5.1_ _ _ _ " 6.2_+ 0.3 ~ Type 2diabetes 6.7_+ 0.2 ~ 6.8_+ 0.2" 6.8_+ 0.3" a 7.0_+ 0.3" 6.8_+ 0.3 a 6.4_+ 0.3" Plasma IRI (m U/l) Nn-bese healthy subjects 14.6_ _ _ _ _+ 2.7 bese nrminsulinaemic patients _ _+ 3.8 patients NGT 24.1_+ 1.5 ~ 78.4_+ 8.3 ~ ~ " 102.8_+ 8.4" ~ IGT ~ 101.4_+ 6.9" 92.2_+ 7.6" 105.8_+ 7.2" 118.6_+ 8.7" 91.2_+ 6.9 ~ " Type 2 diabetes ~ ~ 78.1_+ 5.6 ~ ~ 108.6_+ 9.2 ~ ~ ~ Plasma IRG (ng/l) Nn-bese healthy subjects 59.7 _ _ ] 93.3 _ bese nrminsuiinaemic patients 65.0 _ _ _ _ patients NGT 60.5+_ _ _ IGT ~ ~ _ 48.3 ~ ~ " _ ~ ~ Type 2 diabetes ~ ~ ~ _ ~ " ~ ~ NGT, nrmal glucse tlerance; IGT, impaired glucse tlerance; IRI, immunreactive insulin; IRG, immunreactive glucagn. Results are expressed as mean + SEM. "~ p < 0.05 as cmpared with nn-bese healthy subjects Experiment (2): Infusin f high dse insulin during intravenus arginine infusin while bld glucse levels were clamped at values simulating thse f the arginine infusin test with n insulin Each patient frm the bese hyperinsulinaemic IGT and Type 2 diabetic grups was evaluated. In this experiment, during arginine infusin, the same rates f insulin infusin required t vercme insulin resistance were perfrmed by AER Bld glucse was cntinuusly mnitred and clamped at the levels bserved in the arginine infusin test with n insulin infusin by infusing glucse with the aid f the AEP [2]. Methd f bld sampling and hrmne assays Bld samples fr hrmne determinatins were btained frm the antecubital vein, at 0, 5, 10, 15, 30, 45, 60, 90 and 120 rain after initiatin f the arginine infusin. Plasma IRI was measured by radiimmunassay using the duble-antibdy technique [9]. Fr glucagn determinatin, bld was immediately placed int heparinized tubes cntaining 0.6 ml EDTA-Trasyll slutin (Trasyll, Bayer, Leverkusen, FRG, 5000 U/ml; Na2EDTA: 1.2 g/l). The bld sampies were centrifuged fr 15 rain at 3000 rev/min, and the plasma samples were stred at - 40~ until assay. Plasma immunreactive glucagn (IRG) was measured by radiimmunassay using AL 123 Daiichi Radiistpe Institute, Tky, Japan [10], the antibdy specific fr pancreatic glucagn. Bld glucse was measured by the glucse xidase methd (AutAnalyzer, Technicn, N. Y., USA). Statistical analysis Results were shwn as mean _+ SEM. Student's t-tests were used fr statistical analysis. Results Insulin resistance in bese hyperinsulinaemic patients As shwn in Figure 1, the bese hyperinsulinaemic IGT and Type 2 diabetic grups shwed the delayed hyper-respnse patterns f plasma IRI after an ral glucse lad. IRG respnses after ral glucse lad were suppressed in the bese hyperinsulinaemic NGT, bese nrminsulinaemic patients and nn-bese healthy subjects, but tended t rise in bth bese hyperinsulinaemic IGT and Type 2 diabetic grups. The mean values f glucse metablized (M) in the bese hyperinsulinaemic NGT, IGT and Type 2 diabetic grups were significantly lwer than thse f the bese nrminsulinaemic patients and nn-bese healthy subjects ( , and vs and mg.kg -1.min -1, respectively, p < 0.01, Table 1) and there was n significant difference between the bese hyperinsulinaemic IGT and Type2 diabetic grups. Glucagn respnse t arginine in bese hyperinsulinaernic patients As shwn in Table 2, bld glucse, plasma IRI and plasma IRG respnses in bese nrminsulinaemic patients were nt statistically different frm thse in nn-bese healthy subjects. In the bese hyperinsulinaemic NGT grup, bld glucse cncentratins and IRG respnses

4 804 T. Hamaguchi et al.: Glucagn respnse in hyperinsulinaemic Type 2 diabetes Arginine i.v. 0.5g. kg 4. 30min -~ Nrmalizatin f exaggerated glucagn respnse t arginine in bese hyperinsulinaemic patients E 8 _= 5 " m g 2 _e 10 E 0_ (..5 Q: E. 2 0 r I t I t i I t 9 """-::-:: ::::':::::::;i:i:!:i:::~-:i:i:i:!:i:i~:iiiiii!i!iii~iiiii ii!il iiiiili ~iliii i iii!iiiiiiiii i iiiiii i :i:i:: I I i I, i I I [ I min Fig.2. Bld glucse, plasma immunreactive insulin (IRI) and glucagn (IRG) respnses during and after 30-min arginine infusin in six bese hyperinsulinaemic patients with impaired glucse tlerance under high dse insulin infusin using the artificial endcrine pancreas (~---~) and withut insulin infusin (H). The mean values in 10 nn-bese healthy subjects are als depicted (shaded area). IIR, insulin infusin rate. Mean _+ SEM, * p < 0.05 vs nnbese healthy subjects t arginine stimulatin were nt statistically different frm thse in nn-bese healthy subjects and bese nrminsulinaemic patients. In the bese hyperinsulinaemic IGT grup, the mean bld glucse cncentratins at 30, 45 and 60 rain were statistically higher than the crrespnding values f nnbese healthy subjects. Plasma IRG values rse rapidly frm ng/1 at 0 min t ng/1 at 30 min, and were significantly higher than thse f nnbese healthy subjects. In the bese hyperinsulinaemic Type 2 diabetic grup, the mean bld glucse cncentratins at all sampling pints were significantly higher than thse f nn-bese healthy subjects. Fasting plasma IRG levels were significantly higher than thse f nn-bese healthy subjects, but nt different frm thse f the bese hyperinsulinaemic IGT grup. Plasma IRG rse rapidly frm ng/1 at 0 min t ng/1 at 30 min (Table 2). In the bese hyperinsulinaemic IGT grup, after infusing high dse insulin, bld glucse respnses were nrmalized as shwn in Figure 2. The mean plasma IRI levels were increased t mU/1 at 5min and mu/1 at 30 min. The amunt f insulin infused during 30 min f arginine infusin was IU. By infusing high dse insulin, the exaggerated IRG respnse was nrmalized. In the bese hyperinsulinaemic Type 2 diabetic grup, after infusing high dse insulin, bld glucse respnses were als nrmalized. The mean plasma IRI levels were increased t mU/1 at 5min and mu/1 at 30 rain. During 30 min f arginine infusin, IU f insulin was infused. The insulin infusin als nrmalized the IRG respnse (Fig. 3). In the bese hyperinsulinaemic IGT and Type2 diabetic grups, bld glucse cncentratins were clamped while high dse insulin was infused at levels similar t thse bserved in the arginine infusin test with n insulin infusin. Even under these cnditins, the exaggerated IRG respnses in bth grups were again nrmalized. Discussin It is widely accepted that the excessive glucagn secretin in respnse t arginine stimulatin ccurs in hypinsulinaemic diabetic patients. Similar abnrmal glucagn secretin t arginine has been reprted in bese hyperinsulinaemic patients [3], but the ppsite results have als been reprted [11,12]. In these reprts, the degrees f glucse intlerance and besity f the patients were nt taken int cnsideratin. We examined 120 bese patients (bdy mass index > 27 kg/m 2) and fund that 60, 30 and 10% were hyperinsulinaemic, nrminsulinaemic and hypinsulinaemic, respectively. In the hyperinsulinaemic bese patients, 60, 32 and 8% shwed NGT, IGT and Type 2 diabetes, respectively. An excessive glucagn secretin during arginine infusin was fund nly in the bese hyperinsulinaemic patients with IGT and Type 2 diabetes, but nt in patients with NGT. These results suggested that exaggerated glucagn respnses t arginine were relevant t the degree f glucse intlerance in bese hyperinsulinaemic patients. Kawamri et al. [1, 2] demnstrated that in hypinsulinaemic Type i and Type 2 diabetic patients, the nrmalizatin f exaggerated glucagn respnse t arginine was achieved when plasma insulin cncentratins simulated thse f healthy subjects. Recently, Paliss et al. [13, 14] reprted that the arginine-induced hyperglucagn respnse in Type 1 diabetic patients was reduced greatly when insulin was administered in a pulsatile manner in an attempt t reprduce the pulsatile physilgical release f insulin. These results supprt the cncept that the exaggerated glucagn secretin t arginine in hypinsulinaemic patients is secndary t their insulin deficiency. Hwever, it is nt yet clear whether the exaggerated glucagn respnse bserved in bese hyperinsulinaemic

5 T. Hamaguchi et al.: Glucagn respnse in hyperinsulinaemic Type 2 diabetes 805 E 8 m e- 5 Arginine i.v. 0.5g. kg ~. 30min -~ 0- I ~2.5 ~_~.L."///////tx;~, g _ ~ (~400 h ================================================ ::::::::::::::::::::::::::::::::::::::::::::::::::: 5 1 I,I I I t 9 ================================================ *.. I I I 'I min Fig. 3. Bld glucse, plasma immunreactive insulin (IRI) and glucagn (IRG) respnses during and after 30-rain arginine infusin in six bese hyperinsulinaemic patients with Type 2 (nn-insulin-dependent) diabetes mellitus under the high dse insulin infusin using the artificial endcrine pancreas (--) and withut insulin infusin (9 e). Symbls and abbreviatins used are the same as thse used in Figure 2 patients can be nrmalized r nt. Elahi et al. [15] have shwn that hyperglucagnaemia in insulin resistant bese subjects declined during euglycaemic clamps, whereas Starke et al. [16] demnstrated in dgs the independency f direct insulin actin n Alpha cells t the ambient glucse cncentratin. There is als evidence suggesting that the exaggerated glucagn respnse t arginine in bese patients may be independent f the plasma insulin cncentratins [4]. This study was, therefre, designed t determine if the glucagn hyper-respnsiveness t arginine in bese hyperinsulinaemic patients wuld be imprved by a dsage f infused insulin high enugh t vercme insulin resistance. It was fund that in bese hyperinsulinaemic patients with IGT and Type 2 diabetes, a previusly exaggerated glucagn respnse t arginine culd be nrmalized by infusing insulin at a high enugh dse t vercme insulin resistance. The abnrmal plasma glucse respnse was als nrmalized. T nrmalize the abnrmal glucagn respnse, insulin dses f and IU were required in bese hyperinsulinaemic IGT and Type 2 diabetic patients, respectively (cmpared t IU in hypinsulinaemic Type 1 diabetic patients [1, 2]), reaching peak IRI levels f three t fur-fld higher than thse bserved in nn-bese healthy subjects. Supplemented insulin dses were inversely crrelated with the values f glucse metablized (M), as an index f insulin resistance measured by the euglycaemic clamp methd. T clarify whether r nt nrmalizatin f glycaemic excursins cntributes t the nrmalizatin f the exaggerated glucagn respnse t arginine in these patients, bld glucse was clamped while high dse insulin was infused at the same levels as thse bserved in the arginine infusin test with n insulin infusin. As a result, nrmalizatin f the exaggerated plasma glucagn respnse was achieved, whether hyperglycaemia existed r nt. These results clearly demnstrate that the exaggerated Alphacell functin in bese hyperinsulinaemic patients with glucse intlerance is als secndary t the reduced insulin actin n the pancreatic Alpha cell. We als fund that in sme f the bese hyperinsulinaemic IGT and Type 2 diabetic patients studied, paradxic glucagn rises during GTT culd be nrmalized by the insulin infused at a high enugh dse t vercme insulin resistance (data nt shwn). These results were als supprted by the fact that in bese hyperinsulinaemic patients wh succeeded in weight reductin, the previusly elevated plasma glucagn respnses t arginine and paradxic rises in glucagn during GTT were als reduced t the levels f thse in nn-bese healthy subjects, accmpanied by significant imprvements in bld glucse and IRI respnses (data nt shwn). The exact mechanism f nrmalizatin f an abnrmal glucagn respnse t arginine infusin has nt been elucidated. Samls et al. [17-19] suggested the invlvement f a paracrine mechanism by which insulin culd suppress glucagn in viv and in vitr. Since then, the cncept f nrmalizing an abnrmal glucagn respnse t arginine using insulin has been discussed [16, 20-24]. The results f Asplin et al. [25] als supprt this cncept. Asplin's study stated that, in nn-bese nn-diabetic patients, the verall effect n the pancreatic Alpha cell was a prgressively greater acute glucagn respnse t arginine during the insulin infusins, prbably due t a lesser Alpha-cell suppressin by paracrine Beta-cell activity. Hwever, in ur present studies with bese hyperinsulinaemic IGT and Type 2 diabetic grups, high dse insulin infusin nrmalized the exaggerated glucagn respnse t arginine. Plasma C-peptide levels, hwever, during high dse insulin were suppressed, accmpanied with the decrease in bld glucse (fasting and peak levels f plasma C-peptide levels in bese hyperinsulinaemic IGT patients: and nml/1 in arginine infusin test withut insulin infusin, nml/1 and 3.0 _+ 0.6 nml/1 in arginine and high dse insulin infusins, respectively). Hwever, in the experiment with bld glucse cncentratins clamped at levels similar t thse f the arginine infusin test withut insulin infusin, fasting and peak levels f plasma C-peptide were 1.0_+0.1 nml/1 and nml/1. The same trend was bserved in the bese hyperinsulinaemic Type 2 diabetic grup. These present findings d nt exclude the pssibility f a para-

6 806 T. Hamaguchi et al.: Glucagn respnse in hyperinsulinaemic Type 2 diabetes crine mechanism between islet cells. With the aid f the euglycaemic clamp technique, further studies n the glucagn respnses t arginine with a high dse f insulin infusin in cntrl grups, whether bese nrminsulinaemic r bese hyperinsulinaemic NGT grups are necessary t clarify the pssibility f the paracrine mechanism fr nrmalizatin f abnrmal glucagn respnses t arginine. The regulatry mechanism f insulin n the Alpha cell is believed t be mediated by specific cell surface receptrs, but this has nt been investigated thrughly. ur present results indirectly favur such a cncept. Firstly, a dsage f infused insulin high enugh t vercme insulin resistance culd nrmalize the glucse respnse as well as the glucagn respnse t arginine infusin. Secndly, insulin dses high enugh t vercme insulin resistance were fund t be inversely crrelated with the peripheral insulin resistance measured by the euglycaemic clamp technique. Patel et al. [26] demnstrated that the specific autradigraphic grains assciated with radiactively labelled insulin were fund n Alpha cells. Hwever, by using receptr assay techniques, Van Schravendijk et al. [27] reprted that n specific binding f insulin was detected n purified pancreatic Alpha cells, because f lw receptr assay sensitivity and pssible cellular damage inflicted by the cell islatin technique. Quantificatin and characterizatin f insulin receptrs n the Alpha cell remain t be fully understd, especially cncerning the pssible physilgical rle. In cnclusin, ur study shws that the exaggerated Alpha-cell respnse t arginine infusin in bese hyperinsulinaemic patients with glucse intlerance is secndary t the reduced insulin actin n the pancreatic Alpha cell, and that expressin f insulin actin is very imprtant t nrmalize these abnrmalities. These findings are cnsistent with ur earlier reprts n the nrmalizatin f the exaggerated glucagn respnse t arginine [1, 2] and f the paradxic rise f glucagn t an ral glucse lad [28] in hypinsulinaemic Type i and Type 2 diabetic patients. Acknwledgements. We thank Dr. K. Kisanuki and Dr. K. Nishida fr their skillful wrk. References 1. Kawamri R, Shichiri M, Kikuchi M, Yamasaki Y, Abe H (1980) Perfect nrmalizatin f excessive glucagn respnses t intravenus arginine in human diabetes mellitus with the artificial beta-cell. Diabetes 29: Kawamri R, Shichiri M, Kikuchi M, Yamasaki Y, Abe H (1985) The mechanism f exaggerated glucagn respnse t arginine in diabetes mellitus. Diab Res Clin Pract 1: Kalkhff RK, Gssain VV, Matute ML (1973) Plasma glucagn in besity. Respnse t arginine, glucse and prtein administratin. N Engl J Med 289: Raskin R Aydin I, Unger RH (1976) Effect f insulin n the exaggerated glucagn respnse t arginine stimulatin in diabetes mellitus. Diabetes 25: Diabetes mellitus, reprt f a WH study grup (1985) Technical reprt series 727, WH, Geneva 6. DeFrnz RA, Tbin JD, Andres R (1979) Glucse clamp technique: a methd fr quantifying insulin secretin and resistance. Am J Physi1237:E214-E Kawamri R, Shichiri M, Griya Y, Yamasaki Y, Shigeta Y, Abe H (1978) Imprtance f insulin secretin based n the rate f change in bld glucse cncentratin in glucse tlerance, assessed by the artificial beta cell. Acta Endcrin187: Griya Y, Kawamri R, Shichiri M, Abe H (1979) The develpment f an artificial beta cell system and its validatin in depancreatized dgs: the physilgical restratin f bld glucse hmestasis. Med Prgr Technl 6:9% Hales CN, Randte PJ (1963) Immunassay f insulin with insulin-antibdy precipitate. Bichem J 88: Nishin T, Kdaira T, Shin Set al. (1981) Glucagn radiimmunassay with use f antiserum t glucagn C-terminal fragment. Clin Chem 27: Schade DS, Eaten RP (1974) Rle f insulin and glucagn in besity. Diabetes 23: Santiag JV, Haymnd MW, Clarke WL, Pagliara AS (1977) Glucagn, insulin, and glucse respnses t physilgic testing in nrmal and massively bese adults. Metablism 26: Paliss G, Sgambat S, Trella R et al. (1988) Pulsatile insulin delivery is mre efficient than cntinuus infusin in mdulating islet cell functin in nrmal subjects and patients with type 1 diabetes. J Clin Endcrinl Metab 66: Paliss G, Scheen AJ, Albert A, Lef~bvre PJ (1989) Effect f pulsatile delivery f insulin and glucagn in humans. Am J Physi1257:E686-E Elahi D, Nagulesparan M, Hershcpf RJ et al. (1982) Feedback inhibitin f insulin secretin by insulin: relatin t the hyperinsulinemia f besity. N Engl J Med 306: Starke A, Imamura T, Unger RH (1987) Relatinship f glucagn suppressin by insulin and smatstatin t the ambient glucse cncentratin. J Clin Invest 79: Samls E, Harrisn J (1976) Remarkable ptency f smatstatin as a glucagn suppressant. Metablism [Suppl 1] 25: Samls E, Stagner JI (1988) Intra-islet regulatin. Am J Med 85 [Suppl 5A]: Samls E, Stagner JI (1990) Islet smatstatin-micrvascular, paracrine, and pulsatile regulatin. Metablism [Suppl 2] 39: Weir GC, Atkins RE Martin DB (1976) Glucagn secretin frm the perfused rat pancreas fllwing acute and chrnic streptztcin. Metablism 25: Lef~bvre PJ, Luyckx AS (1979) Glucagn and diabetes: a reappraisal. Diabetlgia 16: Lef~bvre PJ (1983) Glucagn II, Handbk f experimental pharmaclgy, Vl 66. Springer, Berlin Heidelberg New Yrk 23. Maruyama H, Hisatmi A, rci L, Grdsky GM, Unger RH (1984) Insulin within islets is a physilgic glucagn release inhibitr. J Clin Invest 74: Palmer JR McCullck DK, Raghu PK (1985) Recgnitin f hyp- and hyperglycemia by pancreatic A-cell is dependent n the B-cell. Diabetes [Suppl 1] 34:99 Abstract 25. Asplin CM, Paquette TL, Palmer JP (1981) In viv inhibitin f glucagn secretin by paracrine beta cell activity in man. J Clin Invest 68: Patel YC (1982) Quantitative electrn micrscpic autradigraphy f insulin, glucagn, and smatstatin binding sites n islets. Science 217: Van Schravendijk CFH, Friers A, Hghe-Peters EL et al. (1985) Pancreatic hrmne receptrs n islet cells. Endcrinlgy 117: Shichiri M, Kawamri R, Abe H (1979) Nrmalizatin f the paradxic secretin f glucagn in diabetics wh were cntrlled by the artificial beta cell. Diabetes 28: Received: 13 May 1991 and in revised frm: 3 August 1991 Dr. T. Hamaguchi Department f Metablic Medicine Kumamt University Medical Schl 1-1-1, Hnj Kumamt, 860 Japan