261 Horm. Metab. Res. 11 (1979) 261-265
Secretory B-Cell Activity in Insulin Dependent Maturity-Onset Diabetic U. Krause, G. Faisst*, U. Cordes and J. Beyer
Summuy at least not convincing (Heding 1972). Determination of serum C-Peptide levels seems to be a more In 10 insulin dependent maturity onset diabetics we found elevated basal C-Peptide levels (4.78 ± 0.5 ng/mL Normal valuable tool for measurement of the endoerine range 1.1-3.6 ng/ml), which could be suppressed by insulin panereas funetion. Different radioimmunoassay sysinjection to the same extent as in sulfonylurea treated diabetems for C-Peptide have been developed in the past ties could be demonstrated. C-Peptide immunoreactivity in in the laboratories of Beischer (Beischer, Raptis, these patients therefore seems to be newly secreted rather Keller, Heinze, Schröder and Pfeiffer 1975), Heding than accumulated material Since adrenalectomized patients could be suppressed in the same way, it is Iikely, that cate(Heding 1975), Kaneko (Kaneko, Olea, Munemura, cholamines are not the major factor in the mechanism of Oda, Yamashita, Suzuki, Yanaihara, Hashimoto and suppression. Therefore only decrease of bloodsugar levels Yanaihara 1974) and Rubenstein (Rubenstein, Clark. seems to be accountable for the decrease of C-Peptide levels. Melani and Steiner 1969). In previous studies we and High C-Peptide levels in insulin dependent maturity onset diabetics which cannot be stimulated but suppressed may other investigators reported on some maturity onset be explained by a lass of g1ucoreceptor molecules. insulin dependent diabetics having normal or even
Key-Worda: C·Peptide - Glucoreceptor - Adrenalectomy Rtlidual Secretion - Insulin Induced Hypoglycaemilz
Introduction Tbe proinsulin moleeule is converted in the B-cell by specific proteases to insulin and C-Peptide (Kemmler, Peterson, Rubenstein and Steiner 1972; Kemmler, Peterson and Steiner 1971; Kemmler and Steiner 1970). Thus C-Peptide is found to be seereted in equimolaI amounts with insulin into the bloodcireulation (Rubenstein, Block, Sta", Melani and Steiner 1972; Heding 1975). In the peripheral blood however we found a three to fivefold level of C-Peptide on a molar basis (Krause, Cordes and Beyer 1977) due to the different degradation of the two proteins (Faber, Markussen and Naithani 1976; Kuzuya and Matsuda 1976). Seeretion of C-Peptide is therefore an indieator of the endoerine pancreas function. In a radioimmunological assay system CPeptide does not crossreact with insulin (Yanaihara, Hashimoto, Yanaihara, Saleagami, Stein er and Rubenstein 1974). This enab1es the measurement of endoerine panereas function even in insulin dependent diabetics. A few attempts have been made for determination of insulin levels in sera of such patients, but the methods were rather complicated and the results "'Part of doctoral thesis, Mainz 1978, Germany Received: 18 Jan. 1978 0018-5043/79
0432-0261
Accepted: 29 Aug. 1978 S 03.00
elevated basal C-Peptide levels, indicating that there must be a residual function of the B-cells (Block, Mako. Steiner and Rubenstein 1972; Heding and Rasmussen 1975; Beischer, Keller, Maas, Schiefer and pteiffer 1976; Krause, Beyer, Cordes and Happ 1976). In some of these patients C-Peptide secretion is stimulated by intravenous injection of glibenclamide glucose (Pfeiffer and Raptis 1972), while others showed an uninfluenced secretion pattern. One explanation for high C-Peptide levels, which are not stimulated may be a delayed degradation and elimination of the peptide by these patients. This is sureIy true for individuals with renal funetion impairment, sinee C-Peptide is mainly degraded and eliminated in the hidneys (Rubenstein et al. 1972). Another explanation may be the binding of proinsulin to cireulating insulin antibodies which proteet proinsulin from degradation. Accumulated proinsulin may then mimic high C-Peptide levels beeause of a eertain crossreaction in the C-Peptide assay (Yanaihara et al. 1974). In this ease any regulation of C-Peptide seeretion should not be possible. In metabolie normal subjects suppression of C-Peptide seeretion is achieved by insulin induced hypoglycemia. The aim of this study was to clarify whether insulin dependent diabetics with normal or eIevated C-Peptide levels can be suppressed by insulin injeetion. Furthermore we want to elucidate if epi-
© 1979 Georg Thieme Publishers
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Abteilung tür Klinische Endokrinologie. 11. Med. Klinik und Poliklinik der Universität Mainz. Mainz. Germany
262
U. Krause, G. Faisst, U. Cordes and J. Beyer
After an overnight Casting period bloodsugar was lowered in Ofo 32 metabolie normal subjects, 10 insulin dependent maturity onset diabeties, 10 sulfonylurea treated maturity-onset dia- 100 beties, and 6 bilaterally adrenaleetomized non-diabeties by LV. injeetion of 0.15 I.U. insulin (MC-Aetrapid, Novo) per kg bodyweight exeept the insulin dependent patients whieh 90 got the 1.5-fold dose of their daily requirement. None of the patients had any signs of renal dysfunetion as assessed 80 by measurement of serum urea and creatinine levels and ereatinine elearance as weil. Only one of the insulin dependent diabetics had eireulating insulin antibodies as assessed 70 by a simple radioaetive ineubation method. Insulin dependeney was diagnosed by means of the g1ibenelamide-glucose test (Pfeiffer and Raptis 1972). Adrenaleetomjzed patients 60 had undergone adrenaleetomy because of Cushing's disease and were substituted with cortisone. Cortisol levels as assessed by radioimmunoassay were within the normal range. Ven50 ous blood was used for determination of bloodsugar and CPeptide. Bloodsugar was measured enzymatically (GODmethod) in an automatie analyzer. Reagents were from Boeil- 40 ringer, Mannheim. C-Peptide was measured radioimmunologicaUy with a double antibody method as described by Kane30 ko et aJ. (1974) with slight modifications. Reagents were from Byk-Mallinekrodt. Standards were run in triplicate, sampies and eontrol sera in duplieate. Intraassay varianee 20 was 4,4% and interassay varianee was 11.5% under our eonditions. Minimum deteetion limit was 0.3 ng/mI.
..- -.. C'Peptide
10 .----•••• b lood'g I ucos e
Results In sera of six totally duodenopancreatectomized patients we were unable to measure any C-Peptide even after stimulation with 0.5 g arginine/kg bodyweight given intravenously (Thiel, Krause, Mangold, Wolf and Beyer 1977). Table 1 summarizes the results of C-Peptide and bloodsugar determinations du ring the experiments. Starting bloodsugar levels of normal subjects were the same like of adrenalectomized non-diabetic patients, whereas the C-Peptide starting levels were higher in adrenaleetomized patients than in normals. Sulfonylurea treated patients exhibit highest C-Peptide starting levels. Insulin dependent patients seem
o
60
30
90
120
Fig. 1 Bloodsugar and C-Peptide decrease after Lv. injection of 0.15 I.U. insulin per kg bodyweight to metabolie normal persons
After insulin injeetion, metabolie healthy subjects show a rapid deerease of bloodsugar and C-Peptide as weIl during the first 30 minutes (Fig. 1). After 30 minutes bloodsugar increases whereas C-Peptide shows a further decrease. In the early phase the two eurves are nearly eongruent. Fifty percent of the basal C-Peptide level is reaehed after 29 minutes and of the bloodsugar level after 26 minutes.
Thble 1 C·Peptide (upper Iines) and Bloodglucose (Iower lines) deerease after intravenous insulin injeetion (± S.E.M.) Time
0
Insulin dependent diabetics Sulfonylurea treated diabetics
4.78 197 5.27 138
± ±
Adrenaleetomized non diabetics Metabolie heaIthy subjeets
10
20
4.49 168
± ±
0.5 19 0.5 10
3.72 85
± ±
0.8 11
3.06 61
± ±
0.7 15
2.40 84
± ±
0.2 2
2.62 65
± ±
0.3 3
± ±
0.5 19
4.14 ± 0.6 119 ± 22 4.45 '± 0.4 96 ± 10
30 ± ±
2.62 ± 0.5 44 ± 10
3.91 101 3.98 67 2.14 37
± ±
0.6 18 0.4 13 0.5 8
1.91 ± 0.2 41 ± 3
1.13 36
± ±
0.1 2
± ±
120
90
60
3.22 74
± ±
004 11
1.94 39
± ±
0.3 4
3.11 ± 0.5 58 ± 13 2.6 ± 0.4 65 ± 8 1.8 ± 0.2 59 ± 4
0.8 51
± ±
0.1 2
0.73 58
3.46 ± 0.5 65 ± 16
± ±
0.1 2
min.
2.83 ± 0.4 ng/ml 25 ± 8 mg/dl 2.46 ± 0.4 ng/ml 81 ± 7 mg/dl 1.64 61
± ±
0.3 ng/ml 8 mg/dl
0.74 69
± ±
0.1 ng/ml 2 mg/dl
Downloaded by: University of Illinois. Copyrighted material.
nephrine is involved in the mechanism of suppression to have a little lower basal C-Peptide levels, but because catecholamines cause inhibition of insulin se- there is no significant differenee to the sulfonylurea treated patients. Fasting bloodsugar levels are elevatcretion in vitro and in vivo (Vance, Buchanan and ed in the group of sulfonylurea treated patients, and Williams 1971). higher in the insulin dependent diabeties. Methods and Materials
Beta-Cell Activity and Insulin Dependent Maturity-Onset Diabetes
Ofo 100
90
Ofo
n'S
100
\,
90
70
\\A~
60
\1\
80
80
70
I/'i \~_& ~~A 1// I
50
263
••
60 50
\ ............................... \
40
I
30
30
20
20 &--A
10
C' Pept i de
&--&
10
•.••••• -. blood-glucose
..... ---. blood-glucose
o
30
60
C'Peptide
90
120
Fig. 2 Bloodsugar and C-Peptide decrease after Lv. injection of 0.15 i.V. insulin per kg bodyweight to bilaterally adrenalectomized non diabetic patients
o
30
60
90
120
Fig. 4 Bloodsugar and C-Peptide decrease after i.v. injection of 0.15 i. V. insulin per kg bodyweight to sulfonylurea treated maturity onset diabetics
n'\O
0/0
Bilaterally adrenalectomized patients behave like normals during the first 30 minutes (Fig. 2). Later on bloodsugar shows a delayed inerease in eomparison to the normals. Insulin dependent maturity-onset diabeties show the same rapid decrease of bloodsugar as normals and non diabetie adrenaleetomized patients did (Fig. 3). Fifty percent of the starting bloodsugar level is reached at 30 minutes. There is however a dear dissociation between the bloodsugar curve and the C-Peptide curve. Fifty percent level for C-Peptide is reaehed after 120 minutes.
100
90 80
70 60
Sulfonylurea treated patients show during the first 30 minutes the same rapid bloodsugar decrease with a fifty percent level at 29 minutes (Fig. 4). C-Peptide eurve is intermediate between normals and insulin dependent diabeties. Fifty percent CPR-Ievel is reached after about 87 minutes. Bloodsugar inereases after about 90 minutes.
50
40 30
20
Discussion 10
C-Pepllde
CPR-Ievels in insulin dependent diabeties have been attributed to antibody-bound proinsulin-Iike material (Fink, Cresto, Gutman, Lalline, Rubenstein and Recant 1974). However Beischer, Raptis, Keller, Maas, 30 60 120 o 90 Fig. 3 Bloodsugar and C-Peptide decrease in maturity onset Beischer, Feilen andPfeiffer (1978) found high amounts insulin dependent diabetics after i.v. injection of thc 1.5-fold of proinsulin-like material only in juvenile type diabedose of insulin of their daily requirement ties du ring a remission phase. We and other investiga&--&
.-------. bl ood -g I u cos e
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40
264
U. Krause, G. Faisst, U. Cordes and J. Beyer
tors eould show in the past, that C-Peptide levels in several insulin dependent maturity onset diabeties eould be stimulated by food intake and some other agents like glibenclamide-glueose (Krause et al. 1976) and glucagon as weil (Faber and Binder 1977). Such stimulation eannot be explained by antibody-bound proinsulin. If in insulin treated diabetics normal or high C-Peptide levels should be mimiced by antibodybound proinsulin-like material, these antibodies must have a much higher affmity to proinsulin-like material than to insulin itself, because otherwise exogenous insulin will eause an in vivo displacement from the antibodies, and aeeumulation cannot take place. Or proinsulin-like material must be secreted in such an excess that daily insulin doses will not
ty onset diabeties, seerete high amounts of CPR. Sinee we do not know, if it is really C-Peptide or proinsulin· like material, whieh crossreacts in our assay system, we eannot deeide, if the missing biological effeet of the seereted material is due to a peripheral resistance or to a missing biological poteney.
Acknowledpment The authors are greatly indebted for skillful technical assistance to Mn. I. Bemer and to Mn. I. Knoll.
References
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Beischer, W., S. Raptis, L. Keller, M. Maas, B. BelIeher, K. Feilen, E.F. Pfeiffer: Humanes C-Peptid, Teil 111. Sekretionsdynarnik der Beta-Zellen erwachsener Diabetiker nach Glibenclamid-Glucose Lv. Klin. Wschr. 56: 111120 (1978) Beischer, W., L. Keller, M. Maas, E. Schiefer, E.F. Pfelffer: Human C-Peptide, part I: radioimmunoassay. Klin. Wschr. 54: 709-715 (1976) Belseher, W., S. Raptis, L. Keller, E. Helnze, K.E. Sehröder, E.F. Pfeiffer: Characterization of residual beta-cell funetion in diabetes by'a new C-peptide radioimmunoassay. Diabetologia 11: 332 (1975) Block, M.B., M.E. Mako, D.F. Steiner, A.H. Rubenstein: Circulating C-peptide immunoreactivity-studies in normal and diabetic patients. Diabetes 21: 1013-1026 (1972) BotteTmll~n, P., Th. Zilker, R.. Er'!'ler: C-Peptide secret.ion accordmg to glucose and msulin levels (abstract). Dlllbetologia 12: 382 (1976) Faber, O.K., C. Binder: C-Peptide response to glucagon. A test for the residual beta-cell function in diabetes mellitus. Diabetes 26: 605-610 (197.1). ... Fab.er, O.K., ": Marku~sen, V.K. Naltham: C-Peptlde kmetlcs . " . m man. Dlllbetologlll 12: 389 (1976) (abstract) mg a stop of C-Peptlde seeretlon (Bottermann, ZrlFink, G., J.c. Cmto, R.A. Gutman, R.L. Lavlne, A.H. Ru. ker and Ermler 1976). Moreover, reeent results from benstein, L. Recant: Plasma proinsulin-like material in Schatz and Pfeiffer (1977) indieate, that insulin seereinsulin treated diabetics. Horm. Metab. Res. 6: 439 tion from isolated islets of Langerhans is not sup(~974) . . Hedmg, ~. G., .S.M. ~asmus~en: Hu~n C-peptlde m normal Pressed by exogenous insulin in eoncentrations up and dlllbetlC subJects. DlllbetologJa 11: 201-206 (1975) to 5 mV/mI. Heding, L.G.: Radioimmunological determination of human C-Peptide in serum. Diabetologia 11: 541-548 (1975) From our results there is strong evidence, that epiHeding, I.G.: Determination of total serum insulin (lRI) in nephrine is not involved in the mechanism since insulin treated diabetic patients. Diabetologia 8: 260adrenalectomized patients behave like normals con266 (1972) lkeda, Y., N. Tajima, N. Minami, Y. lde, M. Abe: Pancreaceming the CPR-suppression. tic b-cell function of young diabetics assessed by C-Pe~ The different behaviour of CPR-decreases at identitide immunoreactivity. Jikeikai Med. J. 24: 113-122 cal bloodglucose fall may be at least partially ex(1977) Kaneko, T., H. Oka, M. Munemura, T. Od4, K. Yamashlta, plained by the alte red sensitivity of the B-ceil to S. Suzukl, N. Yanaihara, T. Hashlmoto, C. Yanaihara: changes in bloodglueose levels in diabetics. A loss of Radioimmunoassay of human proinsulin C-Peptide using glucoreceptor molecules would fit this fmding (Krausynthetic human connecting peptide. Endocrin. jap. 21: se, Puchinger and Wacker 1973; Mayer and Pfeiffer 141-145 (1974) 1972). Kemmler, W., J.D. Peterson, A.H. Rubensteln, D.F. Steiner: On the biosynthesis, intracellular transport and mechanOur finding, that there is a further C-Peptide decrease ism of conversion of proinsulin to insulin and C-Peptide. while bloodglucose is already inereasing is surprising. Diabetes 21 , Suppl. 2: 572-582 (1972) Kemmler, W., J.D. Petersen, D.F. Steiner: Studie! on the conWe can only speeulate that there exists a protective version of proinsulin to insulin. Conversion in vitro with mechanism whieh inhibits insulin secretion after hytrypsin and carboxypeptidase b. J. bio!. Chern. 246: 6786poglycemia. This point needs further investigation. 6790 (1971) An open question is the biological signifieance of Kemmler. w., D.F. Steiner: Conversion of proinsulin to insuliO in a subcellular fraction frorn rat islet. Biochem. Bioour fmding, that there are insulin dependent maturiphys. Res. Commun. 41: 1223-1230 (1970)
displaee significant amounts of proinsulin-like material from the antibodies. Our insulin dependent patients, exeept one, did not have significant insulin antibody levels. None of them had signs of renal fallure. Therefore it is most likely that normal or high C-Peptide levels in these patients represent newly seereted rather than aeeumulated material. Our finding, that CPR-Ievels in insulin dependent maturityonset diabeties ean be lowered by insulin indueed M · bl00dglueose deerease support thi s assumptlon. oreover, this residual funetion seems to exert metabolie effeets as reported by Faber and Binder (1977) and reeently by Ikeda, Tajima, Minami, Ide andAbe(1977). fu th tor on e t The me · The~ fimdmgs. r er supp.or u e .c p . ehanlsm by whieh suppresslOn ceurs IS not known exactly. Since C-Peptide levels and bloodsugar levels are strongly eorrelated over a wide range it is probable that low bloodglueose is the only factor eaus-
Hyperthyroidism, Insulin and Metabolism
Rubenstein. A.H., J.L. Clark, F. Melani, D.F. Steiner: Secretion of proinsulin C-peptide by pancreatic betacell and its circulation in blood. Nature 244: 697 -699 (1969) Rubenstein, A.H.• M.B. Block, J. Sta", F. Melani. D.F. Steiner: Proinsulin and C-peptide in blood. Diabetes 21: Suppl. 2,661-672 (1972) Schatz, H., E.F. Pfeiffer: Release of immunoreactive and radioactively prelabelled endogenous (pro )insulin from isolated rat islets in the presence of exogenous insulin. J, Endocrinol. 74: 243-249 (1977) Thiel, M., U. Krause, G. Mangold. E. Wolf, J. Beyer: Untersuchungen zur Sekretion des Enteroglukagon bei Patienten nach totaler Duodenopankreatektomie. Verh. d. Deutschen Ges. f. innere Medizin 83: 1399-1402 (1977) Vance. J.E.• K.D. Bcchanan, R.H. Williams: Glucagon and insulin release. Influence of drugs affecting the autonomic nervous system. Diabetes 20: 78-82 (1971) Yanaihara. N., ,T. Hashimoto, C. Yanaihara, M. Sakagami, D. F. Stein er, A.H. Rubenstein: Synthesis of human connecting peptide derivatives and their immunological properties. Biochem. Biophys. Res. Commun. 59: 11241130 (1974)
Requests for reprints should be addressed to: Dr. Ulrich Krause, Abteilung f. Klin. Endokrinologie, Bau 5, Langenbeckstr.l, 0-6500 Mainz (Germany)
Horm. Metab. Res. 11 (1979) 265-270
Serum Insulin and Free Fatty Acid Responses to Siow and Quick Graded Hyperglycaemias in Hypothyroid Oogs* Aurora Renauld**, L.L. Andrade***, R.C. Sverdlik, R.R. Rodrfguez** and D.M. Lindental Institute of Physiology. Buenos Aires University Medical School, Buenos Aires, Argentina
Summary The effects of thyroidectomy by radioiodine on blood sugar, serum immunoreactive insulin and serum free faUy acid levels in dogs, in the course of glucose infusion tests whether involving glucose priming-dose (B tests) or not (A tests) were studied. The basal levels of blood sugar and serum insulin were not affected by hypothyroidism; circulating free fatty acids had -The present study was partly reported in abstract from (XI Congreso Latinoamericano de Ciencias Fisiologicas,
Mendoza, Argentina, July 29 - Agust 4, 1973; VIII Congreso Panamericano de EndocrinologIa, Buenos Aires, Argentina, October 27 - November 2, 1974). The study was sponsored by the "Consejo Nacional de Investigaciones CientIficas y Tecnicas", Argentina, Res. Grant no. 2304-d. "Established Investigator at this Council. -- Post-graduated Fellow, University of Buenos Aires, Argentina. Received: I Sept. 1976
Accepted: 2S Aug. 1978
oolB-5043/79
103.00
0432-0265
hardly risen. In the hypothyroid group, the blood sugar promes curing both tests were somewhat higher than normal, and hyperglycaemia was maintained for longer. These abnormalities were restored by Thyroxine therapy. Hypothyroidism brought about a considerable alteration of the dynamics of serum insulin response to glucose. Early during the continuous stimulation, serum insulin levels were normal (A tests) or somewhat higher than normal (B tests). Late during the stimulation or, even, after interruption of glucose infusion quite an intense insulin response was tardiIy observed, which was the major effect of hypothyroidism on the insulin response to continuous stimulation by glucose. The abnormalities of the serum insulin profile were corrected by thyroxine therapy. Foot Note: The following abbreviations will be used throughout the text: BS, blood sugar; IRI, immuno-reactive insulin; FFA, free fatty acids; BEI, butanol-extractable iodine; PBI, protein-bound iodine; T4, thyroxine.
© 1979 Georg Thieme Publishers
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Krause, u., J. Beyer, U. Cordes, J. Happ: Serum-C-Peptidspiegel und Serllminsulin bei Patienten mit Hypoglykämien sowie unterschiedlichen Ober- und Unterfunktionszuständen des Inselsystems. IV. Internationales Donausymposium über Diabetes meUitus, Dubrovnik 1975 Krause, u., U. Cordes, J. Beyer: C-Peptid, Sekretion und Stoffwechsel bei unterschiedlichen Funktionszuständen und Störungen der B-Zellen der Langerhansschen Inseln. Dtsch. med. Wschr. 102: 785-790 (1977) Krause, u., H. Puchinger, A. Wacker: Inhibition of glucose induced insulin secretion in trypsin treated islets of Langerhans. Horm. Metab. Res. 5: 325-329 (1973) Kuzuya, T., A. Matsuda: Disappearance rate of endogenous human C-peptide from blood. Diabetologia 12: 519-521 (1976) Maier, v., E.F. Pfeiffer: Biosynthese und Sekretion von Insulin nach Behandlung von Langerhansschen Inseln mit Neuraminidase (abstract). Hoppe-Seyler's Z. physiol. Chem. 1546-1547 (1972) Pfeiffer. E.F.. S. Raptis: Controlled extension of oral antidiabetic therapy on former insulin dependent diabetics by means of the combined Lv. glibenclarnide-glucose response test. Diabetologia 8: 41-47 (1972)
265
Secretory B-Cell Activity in Insulin Dependent Maturity-Onset Diabetic U. Krause, G. Faisst*, U. Cordes and J. Beyer
Summuy at least not convincing (Heding 1972). Determination of serum C-Peptide levels seems to be a more In 10 insulin dependent maturity onset diabetics we found elevated basal C-Peptide levels (4.78 ± 0.5 ng/mL Normal valuable tool for measurement of the endoerine range 1.1-3.6 ng/ml), which could be suppressed by insulin panereas funetion. Different radioimmunoassay sysinjection to the same extent as in sulfonylurea treated diabetems for C-Peptide have been developed in the past ties could be demonstrated. C-Peptide immunoreactivity in in the laboratories of Beischer (Beischer, Raptis, these patients therefore seems to be newly secreted rather Keller, Heinze, Schröder and Pfeiffer 1975), Heding than accumulated material Since adrenalectomized patients could be suppressed in the same way, it is Iikely, that cate(Heding 1975), Kaneko (Kaneko, Olea, Munemura, cholamines are not the major factor in the mechanism of Oda, Yamashita, Suzuki, Yanaihara, Hashimoto and suppression. Therefore only decrease of bloodsugar levels Yanaihara 1974) and Rubenstein (Rubenstein, Clark. seems to be accountable for the decrease of C-Peptide levels. Melani and Steiner 1969). In previous studies we and High C-Peptide levels in insulin dependent maturity onset diabetics which cannot be stimulated but suppressed may other investigators reported on some maturity onset be explained by a lass of g1ucoreceptor molecules. insulin dependent diabetics having normal or even
Key-Worda: C·Peptide - Glucoreceptor - Adrenalectomy Rtlidual Secretion - Insulin Induced Hypoglycaemilz
Introduction Tbe proinsulin moleeule is converted in the B-cell by specific proteases to insulin and C-Peptide (Kemmler, Peterson, Rubenstein and Steiner 1972; Kemmler, Peterson and Steiner 1971; Kemmler and Steiner 1970). Thus C-Peptide is found to be seereted in equimolaI amounts with insulin into the bloodcireulation (Rubenstein, Block, Sta", Melani and Steiner 1972; Heding 1975). In the peripheral blood however we found a three to fivefold level of C-Peptide on a molar basis (Krause, Cordes and Beyer 1977) due to the different degradation of the two proteins (Faber, Markussen and Naithani 1976; Kuzuya and Matsuda 1976). Seeretion of C-Peptide is therefore an indieator of the endoerine pancreas function. In a radioimmunological assay system CPeptide does not crossreact with insulin (Yanaihara, Hashimoto, Yanaihara, Saleagami, Stein er and Rubenstein 1974). This enab1es the measurement of endoerine panereas function even in insulin dependent diabetics. A few attempts have been made for determination of insulin levels in sera of such patients, but the methods were rather complicated and the results "'Part of doctoral thesis, Mainz 1978, Germany Received: 18 Jan. 1978 0018-5043/79
0432-0261
Accepted: 29 Aug. 1978 S 03.00
elevated basal C-Peptide levels, indicating that there must be a residual function of the B-cells (Block, Mako. Steiner and Rubenstein 1972; Heding and Rasmussen 1975; Beischer, Keller, Maas, Schiefer and pteiffer 1976; Krause, Beyer, Cordes and Happ 1976). In some of these patients C-Peptide secretion is stimulated by intravenous injection of glibenclamide glucose (Pfeiffer and Raptis 1972), while others showed an uninfluenced secretion pattern. One explanation for high C-Peptide levels, which are not stimulated may be a delayed degradation and elimination of the peptide by these patients. This is sureIy true for individuals with renal funetion impairment, sinee C-Peptide is mainly degraded and eliminated in the hidneys (Rubenstein et al. 1972). Another explanation may be the binding of proinsulin to cireulating insulin antibodies which proteet proinsulin from degradation. Accumulated proinsulin may then mimic high C-Peptide levels beeause of a eertain crossreaction in the C-Peptide assay (Yanaihara et al. 1974). In this ease any regulation of C-Peptide seeretion should not be possible. In metabolie normal subjects suppression of C-Peptide seeretion is achieved by insulin induced hypoglycemia. The aim of this study was to clarify whether insulin dependent diabetics with normal or eIevated C-Peptide levels can be suppressed by insulin injeetion. Furthermore we want to elucidate if epi-
© 1979 Georg Thieme Publishers
Downloaded by: University of Illinois. Copyrighted material.
Abteilung tür Klinische Endokrinologie. 11. Med. Klinik und Poliklinik der Universität Mainz. Mainz. Germany
262
U. Krause, G. Faisst, U. Cordes and J. Beyer
After an overnight Casting period bloodsugar was lowered in Ofo 32 metabolie normal subjects, 10 insulin dependent maturity onset diabeties, 10 sulfonylurea treated maturity-onset dia- 100 beties, and 6 bilaterally adrenaleetomized non-diabeties by LV. injeetion of 0.15 I.U. insulin (MC-Aetrapid, Novo) per kg bodyweight exeept the insulin dependent patients whieh 90 got the 1.5-fold dose of their daily requirement. None of the patients had any signs of renal dysfunetion as assessed 80 by measurement of serum urea and creatinine levels and ereatinine elearance as weil. Only one of the insulin dependent diabetics had eireulating insulin antibodies as assessed 70 by a simple radioaetive ineubation method. Insulin dependeney was diagnosed by means of the g1ibenelamide-glucose test (Pfeiffer and Raptis 1972). Adrenaleetomjzed patients 60 had undergone adrenaleetomy because of Cushing's disease and were substituted with cortisone. Cortisol levels as assessed by radioimmunoassay were within the normal range. Ven50 ous blood was used for determination of bloodsugar and CPeptide. Bloodsugar was measured enzymatically (GODmethod) in an automatie analyzer. Reagents were from Boeil- 40 ringer, Mannheim. C-Peptide was measured radioimmunologicaUy with a double antibody method as described by Kane30 ko et aJ. (1974) with slight modifications. Reagents were from Byk-Mallinekrodt. Standards were run in triplicate, sampies and eontrol sera in duplieate. Intraassay varianee 20 was 4,4% and interassay varianee was 11.5% under our eonditions. Minimum deteetion limit was 0.3 ng/mI.
..- -.. C'Peptide
10 .----•••• b lood'g I ucos e
Results In sera of six totally duodenopancreatectomized patients we were unable to measure any C-Peptide even after stimulation with 0.5 g arginine/kg bodyweight given intravenously (Thiel, Krause, Mangold, Wolf and Beyer 1977). Table 1 summarizes the results of C-Peptide and bloodsugar determinations du ring the experiments. Starting bloodsugar levels of normal subjects were the same like of adrenalectomized non-diabetic patients, whereas the C-Peptide starting levels were higher in adrenaleetomized patients than in normals. Sulfonylurea treated patients exhibit highest C-Peptide starting levels. Insulin dependent patients seem
o
60
30
90
120
Fig. 1 Bloodsugar and C-Peptide decrease after Lv. injection of 0.15 I.U. insulin per kg bodyweight to metabolie normal persons
After insulin injeetion, metabolie healthy subjects show a rapid deerease of bloodsugar and C-Peptide as weIl during the first 30 minutes (Fig. 1). After 30 minutes bloodsugar increases whereas C-Peptide shows a further decrease. In the early phase the two eurves are nearly eongruent. Fifty percent of the basal C-Peptide level is reaehed after 29 minutes and of the bloodsugar level after 26 minutes.
Thble 1 C·Peptide (upper Iines) and Bloodglucose (Iower lines) deerease after intravenous insulin injeetion (± S.E.M.) Time
0
Insulin dependent diabetics Sulfonylurea treated diabetics
4.78 197 5.27 138
± ±
Adrenaleetomized non diabetics Metabolie heaIthy subjeets
10
20
4.49 168
± ±
0.5 19 0.5 10
3.72 85
± ±
0.8 11
3.06 61
± ±
0.7 15
2.40 84
± ±
0.2 2
2.62 65
± ±
0.3 3
± ±
0.5 19
4.14 ± 0.6 119 ± 22 4.45 '± 0.4 96 ± 10
30 ± ±
2.62 ± 0.5 44 ± 10
3.91 101 3.98 67 2.14 37
± ±
0.6 18 0.4 13 0.5 8
1.91 ± 0.2 41 ± 3
1.13 36
± ±
0.1 2
± ±
120
90
60
3.22 74
± ±
004 11
1.94 39
± ±
0.3 4
3.11 ± 0.5 58 ± 13 2.6 ± 0.4 65 ± 8 1.8 ± 0.2 59 ± 4
0.8 51
± ±
0.1 2
0.73 58
3.46 ± 0.5 65 ± 16
± ±
0.1 2
min.
2.83 ± 0.4 ng/ml 25 ± 8 mg/dl 2.46 ± 0.4 ng/ml 81 ± 7 mg/dl 1.64 61
± ±
0.3 ng/ml 8 mg/dl
0.74 69
± ±
0.1 ng/ml 2 mg/dl
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nephrine is involved in the mechanism of suppression to have a little lower basal C-Peptide levels, but because catecholamines cause inhibition of insulin se- there is no significant differenee to the sulfonylurea treated patients. Fasting bloodsugar levels are elevatcretion in vitro and in vivo (Vance, Buchanan and ed in the group of sulfonylurea treated patients, and Williams 1971). higher in the insulin dependent diabeties. Methods and Materials
Beta-Cell Activity and Insulin Dependent Maturity-Onset Diabetes
Ofo 100
90
Ofo
n'S
100
\,
90
70
\\A~
60
\1\
80
80
70
I/'i \~_& ~~A 1// I
50
263
••
60 50
\ ............................... \
40
I
30
30
20
20 &--A
10
C' Pept i de
&--&
10
•.••••• -. blood-glucose
..... ---. blood-glucose
o
30
60
C'Peptide
90
120
Fig. 2 Bloodsugar and C-Peptide decrease after Lv. injection of 0.15 i.V. insulin per kg bodyweight to bilaterally adrenalectomized non diabetic patients
o
30
60
90
120
Fig. 4 Bloodsugar and C-Peptide decrease after i.v. injection of 0.15 i. V. insulin per kg bodyweight to sulfonylurea treated maturity onset diabetics
n'\O
0/0
Bilaterally adrenalectomized patients behave like normals during the first 30 minutes (Fig. 2). Later on bloodsugar shows a delayed inerease in eomparison to the normals. Insulin dependent maturity-onset diabeties show the same rapid decrease of bloodsugar as normals and non diabetie adrenaleetomized patients did (Fig. 3). Fifty percent of the starting bloodsugar level is reached at 30 minutes. There is however a dear dissociation between the bloodsugar curve and the C-Peptide curve. Fifty percent level for C-Peptide is reaehed after 120 minutes.
100
90 80
70 60
Sulfonylurea treated patients show during the first 30 minutes the same rapid bloodsugar decrease with a fifty percent level at 29 minutes (Fig. 4). C-Peptide eurve is intermediate between normals and insulin dependent diabeties. Fifty percent CPR-Ievel is reached after about 87 minutes. Bloodsugar inereases after about 90 minutes.
50
40 30
20
Discussion 10
C-Pepllde
CPR-Ievels in insulin dependent diabeties have been attributed to antibody-bound proinsulin-Iike material (Fink, Cresto, Gutman, Lalline, Rubenstein and Recant 1974). However Beischer, Raptis, Keller, Maas, 30 60 120 o 90 Fig. 3 Bloodsugar and C-Peptide decrease in maturity onset Beischer, Feilen andPfeiffer (1978) found high amounts insulin dependent diabetics after i.v. injection of thc 1.5-fold of proinsulin-like material only in juvenile type diabedose of insulin of their daily requirement ties du ring a remission phase. We and other investiga&--&
.-------. bl ood -g I u cos e
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40
264
U. Krause, G. Faisst, U. Cordes and J. Beyer
tors eould show in the past, that C-Peptide levels in several insulin dependent maturity onset diabeties eould be stimulated by food intake and some other agents like glibenclamide-glueose (Krause et al. 1976) and glucagon as weil (Faber and Binder 1977). Such stimulation eannot be explained by antibody-bound proinsulin. If in insulin treated diabetics normal or high C-Peptide levels should be mimiced by antibodybound proinsulin-like material, these antibodies must have a much higher affmity to proinsulin-like material than to insulin itself, because otherwise exogenous insulin will eause an in vivo displacement from the antibodies, and aeeumulation cannot take place. Or proinsulin-like material must be secreted in such an excess that daily insulin doses will not
ty onset diabeties, seerete high amounts of CPR. Sinee we do not know, if it is really C-Peptide or proinsulin· like material, whieh crossreacts in our assay system, we eannot deeide, if the missing biological effeet of the seereted material is due to a peripheral resistance or to a missing biological poteney.
Acknowledpment The authors are greatly indebted for skillful technical assistance to Mn. I. Bemer and to Mn. I. Knoll.
References
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Beischer, W., S. Raptis, L. Keller, M. Maas, B. BelIeher, K. Feilen, E.F. Pfeiffer: Humanes C-Peptid, Teil 111. Sekretionsdynarnik der Beta-Zellen erwachsener Diabetiker nach Glibenclamid-Glucose Lv. Klin. Wschr. 56: 111120 (1978) Beischer, W., L. Keller, M. Maas, E. Schiefer, E.F. Pfelffer: Human C-Peptide, part I: radioimmunoassay. Klin. Wschr. 54: 709-715 (1976) Belseher, W., S. Raptis, L. Keller, E. Helnze, K.E. Sehröder, E.F. Pfeiffer: Characterization of residual beta-cell funetion in diabetes by'a new C-peptide radioimmunoassay. Diabetologia 11: 332 (1975) Block, M.B., M.E. Mako, D.F. Steiner, A.H. Rubenstein: Circulating C-peptide immunoreactivity-studies in normal and diabetic patients. Diabetes 21: 1013-1026 (1972) BotteTmll~n, P., Th. Zilker, R.. Er'!'ler: C-Peptide secret.ion accordmg to glucose and msulin levels (abstract). Dlllbetologia 12: 382 (1976) Faber, O.K., C. Binder: C-Peptide response to glucagon. A test for the residual beta-cell function in diabetes mellitus. Diabetes 26: 605-610 (197.1). ... Fab.er, O.K., ": Marku~sen, V.K. Naltham: C-Peptlde kmetlcs . " . m man. Dlllbetologlll 12: 389 (1976) (abstract) mg a stop of C-Peptlde seeretlon (Bottermann, ZrlFink, G., J.c. Cmto, R.A. Gutman, R.L. Lavlne, A.H. Ru. ker and Ermler 1976). Moreover, reeent results from benstein, L. Recant: Plasma proinsulin-like material in Schatz and Pfeiffer (1977) indieate, that insulin seereinsulin treated diabetics. Horm. Metab. Res. 6: 439 tion from isolated islets of Langerhans is not sup(~974) . . Hedmg, ~. G., .S.M. ~asmus~en: Hu~n C-peptlde m normal Pressed by exogenous insulin in eoncentrations up and dlllbetlC subJects. DlllbetologJa 11: 201-206 (1975) to 5 mV/mI. Heding, L.G.: Radioimmunological determination of human C-Peptide in serum. Diabetologia 11: 541-548 (1975) From our results there is strong evidence, that epiHeding, I.G.: Determination of total serum insulin (lRI) in nephrine is not involved in the mechanism since insulin treated diabetic patients. Diabetologia 8: 260adrenalectomized patients behave like normals con266 (1972) lkeda, Y., N. Tajima, N. Minami, Y. lde, M. Abe: Pancreaceming the CPR-suppression. tic b-cell function of young diabetics assessed by C-Pe~ The different behaviour of CPR-decreases at identitide immunoreactivity. Jikeikai Med. J. 24: 113-122 cal bloodglucose fall may be at least partially ex(1977) Kaneko, T., H. Oka, M. Munemura, T. Od4, K. Yamashlta, plained by the alte red sensitivity of the B-ceil to S. Suzukl, N. Yanaihara, T. Hashlmoto, C. Yanaihara: changes in bloodglueose levels in diabetics. A loss of Radioimmunoassay of human proinsulin C-Peptide using glucoreceptor molecules would fit this fmding (Krausynthetic human connecting peptide. Endocrin. jap. 21: se, Puchinger and Wacker 1973; Mayer and Pfeiffer 141-145 (1974) 1972). Kemmler, W., J.D. Peterson, A.H. Rubensteln, D.F. Steiner: On the biosynthesis, intracellular transport and mechanOur finding, that there is a further C-Peptide decrease ism of conversion of proinsulin to insulin and C-Peptide. while bloodglucose is already inereasing is surprising. Diabetes 21 , Suppl. 2: 572-582 (1972) Kemmler, W., J.D. Petersen, D.F. Steiner: Studie! on the conWe can only speeulate that there exists a protective version of proinsulin to insulin. Conversion in vitro with mechanism whieh inhibits insulin secretion after hytrypsin and carboxypeptidase b. J. bio!. Chern. 246: 6786poglycemia. This point needs further investigation. 6790 (1971) An open question is the biological signifieance of Kemmler. w., D.F. Steiner: Conversion of proinsulin to insuliO in a subcellular fraction frorn rat islet. Biochem. Bioour fmding, that there are insulin dependent maturiphys. Res. Commun. 41: 1223-1230 (1970)
displaee significant amounts of proinsulin-like material from the antibodies. Our insulin dependent patients, exeept one, did not have significant insulin antibody levels. None of them had signs of renal fallure. Therefore it is most likely that normal or high C-Peptide levels in these patients represent newly seereted rather than aeeumulated material. Our finding, that CPR-Ievels in insulin dependent maturityonset diabeties ean be lowered by insulin indueed M · bl00dglueose deerease support thi s assumptlon. oreover, this residual funetion seems to exert metabolie effeets as reported by Faber and Binder (1977) and reeently by Ikeda, Tajima, Minami, Ide andAbe(1977). fu th tor on e t The me · The~ fimdmgs. r er supp.or u e .c p . ehanlsm by whieh suppresslOn ceurs IS not known exactly. Since C-Peptide levels and bloodsugar levels are strongly eorrelated over a wide range it is probable that low bloodglueose is the only factor eaus-
Hyperthyroidism, Insulin and Metabolism
Rubenstein. A.H., J.L. Clark, F. Melani, D.F. Steiner: Secretion of proinsulin C-peptide by pancreatic betacell and its circulation in blood. Nature 244: 697 -699 (1969) Rubenstein, A.H.• M.B. Block, J. Sta", F. Melani. D.F. Steiner: Proinsulin and C-peptide in blood. Diabetes 21: Suppl. 2,661-672 (1972) Schatz, H., E.F. Pfeiffer: Release of immunoreactive and radioactively prelabelled endogenous (pro )insulin from isolated rat islets in the presence of exogenous insulin. J, Endocrinol. 74: 243-249 (1977) Thiel, M., U. Krause, G. Mangold. E. Wolf, J. Beyer: Untersuchungen zur Sekretion des Enteroglukagon bei Patienten nach totaler Duodenopankreatektomie. Verh. d. Deutschen Ges. f. innere Medizin 83: 1399-1402 (1977) Vance. J.E.• K.D. Bcchanan, R.H. Williams: Glucagon and insulin release. Influence of drugs affecting the autonomic nervous system. Diabetes 20: 78-82 (1971) Yanaihara. N., ,T. Hashimoto, C. Yanaihara, M. Sakagami, D. F. Stein er, A.H. Rubenstein: Synthesis of human connecting peptide derivatives and their immunological properties. Biochem. Biophys. Res. Commun. 59: 11241130 (1974)
Requests for reprints should be addressed to: Dr. Ulrich Krause, Abteilung f. Klin. Endokrinologie, Bau 5, Langenbeckstr.l, 0-6500 Mainz (Germany)
Horm. Metab. Res. 11 (1979) 265-270
Serum Insulin and Free Fatty Acid Responses to Siow and Quick Graded Hyperglycaemias in Hypothyroid Oogs* Aurora Renauld**, L.L. Andrade***, R.C. Sverdlik, R.R. Rodrfguez** and D.M. Lindental Institute of Physiology. Buenos Aires University Medical School, Buenos Aires, Argentina
Summary The effects of thyroidectomy by radioiodine on blood sugar, serum immunoreactive insulin and serum free faUy acid levels in dogs, in the course of glucose infusion tests whether involving glucose priming-dose (B tests) or not (A tests) were studied. The basal levels of blood sugar and serum insulin were not affected by hypothyroidism; circulating free fatty acids had -The present study was partly reported in abstract from (XI Congreso Latinoamericano de Ciencias Fisiologicas,
Mendoza, Argentina, July 29 - Agust 4, 1973; VIII Congreso Panamericano de EndocrinologIa, Buenos Aires, Argentina, October 27 - November 2, 1974). The study was sponsored by the "Consejo Nacional de Investigaciones CientIficas y Tecnicas", Argentina, Res. Grant no. 2304-d. "Established Investigator at this Council. -- Post-graduated Fellow, University of Buenos Aires, Argentina. Received: I Sept. 1976
Accepted: 2S Aug. 1978
oolB-5043/79
103.00
0432-0265
hardly risen. In the hypothyroid group, the blood sugar promes curing both tests were somewhat higher than normal, and hyperglycaemia was maintained for longer. These abnormalities were restored by Thyroxine therapy. Hypothyroidism brought about a considerable alteration of the dynamics of serum insulin response to glucose. Early during the continuous stimulation, serum insulin levels were normal (A tests) or somewhat higher than normal (B tests). Late during the stimulation or, even, after interruption of glucose infusion quite an intense insulin response was tardiIy observed, which was the major effect of hypothyroidism on the insulin response to continuous stimulation by glucose. The abnormalities of the serum insulin profile were corrected by thyroxine therapy. Foot Note: The following abbreviations will be used throughout the text: BS, blood sugar; IRI, immuno-reactive insulin; FFA, free fatty acids; BEI, butanol-extractable iodine; PBI, protein-bound iodine; T4, thyroxine.
© 1979 Georg Thieme Publishers
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Krause, u., J. Beyer, U. Cordes, J. Happ: Serum-C-Peptidspiegel und Serllminsulin bei Patienten mit Hypoglykämien sowie unterschiedlichen Ober- und Unterfunktionszuständen des Inselsystems. IV. Internationales Donausymposium über Diabetes meUitus, Dubrovnik 1975 Krause, u., U. Cordes, J. Beyer: C-Peptid, Sekretion und Stoffwechsel bei unterschiedlichen Funktionszuständen und Störungen der B-Zellen der Langerhansschen Inseln. Dtsch. med. Wschr. 102: 785-790 (1977) Krause, u., H. Puchinger, A. Wacker: Inhibition of glucose induced insulin secretion in trypsin treated islets of Langerhans. Horm. Metab. Res. 5: 325-329 (1973) Kuzuya, T., A. Matsuda: Disappearance rate of endogenous human C-peptide from blood. Diabetologia 12: 519-521 (1976) Maier, v., E.F. Pfeiffer: Biosynthese und Sekretion von Insulin nach Behandlung von Langerhansschen Inseln mit Neuraminidase (abstract). Hoppe-Seyler's Z. physiol. Chem. 1546-1547 (1972) Pfeiffer. E.F.. S. Raptis: Controlled extension of oral antidiabetic therapy on former insulin dependent diabetics by means of the combined Lv. glibenclarnide-glucose response test. Diabetologia 8: 41-47 (1972)
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