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Fasting and Skeletal Muscle Enzymes in Obese Men Horm. metabol. Res.

15 (1983) 271

Rath, R., K. Vondra: Short-term fasting in the treatment of obesity. Vondra, K., R. Rath: Obesity and thyroid function. 2. The effect
Nahrung 21: 193-197 (1977) of prolonged caloric restriction on Achilles tendon reflex values.
Rath, R., Z. Slabochova, K. Vondra: Immunoreactive insulin in Endokrinologie 66: 332-336 (1975)
obesity of adult women. Intemat J. of Obesity 1: 279-286 Vondra, K., R. Rath, A. Bass: Skeletal muscle HK activity and
(1977) fasting FFA blood level in man. Horm. Metab. Res. 8: 323 (1976)
Vondra, K., R. Rath: Obesity and thyroid function. 1. Values of Vondra, K., R. Rath, A. Bass, L. Kukla, Z. Slabochova: Effect of
the Achilles tendon reflex. Endokrinologie 62: 310-320 (1973) protracted intermittent fasting on the activities of enzymes in-
Vondra, K., R. Rath, Z. Kroupa: Improved needle for muscle volved in energy metabolism, and on the concentration of glyco-
biopsy. Klin. Wschr. 52: 747-748 (1974) gen, protein a DNA in skeletal muscle of obese women. Nutr.
Vondra, K., R. Rath, A. Bass: Activity of some enzymes of energy Metab. 20: 329-337 (1976)
metabolism in striated muscle of obese subjects with respect Vondra, K., A. Bass, V. Brodan, E. Kuhn, M. Andel, A. Veselkova,
to body composition. Horm. Metab. Res. 7: 475-480 (1975) V. Vitek: Activities of muscle energy supplying enzymes after
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(1982)31:311-314(1982)

Requests for reprints should be addressed to: MUDr. K. Vondra, CSc, Department of Medicine I of the Institute for Clinical and Experimental
Medicine, Videnska 800, 146 22 Prague 4 (Czechoslovakia)

Horm. metabol. Res. 15 (1983) 271-274


©Georg Thieme Verlag Stuttgart • New York

Different Potencies of Biosynthetic Human and Purified Porcine Insulin

K.J. Schliiter, F. Enzmann* and L. Kerp


Zentrum fiir Innere Medizin, Abteilung fiir Klinische Endokrinologie, Universitat Freiburg, and
* E l i Lilly, Bad Homburg, Germany

Summary
Introduction
The glucose clamp technique was used to compare the biological
The biological activity of insulin has been assessed by intra-
activity of purified porcine insulin and Biosynthetic Human venous bolus injection, continuous infusion, and applica-
Insulin (BHI). An intravenous bolus of 0.1 U/kg BW was tion of a single subcutaneous dose. Studies show that
injected in eight male volunteers, and the glucose was intravenous bolus injection produce counterregulatory
clamped at baseline values (euglycemic clamp).
hormonal responses which modify the metabolism of
Serum insulin, serum C-peptide and plasma glucose did not glucose. Since differences in the hormonal responses to
differ between porcine and human insulin. The insulin in-
duced glucose consumption differed significantly ( p < 0.007)
human and porcine insulin have been reported, namely that
between purified porcine insulin (50.5 ± 5.2 [SEM] g/2h) and human insulin produced a lower output of growth hormone,
Biosynthetic Human Insulin (63.5 ± 4 . 5 g / 2 h ) . Purified Cortisol, and epinephrine (SchViter, Petersen, Borsche,
porcine Insulin induced a hormonal response w i t h significantly Hobitz and Kerp 1981; SchViter, Petersen and Kerp 1982),
( p < 0.05) elevated concentrations of serum growth hormone
(12.1 ± 0.25 ng/ml) and serum Cortisol (161.4 ± 28.6 ng/ml),
it is difficult to compare the effects on glucose metabolism.
which were not observed following Biosynthetic Human With the glucose clamp technique, essentially normal fasting
Insulin (serum growth hormone: 2.6 ± 0.2 n g / m l ; serum plasma glucose concentrations can be maintained (Pfeiffer,
Cortisol: 117.3 ± 14.8 ng/ml). The data confirm earlier results
indicating hormonal and metabolic differences between
Thum and Clemens 1974; DeFronzo, Tobin and Andres
human and porcine insulin. 1979;Nosadini, Noy, Kurtz and Alberti 1981). Hormonal
responses are minimal under these experimental conditions.
Key-Words: Biosynthetic Human Insulin — Glucose Clamp The effect of Biosynthetic Human Insulin (BHI) and puri-
Technique — Glucose Requirement — Growth Hormone — fied porcine insulin (PPI) on glucose metabolism were meas-
Cortisol
ured using the euglycemic clamp technique to avoid inter-
ference of other hormones.

Subjects and Methods


Informed consent was obtained from 8 male volunteers (age: 24.2
±1.3 years [mean ± SEM], body weight: 75.1 ± 1.3 kg; height: 185.6
Received: 15 March 1982 Accepted: 25 Aug. 1982 ±3.9 cm) without family or personal history of diabetes. Laboratory
272 Horm. metabol. Res. 15 (1983) K.J. Schliiter, F. Enzmann and L. Kerp
chemistry, ECG, physical examination and oral glucose tolerance
tests (100 g) were normal.
In a randomized study, each subject received both Biosynthetic
Human Insulin (BHI) (Eli Lilly, Indianapolis, CT 4969-1B) and
identical formulated purified pork insulin (PPI) (Eli Lilly, Indiana-
polis, CT 4970-OA) intravenously (bolus: 0.1 U/kg BW). The com-
pound of BHI used in this experiment was identical to the BHI (Lot
615-70N-174-9) used in the USP-rabbit hypoglycemia assay. The
biological activity in rabbits was 27.5 ±1.7 units/mg, which was
160 nmoles/mg for BHI and 159 nmoles/mg for PPI.
The tests were performed at one week intervals in this study. One
hour before the administration of insulin, three catheters were in-
serted into antecubital veins. The glucose-controlled insulin infusion
system (Biostator, Life Science Instruments, Miinchen, FRG) was
calibrated for continuous blood glucose monitoring and glucose in-
fusion. The plasma glucose concentration was clamped at individual
fasting baseline levels (± 0.25 mMol/1). The Biostator was pro-
grammed to maintain the individual (76—96 mg/dl) euglycemia of
the subjects. Glucose (40 g/100 ml) was infused through the three
channels (saline-, glucose-, and optional channel) of the Biostator.
The maximum glucose infusion by the Biostator was 2.4 g/min.
After an hour's rest, a bolus of insulin (0.1 U/kg BW) was injected
intravenously. Venous blood samples were obtained at-15, 0, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105 and
120 minutes.
Plasma glucose was determined by the glucose oxidase technique with
a Beckmann glucose analyzer (Beckmann Instruments, Inc., Fullerton,
Calif.).
The following radioimmunoassays were carried out: serum insulin
(Phadebas-Insulintest, Pharmacia-Diagnostics, AB, Uppsala, Sweden)
(the cross-reaction of PPI and BHI with the antibody used in this
RIA was identical in the range from 10 MU/ml to 320 MU/ml), serum
Fig. 1 Plasma glucose (mg/ml), serum insulin (MU/ml), serum
C-peptide (Riamat, C-peptide assay, Byk-Mallinckrodt, Diezenbach,
C-peptide (ng/ml) after an i.v. bolus (0.1 U/kg BW, given at 0
FRG), serum Cortisol (Cortisol-Ria, Travenol, Cambridge, Mass.),
minutes) of Biosynthetic Human Insulin (• •) and purified
and serum growth hormone (Serono, Freiburg, FRG).
pork insulin (o—o) during euglycemic clamp in eight male
Interassay variations were reduced by using the same immunoassay volunteers. The values at each time interval represent the mean
for all samples of an individual subject. Intra-assay error, measured ± SEM of eight samples. There was no statistical significance of
as coefficient of variation, was below 5% in all cases. differences between the porcine and human group.
The data are expressed as mean ± SEM. Areas under the concentra-
tion-time curves were calculated according to the equation:
*f ( x n+l- x n> (vn+l + yn)

L
Wilcoxons test for paired differences was used.

Results
Eight male volunteers received either Biosynthetic Human
Insulin (BHI) (0.1 U/kg BW) or purified porcine insulin
(PPI) during an euglycemic clamp study. Plasma glucose,
serum insulin, serum C-peptide, serum growth hormone,
and serum Cortisol concentrations obtained from the
venous blood samples are shown in Fig. 1 and Fig. 2.
The individual fasting plasma glucose values (BHI: 89.1 ±
1.6 mg/dl;PPI: 90.1 ± 2.7 mg/dl) were maintained through-
out the clamping procedure, but those following Bio-
synthetic Human Insulin were slightly lower than those
following purified porcine insulin. Throughout the experi-
ment the maximum blood glucose fluctuation was 20 mg/dl
and the coefficient of variation of the plasma glucose values Fig. 2 Serum growth hormone and serum Cortisol after an i.v.
was below 15%. bolus (0.1 U/kg BW, given at 0 minutes) of Biosynthetic Human
Insulin (• • ) and purified porcine insulin (o—o) during
Serum insulin concentrations did not differ during the test euglycemic clamp in eight male volunteers. The values at each time
period. Following the intravenous administration identical interval represent the mean ± SEM of eight samples.
X, values that differ significantly from the human insulin group at
peak values (BHI: 309.3 ± 61.0 juU/ml; PPI: 335.2 ± 64.2 that time interval (p< 0.05 by paired Wilcoxon's test).
/xU/ml) of insulin were obtained.
Potencies of Biosynthetic Human and Purified Porcine Insulin Horm. metabol. Res. 15 (1983) 273

Serum C-peptide levels following Biosynthetic Human Chiasson, Keller and Rubenstein 1978; Beischer, Schmid,
Insulin and purified porcine insulin did not differ signifi- Kerner, Keller and Pfeiffer 1978) may be due to this tech-
cantly. A suppression of endogenous insulin secretion by nical difference.
exogenous insulin was not observed (Fig. 1). A significant A small but significant rise in serum growth hormone and
(p< 0.05) elevation of serum growth hormone concentra- serum Cortisol concentrations was observed following the
tion (from 2.8 ± 1.7 ng/ml at 0 minutes to 12.1 ± 6.2 ng/ml injection of a bolus of purified porcine insulin, which was
at 30 minutes) occurred after the administration of puri- not observed after Biosynthetic Human Insulin. This can-
fied porcine insulin. BHI did not produce any fluctuation not be attributed to differences in plasma glucose or to
of serum growth hormone (2.5 ± 1.9 ng/ml at 0 minutes plasma glucose concentrations because plasma glucose was
and 2.5 ± 2.0 ng/ml at 30 minutes). clamped at individual fasting glucose levels. The secretion
Serum Cortisol levels were also elevated (A serum Cortisol of growth hormone and of Cortisol appears to be a response
38.8 ± 8.1 ng/ml) after purified porcine insulin but not to the heterologous insulin.
after human insulin (A serum Cortisol 23.1 ± 4.5 ng/ml) Significantly more exogenous glucose was required to
(p<0.05). compensate the hypoglycemic effect of Biosynthetic Human
The amount of glucose required to compensate the effect Insulin in comparison to purified pork insulin (+ 30.6 ± 7.6
of exogenous insulin was significantly (2p< 0.007) higher percent). In five insulin-dependent diabetic subjects BHI
after Biosynthetic Human Insulin (63.5 ± 4.5 g/2h) than was more (but not significantly) effective than natural pork
after purified porcine insulin (50.4 ±5.2 g/2h). insulin (Klier, Kerner, Torres and Pfeiffer 1981). This dif-
Table 1 shows the individual glucose requirements over the ference which is in contrast to a previous study (Massi-
two-hour periods for each subject. Benedetti, Burrin, Capaldo and Alberti 1981) with the
insulin infusion technique, can be explained by the small in-
crements of endogenous Cortisol and growth hormone ob-
served after porcine insulin. Cortisol and growth hormone
produced hyperglycemia by decreasing both hepatic and
Discussion
extrahepatic sensitivity to insulin (Rizza, Mandarino and
The glucose clamp technique has been used to assess the Gerich 1981; Rizza, Mandarino, Westland and Gerich
biological activity of insulin by continuous infusion (De 1981).
Fronzo, Tobin and Andres \919;Pfeiffer, Thum and
Clemens 1974;Nasadini, Noy, Kurtz and Alberti 1981; On the other hand a significantly inhibited hepatic glucose
Massi-Benedetti, Burrin, Capaldo and Alberti 1981). In this production has been observed after porcine insulin in com-
study, however, a bolus injection of insulin was used. The parison to semisynthetic human insulin (Mutter, Keller and
peak serum insulin concentrations (about 300 /iU/ml in Berger 1982). The weaker suppression of hepatic glucose
comparison to values obtained by continuous infusion of production following porcine insulin, caused by Cortisol
about 50—100 ^U/ml) (Massi-Benedetti, Burrin, Capaldo and growth hormone secretion, might explain the comparab-
and Alberti 19Sl;Dobeme, Schulz and Reaven 1981) per- ly low glucose requirement following porcine insulin in-
sisted for only a short period while continuous infusion jection in comparison to human insulin. The results indi-
results in prolonged elevated levels. The fact that we have cate that homologous insulin produces effects which are
not observed a suppression of endogenous insulin secretion different from those produced by heterologous insulin in
as judged by serum C-peptide (Liljenquist, Horwitz, Jennings, man.

Acknowledgements
Table 1 Glucose requirement (g/2h) after a bolus injection (0.1 U/kg
The authors gratefully acknowledge the skilled technical assistance
BW) of Biosynthetic Human Insulin (BHI) and purified pork insulin
of Mrs. Heike Vorwerck and thank Prof. Dr. John A. Galloway
(PPI) during euglycemic clamp.
(University of Indianapolis) for his comments on the manuscript.
glucose requirement
g/2h
Subject BHI PPI References
No. Beischer, W., M. Schmid, W. Kerner, L. Keller, E.F. Pfeiffer: Does
insulin play a role in the regulation of its own secretion? Horm.
Metab. Res. 10: 168-169 (1978)
De Fronzo, R.A., J.D. Tobin, R. Andres: Glucose clamp technique:
a method for quantifying insulin secretion and resistance. Am.
J. Physiol. 237 (3): E214-E223 (1979)
Doberne, L., M.S. Greenfield, B. Schulz, M. Reaven: Enhanced
glucose utilization during prolonged glucose clamp studies.
Diabetes 30: 829-835 (1981)
Klier, M., W. Kerner, A.A. Torres, E.F. Pfeiffer: Comparison of the
biologic activity of Biosynthetic Human Insulin and natural
pork insulin in juvenile-onset diabetic subjects assessed by the
glucose controlled insulin infusion system. Diabetes Care 4:
*Significance of mean differences, p< 0.007. 193-195 (1981)
274 Horm. metabol. Res. 15 (1983) K J. Schliiter, F. Enzmann and L. Kerp

Liljenquist, J.E., D.L. Horwitz, A.S. Jennings, J.-L. Chiasson, Rizza, R., L. Mandarino, J. Gerich: Dose-response characteristics
U. Keller, A.H. Rubenstein: Inhibition of insulin secretion by for the effects of insulin on production and utilization of
exogenous insulin in normal man as demonstrated by C-peptide glucose in man. Am. J. Physiol. 240: 630-639 (1981)
assay. Diabetes 27: 563-570 (1978) Rizza, R., L. Mandarino, R. Westland, J. Gerich: Growth hormone
Massi-Benedetti, M., J.M.Burrin, B. Capaldo, K.GM.M. Alberti: induced insulin resistance in man: Postreceptor impairment in
A comparative study of the activity of biosynthetic human hepatic and peripheral tissue sensitivity to insulin. Diabetes 30:
insulin and pork insulin using the glucose clamp technique in 38(1981)
normal subjects. Diabetes Care 4:163-167 (1981) Schliiter, K.J., K.-G. Petersen, A. Borsche, H. Hobitz, L. Kerp: Effects
Miiller, R., U. Keller, W. Berger: Comparison of semisynthetic and of fully synthetic human insulin in comparison to porcine insulin
porcine insulin in man. J. Clin. Invest. 12: 281 (1982) in normal subjects. Horm. Metab. Res. 13: 657-659 (1981)
Nosadini, R., G. Noy, A.B. Kurtz, K.G.M.M. Alberti: Differential Schliiter, K.J., K.-G. Petersen, A., L. Kerp: Unterschiedliche Wir-
response to infusions of highly purified and conventional bovine kung von Human- und Schweineinsulin. In: Neue Insuline, eds.
and porcine insulins. Diabetes 30: 650-655 (1981) Petersen, K.-G., K.J. Schliiter, L. Kerp. Freiburg, Freiburger
Pfeiffer, E.F., Ch. Thum, A.H. Clemens: The artificial beta cell - a Graphische Betriebe, 86-92 (1982)
continuous control of blood sugar by external regulation of
insulin infusion (glucose controlled insulin infusion system).
Horm. Metab. Res. 487: 339-342 (1974)

Requests for reprints should be addressed to: K.J. Schliiter, M.D., Zentrum fur Innere Medizin, Universitat Freiburg, Hugstetter Str. 55,
D-7800 Freiburg (Germany)

Horm. metabol. Res. 15 (1983) 274-278


©Georg Thieme Verlag Stuttgart • New York

125
Clinical Factors Influencing the Absorption of I-NPH Insulin in Diabetic Patients

K. Kdlendorf1, J. Bojsen2 and T. Deckert1


1
Steno Memorial Hospital, Gentofte, and
The Finsen Laboratory, The Finsen Institute, Copenhagen, Denmark

Summary
Introduction
Clinical factors which might influence the absorption of sub-
Earlier studies have shown great inter- and intrasubject
cutaneously injected 125 I-NPH insulin were studied in 101 variations for absorption of intermediate-acting insulins
diabetics. The disappearance curve was monoexponential (Binder 1969; Galloway, Spradlin, Nelson, Wentworth,
after a delay period of 1.5±0.8 h (mean ± SD). Lipohyper- Davidson and Swamer 1981; Lauritzen, Faber and Binder
trophy significantly prolonged insulin absorption (half life
( T 1 / 2 ) = 11.2±3.1 h, p = 0.0001 >. Low bicarbonate levels
1979). However, only the influence of age and diabetes
increased the absorption ( T 1 / 2 3.9±2.3 h , p < 0.05). Lean duration on NPH insulin absorption have been systematical-
diabetics had a faster absorption (6.2 ± 1.9 h) than normal ly studied (Dobson, Robbins, Johnson, Mdalel, Odem, Corn-
weight diabetics (7.5 ± 2.0 h, p < 0.02). Sex, age, diabetes wall and Davis 1967). The aim of this study was to assess
duration and injection depth d i d not influence T 1 / 2 . The some clinical factors which might influence the absorption
half life was significantly inversely correlated to the resting
subcutaneous blood f l o w (r= - 0 . 8 8 2 , p < 0.01). The overall
of I25I-NPH insulin from the subcutaneous tissue in diabetic
interindividual coefficient of variation for insulin absorption patients by using the biotelemetric technique. The absorp-
in nonketotic diabetics was 27.4%. Also considerable intra- tion of insulin was measured to determine the influence
patient day-to-day variation was found (24.5%), and between of lipohypertrophy, ketosis, body weight, sex, age, dia-
different injection sites (30.2%). These variations emphasize betes duration and inter- and intrapatient variation. In some
the drawbacks of conventional insulin therapy in the manage-
ment of insulin-requiring diabetics.
diabetics, also the effect of injection depth and subcutane-
ous bloodflow(SBF) was evaluated.
Key-Words: Insulin Absorption — NPH Insulin — Diabetes
Mellitus — Lipohypertrophy — Ketosis — Body Weight — Sex —
Age — Diabetes: Duration — Subcutaneous Blood-Flow — Material and Methods
125
l-NPH Insulin
One hundred and one diabetic patients were investigated as inpatients
in 194 studies after informed consent was obtained. Group 1 com-
prised 20 patients with insulin-dependent diabetes mellitus (IDDM)
Received: IS March 1982 Accepted: 31 July 1982 with palpable lipohypertrophy at the injection site. Group 2 included