Fasting and Skeletal Muscle Enzymes in Obese Men
15 (1983) 271
Rath, R., K. Vondra: Short-term fasting in the treatment of obesity.
Nahrung 21: 193-197 (1977)
Rath, R., Z. Slabochova, K. Vondra: Immunoreactive insulin in
obesity of adult women. Intemat J. of Obesity 1: 279-286
(1977)
Vondra, K., R. Rath: Obesity and thyroid function. 1. Values of
the Achilles tendon reflex. Endokrinologie 62: 310-320 (1973)
Vondra, K., R. Rath, Z. Kroupa: Improved needle for muscle
biopsy. Klin. Wschr. 52: 747-748 (1974)
Vondra, K., R. Rath, A. Bass: Activity of some enzymes of energy
metabolism in striated muscle of obese subjects with respect
to body composition. Horm. Metab. Res. 7: 475-480 (1975)
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)
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
The glucose clamp technique was used to compare the biological
activity of purified porcine insulin and Biosynthetic Human
Insulin (BHI). An intravenous bolus of 0.1 U/kg BW was
injected in eight male volunteers, and the glucose was
clamped at baseline values (euglycemic clamp).
Serum insulin, serum C-peptide and plasma glucose did not
differ between porcine and human insulin. The insulin in-
duced glucose consumption differed significantly ( p < 0.007)
between purified porcine insulin (50.5 ± 5.2 [SEM] g/2h) and
Biosynthetic Human Insulin (63.5 ± 4 . 5 g / 2 h ) . Purified
porcine Insulin induced a hormonal response w i t h significantly
( p < 0.05) elevated concentrations of serum growth hormone
(12.1 ± 0.25 ng/ml) and serum Cortisol (161.4 ± 28.6 ng/ml),
which were not observed following Biosynthetic Human
Insulin (serum growth hormone: 2.6 ± 0.2 n g / m l ; serum
Cortisol: 117.3 ± 14.8 ng/ml). The data confirm earlier results
indicating hormonal and metabolic differences between
human and porcine insulin.
Key-Words: Biosynthetic Human Insulin — Glucose Clamp
Technique — Glucose Requirement — Growth Hormone —
Cortisol
Received: 15 March 1982 Accepted: 25 Aug. 1982
Horm. metabol. Res.
Vondra, K., R. Rath: Obesity and thyroid function. 2. The effect
of prolonged caloric restriction on Achilles tendon reflex values.
Endokrinologie 66: 332-336 (1975)
Vondra, K., R. Rath, A. Bass: Skeletal muscle HK activity and
fasting FFA blood level in man. Horm. Metab. Res. 8: 323 (1976)
Vondra, K., R. Rath, A. Bass, L. Kukla, Z. Slabochova: Effect of
protracted intermittent fasting on the activities of enzymes in-
volved in energy metabolism, and on the concentration of glyco-
gen, protein a DNA in skeletal muscle of obese women. Nutr.
Metab. 20: 329-337 (1976)
Vondra, K., A. Bass, V. Brodan, E. Kuhn, M. Andel, A. Veselkova,
V. Vitek: Activities of muscle energy supplying enzymes after
5 days complete fasting in young men. Physiol. Bohemoslov.
(1982)31:311-314(1982)
Horm. metabol. Res. 15 (1983) 271-274
©Georg Thieme Verlag Stuttgart • New York
Introduction
The biological activity of insulin has been assessed by intra-
venous bolus injection, continuous infusion, and applica-
tion of a single subcutaneous dose. Studies show that
intravenous bolus injection produce counterregulatory
hormonal responses which modify the metabolism of
glucose. Since differences in the hormonal responses to
human and porcine insulin have been reported, namely that
human insulin produced a lower output of growth hormone,
Cortisol, and epinephrine (SchViter, Petersen, Borsche,
Hobitz and Kerp 1981; SchViter, Petersen and Kerp 1982),
it is difficult to compare the effects on glucose metabolism.
With the glucose clamp technique, essentially normal fasting
plasma glucose concentrations can be maintained (Pfeiffer,
Thum and Clemens 1974; DeFronzo, Tobin and Andres
1979;Nosadini, Noy, Kurtz and Alberti 1981). Hormonal
responses are minimal under these experimental conditions.
The effect of Biosynthetic Human Insulin (BHI) and puri-
fied porcine insulin (PPI) on glucose metabolism were meas-
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
±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
Serum C-peptide levels following Biosynthetic Human
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
exogenous insulin was not observed (Fig. 1). A significant
(p< 0.05) elevation of serum growth hormone concentra-
tion (from 2.8 ± 1.7 ng/ml at 0 minutes to 12.1 ± 6.2 ng/ml
at 30 minutes) occurred after the administration of puri-
fied porcine insulin. BHI did not produce any fluctuation
of serum growth hormone (2.5 ± 1.9 ng/ml at 0 minutes
and 2.5 ± 2.0 ng/ml at 30 minutes).
Serum Cortisol levels were also elevated (A serum Cortisol
38.8 ± 8.1 ng/ml) after purified porcine insulin but not
after human insulin (A serum Cortisol 23.1 ± 4.5 ng/ml)
(p<0.05).
The amount of glucose required to compensate the effect
of exogenous insulin was significantly (2p< 0.007) higher
after Biosynthetic Human Insulin (63.5 ± 4.5 g/2h) than
after purified porcine insulin (50.4 ±5.2 g/2h).
Table 1 shows the individual glucose requirements over the
two-hour periods for each subject.
Discussion
The glucose clamp technique has been used to assess the
biological activity of insulin by continuous infusion (De
Fronzo, Tobin and Andres \919;Pfeiffer, Thum and
Clemens 1974;Nasadini, Noy, Kurtz and Alberti 1981;
Massi-Benedetti, Burrin, Capaldo and Alberti 1981). In this
study, however, a bolus injection of insulin was used. The
peak serum insulin concentrations (about 300 /iU/ml in
comparison to values obtained by continuous infusion of
about 50—100 ^U/ml) (Massi-Benedetti, Burrin, Capaldo
and Alberti 19Sl;Dobeme, Schulz and Reaven 1981) per-
sisted for only a short period while continuous infusion
results in prolonged elevated levels. The fact that we have
not observed a suppression of endogenous insulin secretion
as judged by serum C-peptide (Liljenquist, Horwitz, Jennings, man.
Table 1 Glucose requirement (g/2h) after a bolus injection (0.1 U/kg
BW) of Biosynthetic Human Insulin (BHI) and purified pork insulin
(PPI) during euglycemic clamp.
glucose requirement
g/2h
Subject BHI PPI
No.
*Significance of mean differences, p< 0.007.
Horm. metabol. Res. 15 (1983) 273
Chiasson, Keller and Rubenstein 1978; Beischer, Schmid,
nical difference.
A small but significant rise in serum growth hormone and
serum Cortisol concentrations was observed following the
injection of a bolus of purified porcine insulin, which was
not observed after Biosynthetic Human Insulin. This can-
not be attributed to differences in plasma glucose or to
plasma glucose concentrations because plasma glucose was
clamped at individual fasting glucose levels. The secretion
of growth hormone and of Cortisol appears to be a response
to the heterologous insulin.
Significantly more exogenous glucose was required to
compensate the hypoglycemic effect of Biosynthetic Human
Insulin in comparison to purified pork insulin (+ 30.6 ± 7.6
percent). In five insulin-dependent diabetic subjects BHI
was more (but not significantly) effective than natural pork
insulin (Klier, Kerner, Torres and Pfeiffer 1981). This dif-
ference which is in contrast to a previous study (Massi-
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
extrahepatic sensitivity to insulin (Rizza, Mandarino and
Gerich 1981; Rizza, Mandarino, Westland and Gerich
1981).
On the other hand a significantly inhibited hepatic glucose
production has been observed after porcine insulin in com-
parison to semisynthetic human insulin (Mutter, Keller and
Berger 1982). The weaker suppression of hepatic glucose
production following porcine insulin, caused by Cortisol
and growth hormone secretion, might explain the comparab-
ly low glucose requirement following porcine insulin in-
jection in comparison to human insulin. The results indi-
cate that homologous insulin produces effects which are
different from those produced by heterologous insulin in
Acknowledgements
The authors gratefully acknowledge the skilled technical assistance
of Mrs. Heike Vorwerck and thank Prof. Dr. John A. Galloway
(University of Indianapolis) for his comments on the manuscript.
References
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:
193-195 (1981)
274 Horm. metabol. Res. 15 (1983)
Liljenquist, J.E., D.L. Horwitz, A.S. Jennings, J.-L. Chiasson,
U. Keller, A.H. Rubenstein: Inhibition of insulin secretion by
exogenous insulin in normal man as demonstrated by C-peptide
assay. Diabetes 27: 563-570 (1978)
Massi-Benedetti, M., J.M.Burrin, B. Capaldo, K.GM.M. Alberti:
A comparative study of the activity of biosynthetic human
insulin and pork insulin using the glucose clamp technique in
normal subjects. Diabetes Care 4:163-167 (1981)
Miiller, R., U. Keller, W. Berger: Comparison of semisynthetic and
porcine insulin in man. J. Clin. Invest. 12: 281 (1982)
Nosadini, R., G. Noy, A.B. Kurtz, K.G.M.M. Alberti: Differential
response to infusions of highly purified and conventional bovine
and porcine insulins. Diabetes 30: 650-655 (1981)
Pfeiffer, E.F., Ch. Thum, A.H. Clemens: The artificial beta cell - a
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)
Clinical Factors Influencing the Absorption of
K. Kdlendorf1, J. Bojsen2 and T. Deckert1
1
Steno Memorial Hospital, Gentofte, and
The Finsen Laboratory, The Finsen Institute, Copenhagen, Denmark
Summary
Clinical factors which might influence the absorption of sub-
cutaneously injected 125 I-NPH insulin were studied in 101
diabetics. The disappearance curve was monoexponential
after a delay period of 1.5±0.8 h (mean ± SD). Lipohyper-
trophy significantly prolonged insulin absorption (half life
( T 1 / 2 ) = 11.2±3.1 h, p = 0.0001 >. Low bicarbonate levels
increased the absorption ( T 1 / 2 3.9±2.3 h , p < 0.05). Lean
diabetics had a faster absorption (6.2 ± 1.9 h) than normal
weight diabetics (7.5 ± 2.0 h, p < 0.02). Sex, age, diabetes
duration and injection depth d i d not influence T 1 / 2 . The
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
interindividual coefficient of variation for insulin absorption
in nonketotic diabetics was 27.4%. Also considerable intra-
patient day-to-day variation was found (24.5%), and between
different injection sites (30.2%). These variations emphasize
the drawbacks of conventional insulin therapy in the manage-
ment of insulin-requiring diabetics.
Key-Words: Insulin Absorption — NPH Insulin — Diabetes
Mellitus — Lipohypertrophy — Ketosis — Body Weight — Sex —
Age — Diabetes: Duration — Subcutaneous Blood-Flow —
125
l-NPH Insulin
Received: IS March 1982 Accepted: 31 July 1982
K J. Schliiter, F. Enzmann and L. Kerp
Rizza, R., L. Mandarino, J. Gerich: Dose-response characteristics
for the effects of insulin on production and utilization of
glucose in man. Am. J. Physiol. 240: 630-639 (1981)
Rizza, R., L. Mandarino, R. Westland, J. Gerich: Growth hormone
induced insulin resistance in man: Postreceptor impairment in
hepatic and peripheral tissue sensitivity to insulin. Diabetes 30:
38(1981)
Schliiter, K.J., K.-G. Petersen, A. Borsche, H. Hobitz, L. Kerp: Effects
of fully synthetic human insulin in comparison to porcine insulin
in normal subjects. Horm. Metab. Res. 13: 657-659 (1981)
Schliiter, K.J., K.-G. Petersen, A., L. Kerp: Unterschiedliche Wir-
kung von Human- und Schweineinsulin. In: Neue Insuline, eds.
Petersen, K.-G., K.J. Schliiter, L. Kerp. Freiburg, Freiburger
Graphische Betriebe, 86-92 (1982)
Horm. metabol. Res. 15 (1983) 274-278
©Georg Thieme Verlag Stuttgart • New York
125
I-NPH Insulin in Diabetic Patients
Introduction
Earlier studies have shown great inter- and intrasubject
variations for absorption of intermediate-acting insulins
(Binder 1969; Galloway, Spradlin, Nelson, Wentworth,
Davidson and Swamer 1981; Lauritzen, Faber and Binder
1979). However, only the influence of age and diabetes
duration on NPH insulin absorption have been systematical-
ly studied (Dobson, Robbins, Johnson, Mdalel, Odem, Corn-
wall and Davis 1967). The aim of this study was to assess
some clinical factors which might influence the absorption
of I25I-NPH insulin from the subcutaneous tissue in diabetic
patients by using the biotelemetric technique. The absorp-
tion of insulin was measured to determine the influence
of lipohypertrophy, ketosis, body weight, sex, age, dia-
betes duration and inter- and intrapatient variation. In some
diabetics, also the effect of injection depth and subcutane-
ous bloodflow(SBF) was evaluated.
Material and Methods
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)
with palpable lipohypertrophy at the injection site. Group 2 included