Different Potencies Of Biosynthetic Human And Purified Porcine Insulin

Fasting and Skeletal Muscle Enzymes in Obese Men 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)

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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 involved in energy metabolism, and on the concentration of glycogen, 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)

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 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 induced 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

Introduction The biological activity of insulin has been assessed by intravenous bolus injection, continuous infusion, and application 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 purified porcine insulin (PPI) on glucose metabolism were measured using the euglycemic clamp technique to avoid interference of other hormones. Subjects and Methods

Received: 15 March 1982

Accepted: 25 Aug. 1982

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

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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, Indianapolis, CT 4970-OA) intravenously (bolus: 0.1 U/kg BW). The compound 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 inserted into antecubital veins. The glucose-controlled insulin infusion system (Biostator, Life Science Instruments, Miinchen, FRG) was calibrated for continuous blood glucose monitoring and glucose infusion. The plasma glucose concentration was clamped at individual fasting baseline levels (± 0.25 mMol/1). The Biostator was programmed 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 C-peptide (Riamat, C-peptide assay, Byk-Mallinckrodt, Diezenbach, FRG), serum Cortisol (Cortisol-Ria, Travenol, Cambridge, Mass.), and serum growth hormone (Serono, Freiburg, FRG). Interassay variations were reduced by using the same immunoassay for all samples of an individual subject. Intra-assay error, measured as coefficient of variation, was below 5% in all cases. The data are expressed as mean ± SEM. Areas under the concentration-time curves were calculated according to the equation: *f

Fig. 1 Plasma glucose (mg/ml), serum insulin (MU/ml), serum C-peptide (ng/ml) after an i.v. bolus (0.1 U/kg BW, given at 0 minutes) of Biosynthetic Human Insulin (• •) and purified pork insulin (o—o) during euglycemic clamp in eight male volunteers. The values at each time interval represent the mean ± SEM of eight samples. There was no statistical significance of differences between the porcine and human group.

( 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 throughout the clamping procedure, but those following Biosynthetic Human Insulin were slightly lower than those following purified porcine insulin. Throughout the experiment the maximum blood glucose fluctuation was 20 mg/dl and the coefficient of variation of the plasma glucose values was below 15%. Serum insulin concentrations did not differ during the test period. Following the intravenous administration identical peak values (BHI: 309.3 ± 61.0 juU/ml; PPI: 335.2 ± 64.2 /xU/ml) of insulin were obtained.

Fig. 2 Serum growth hormone and serum Cortisol after an i.v. bolus (0.1 U/kg BW, given at 0 minutes) of Biosynthetic Human Insulin (• • ) and purified porcine insulin (o—o) during euglycemic clamp in eight male volunteers. The values at each time interval represent the mean ± SEM of eight samples. X, values that differ significantly from the human insulin group at that time interval (p< 0.05 by paired Wilcoxon's test).

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Serum C-peptide levels following Biosynthetic Human Insulin and purified porcine insulin did not differ significantly. A suppression of endogenous insulin secretion by exogenous insulin was not observed (Fig. 1). A significant (p< 0.05) elevation of serum growth hormone concentration (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 purified 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.

Chiasson, Keller and Rubenstein 1978; Beischer, Schmid, Kerner, Keller and Pfeiffer 1978) may be due to this technical 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 cannot 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 difference which is in contrast to a previous study (MassiBenedetti, Burrin, Capaldo and Alberti 1981) with the insulin infusion technique, can be explained by the small increments of endogenous Cortisol and growth hormone observed 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 On the other hand a significantly inhibited hepatic glucose Clemens 1974;Nasadini, Noy, Kurtz and Alberti 1981; production has been observed after porcine insulin in comMassi-Benedetti, Burrin, Capaldo and Alberti 1981). In this parison to semisynthetic human insulin (Mutter, Keller and study, however, a bolus injection of insulin was used. The Berger 1982). The weaker suppression of hepatic glucose peak serum insulin concentrations (about 300 /iU/ml in production following porcine insulin, caused by Cortisol comparison to values obtained by continuous infusion of and growth hormone secretion, might explain the comparababout 50—100 ^U/ml) (Massi-Benedetti, Burrin, Capaldo ly low glucose requirement following porcine insulin inand Alberti 19Sl;Dobeme, Schulz and Reaven 1981) perjection in comparison to human insulin. The results indisisted for only a short period while continuous infusion cate that homologous insulin produces effects which are results in prolonged elevated levels. The fact that we have different from those produced by heterologous insulin in not observed a suppression of endogenous insulin secretion 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 BW) of Biosynthetic Human Insulin (BHI) and purified pork insulin (PPI) during euglycemic clamp.

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.

glucose requirement g/2h Subject No.

BHI

*Significance of mean differences, p< 0.007.

PPI

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)

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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)

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 Wirkung von Human- und Schweineinsulin. In: Neue Insuline, eds. Petersen, K.-G., K.J. Schliiter, L. Kerp. Freiburg, Freiburger Graphische Betriebe, 86-92 (1982)

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

Clinical Factors Influencing the Absorption of

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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 Clinical factors which might influence the absorption of subcutaneously 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). Lipohypertrophy 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 intrapatient 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 management 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

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 systematically studied (Dobson, Robbins, Johnson, Mdalel, Odem, Cornwall 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 absorption of insulin was measured to determine the influence of lipohypertrophy, ketosis, body weight, sex, age, diabetes duration and inter- and intrapatient variation. In some diabetics, also the effect of injection depth and subcutaneous 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 comprised 20 patients with insulin-dependent diabetes mellitus (IDDM) with palpable lipohypertrophy at the injection site. Group 2 included