Effects of fully synthetic human insulin in comparison to porcine insulin in normal subjects

Fully synthetic human insulin (CGP 12'831) was compared to porcine insulin in identical and non-identical formulation by intravenous insulin tolerance tests in 12 volunteers. The half-lives of the three insulins tested did not differ (t 1/2: 5.5 +/- 0.2 minutes), though acid porcine insulin exhibited lower serum peak values. The hypoglycemic effects of the three insulins were identical. Human insulin produced a significantly smaller decrease in serum potassium (2p less than 0.01). The secretion of serum C-peptide was less inhibited by human insulin (2p less than 0.05). The counter-regulatory hormonal response of cortisol and growth hormone was lower after hypoglycemia induced by human insulin (2p less than 0.05). It is suggested that the hormonal effects of hypoglycemia are modified by human insulin and depend in part on the molecular structure of insulin.     

Effects of fully synthetic human insulin in comparison to porcine insulin in normal subjects

Fully synthetic human insulin (CGP 12'831) was compared to porcine insulin in identical and non-identical formula by intravenous insulin tolerance tests in 12 volunteers. The half-lives of the three insulins tested did not differ (t 1/2: 5.5 +/- 0.2 minutes), though acid porcine insulin exhibited lower serum peak values. The hypoglycemic effects of the three insulins were identical. Human insulin produced a significantly smaller decrease in serum potassium (2p less than 0.01). The secretion of serum C-peptide was less inhibited by human insulin (2p less than 0.05). The counter-regulatory hormonal response of cortisol and growth hormone was lower after hypoglycemia induced by human insulin (2p less than 0.05). It is suggested that the hormonal effects of hypoglycemia are modified by insulin and depend in part on the molecular structure of insulin.     

Recombinant human insulin.

Insulin is a well-characterized peptide that can be produced by recombinant DNA technology for human therapeutic use. A brief overview of insulin production from both traditional mammalian pancreatic extraction and recombinant bacterial and yeast systems is presented, and detection techniques, including electrophoresis, are reviewed. Analytical systems for insulin separation are principally based on reversed-phase chromatography, which resolves the deamidation product(s) (desamido insulin) of insulin, proinsulin, and insulin. Process-scale separation is a multistep process and includes ion exchange, reversed-phase, and size exclusion chromatography. Advantages and/or disadvantages of various separation approaches, as described by the numerous literature references on insulin purification, are presented.     

NovoSol Basal: pharmacokinetics of a novel soluble long acting insulin analogue.

OBJECTIVE--To determine the courses of absorption and the interindividual and intraindividual variations in absorption of iodine-125 labelled Ultratard HM and NovoSol Basal injected subcutaneously. DESIGN--Open randomised crossover study. Each patient was tested during two study periods of five days each, during which he or she received a subcutaneous injection of either 125I-NovoSol Basal or 125I-Ultratard HM on four consecutive days. The aim was to detect a reduction in intraindividual standard deviation by a factor of two with a probability 0.95, taking 0.05 as the level of significance. This required 24 degrees of freedom and led to the choice of four courses in each of eight patients. SETTING--Referrals to the diabetes research centre in Hvidøre, Copenhagen. PATIENTS--Eight insulin dependent (type I) diabetics with low or undetectable C peptide concentrations who were receiving a multiple insulin injection regimen. One patient withdrew immediately after recruitment. INTERVENTIONS--After an overnight fast patients received 96 nmol (16 IU insulin) of either 125I-NovoSol Basal or 125I-Ultratard HM injected subcutaneously into the thigh. To ensure that the insulin entered the subcutaneous fat at the same depth, ultrasonography was performed on each patient before the first injection. A different injection site on the thigh was used each day for four days in order to facilitate monitoring of the disappearance of four different depots in each patient. MAIN OUTCOME MEASURE--Residual activity at the injection site was measured roughly every two hours throughout the day. No radioactivity measurements were performed overnight (10 pm till 8 am). The residual radioactivity after the injection on the first day (upper right thigh) was recorded for five days, that after the injection on the second day (upper left thigh) for four days, after the injection on the third day (lower right thigh) for three days, and after the last injection (lower left thigh) for two days. RESULTS--NovoSol Basal was absorbed according to first order kinetics with a mean t50% of 35.3 (SEM 1.4) hours; Ultratard HM was absorbed after a lag phase and the corresponding t50% was 25.5 (2.5) hours. The intraindividual variations in t50% were significantly smaller with NovoSol Basal than with Ultratard HM (18.4% v 44.5%; p less than 0.001). Interindividual variations, however, were not significantly different (25.2% v 36.9%; p = 0.38). The total variation in t50% was substantially smaller with NovoSol Basal than with Ultratard HM (20.3% v 42.8%). CONCLUSIONS--NovoSol Basal seems to be an appreciable advance over Ultratard HM as a soluble insulin preparation for obtaining reproducible 24 hour insulin concentrations in the blood     

Effects of a long-acting somatostatin analogue on pituitary-adrenocortical secretion in normal human subjects

A potent and long-acting somatostatin analogue, SMS 201-995 (SMS) is currently employed for the treatment of various diseases with hypersecretion of hormones such as acromegaly and gastrinoma. The suppressive effects of SMS are also reported on the other pituitary and gastrointestinal hormones. The corticotropic-adrenocortical axis is a crucial hormonal complex in maintaining normal activity and life itself. In this study, the effects of SMS on corticotropic-adrenocortical functions were studied, since the effects of SMS on this hormonal axis are not well established. Seven normal males received a sc injection of 100 micrograms SMS or placebo at 0830 h and 100 micrograms synthetic human corticotropin-releasing hormone (hCRH) intravenously (SMS-hCRH study). Five of the 7 subjects were given an injection of a synthetic (1-24) ACTH (250 micrograms or 63 micrograms) at 0900 h after 100 micrograms SMS or a placebo at 0830 h (SMS-ACTH study). Blood samples were drawn at -30, 0, 15, 30, 60, 90 and 120 min after the hCRH injection for the determination of ACTH and cortisol in the SMS-hCRH study, and cortisol and aldosterone in the SMS-ACTH study. Although significant rises in plasma ACTH and cortisol levels were observed regardless of the preinjection of SMS, their responses to hCRH were significantly lower with the pretreatment with SMS than without SMS. A significant increase in plasma cortisol and aldosterone was observed in response to synthetic ACTH with both ACTH alone and the combined administration of SMS and ACTH, at either dose of ACTH. However, no significant difference in cortisol and aldosterone secretion was detected with and without SMS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitogenic effect of the insulin analogue glargine in malignant cells in comparison with insulin and IGF-I.

The aim of the study was to investigate if the insulin analogue glargine, with an increased affinity for the IGF-I receptor (IGF-IR), affects the cell growth to a larger extent than human insulin in malignant cells expressing IGF-IRs. The breast cancer cell lines MCF-7 and SKBR-3, and the osteosarcoma cell line SaOS-2 were used. Gene expression was determined by real-time RT-PCR and receptor protein quantified by ELISAs. Receptor phosphorylation was assessed by immunoprecipitation and Western blot. Mitogenic effect was determined as (3)H-thymidine incorporation into DNA. The gene expression of insulin receptor (IR) varied between 4.3-7.5 x 10(-3) and the expression of IGF-IR between 7.7-147.7 x 10(-3) in relation to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Insulin receptor and IGF-IR protein varied between 2.0-4.1 ng/mg protein and 2.0-40.4 ng/mg protein, respectively. The IGF-IR was phosphorylated by IGF-I at a concentration of 10(-10)-10(-9) M. All three polypeptides stimulated DNA synthesis in MCF-7, SKBR-3, and SaOS-2 cells. SaOS-2 cells were more sensitive to IGF-I than to insulin and glargine. MCF-7 cells were more sensitive to des(1-3)IGF-I than to IGF-I. In SKBR-3 and SaOS-2 cells, glargine tended to be more potent than human insulin to stimulate DNA synthesis. Our results suggest that glargine, compared to human insulin, has little or no increased mitogenic effect in malignant cells expressing IGF-IRs.     

Insulin receptors in human mononuclear leucocytes. I. Binding and degradation of insulin in normal subjects.

The binding and degradation of 125I-Insulin were investigated in mononuclear leukocytes of normal subjects. The binding data analysis show that the insulin degradation is strictly correlated with the binding of the hormone to its receptors: these data suggest that the binding of insulin to specific receptor is the possible first step for its degradation.     

27-hydroxycholesterol: production rates in normal human subjects.

We attempted to quantitate production of bile acid via the 27-hydroxylation pathway in six human subjects. After bolus intravenous injection of known amounts of [24-14C]cholic acid and [24-14C]chenodeoxycholic acid, each subject underwent a constant intravenous infusion of a mixture of [22, 23-3H]-27-hydroxycholesterol and [2H]-27-hydroxycholesterol for 6;-10 h. Production rate of 27-hydroxycholesterol was calculated from the infusion rate of [2H]-27-hydroxycholesterol and the serum ratio of deuterated/protium 27-hydroxycholesterol, which reached a plateau level by 4 h of infusion. Conversion of 27-hydroxycholesterol to cholic and chenodeoxycholic acids was determined from the 3H/14C ratio of these two bile acids in bile samples obtained the day after infusion. In five of the six subjects, independent measurement of bile acid synthesis by fecal acidic sterol output was available from previous studies. Endogenous production of 27-hydroxycholesterol averaged 17.6 mg/day and ranged from 5.0 to 28.2 mg/day, which amounted to 8.7% (range 3.0;-17.9%) of total bile acid synthesis. On average 66% of infused 27-hydroxycholesterol was converted to bile acid, of which 72.6% was chenodeoxycholic acid.These data suggest that relatively little bile acid synthesis takes place via the 27-hydroxylation pathway in healthy humans. Nevertheless, even this amount, occurring predominantly in vascular endothelium and macrophages, could represent an important means for removal of cholesterol deposited in endothelium.     

Deletion analysis of the human insulin receptor ectodomain reveals independently folded soluble subdomains and insulin binding by a monomeric alpha-subunit

A series of 13 deletions within the extracellular domain of the human insulin receptor delineates the boundaries of subdomains that fold de novo into stable proteins that are efficiently secreted and retain the epitopes required for interaction with two conformation-specific monoclonal antibodies. While most of these proteins fail to bind insulin, a truncation that includes only the alpha-subunit is secreted as a monomer that binds the hormone with an affinity only slightly less than that of the complete heterotetrameric extracellular domain. These results thus demarcate landmarks within the primary sequence which will now guide further analysis of the structure and function of this complex domain of the receptor.     

Recombinant human insulin: X. Simplification of folding of the biotechnological precursor of recombinant human insulin

The folding of biotechnological precursor of human insulin precursor was carried out from solubilized inclusion bodies without a preliminary oxidizing or reducing of Cys residues. The inclusion bodies were dissolved in 8 M urea with addition of 10 mM 2-mercaptoethanol. Hydrophobic cell components were removed from the solution by passing through a neutral weakly hydrophobic sorbent, the solution was five times diluted and refolded upon addition of 0.3 mM cystine for initiation of disulfide rearrangement. The presence of nucleic acids and cell protein impurities does not affect the folding efficiency. The resulting precursor of folded human insulin was purified by metal-chelate affinity chromatography and converted into insulin by two-stage enzymatic cleavage.     

DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains — BALB/c, C3H/He and C57BL/10.     

Biological activity of nasally administered insulin in normal subjects

Nasally administered (IN) insulin has been advocated as a potentially useful alternative to subcutaneously administered regular insulin because of its more rapid onset and time to peak action and its shorter duration of action. This study further defines the pharmacodynamics of IN insulin by using a euglycemic clamp technique to determine the bioavailability of IN insulin as compared with intravenous (IV) insulin, and to ascertain whether multiple sequentially administered doses of IN insulin alter pharmacodynamics. Eight normal volunteers received 2 control doses of IV insulin (0.05 U/kg), and 3 high doses (0.7 U/kg) and 3 low doses (0.35 U/kg) of IN insulin with an absorption enhancer (tauro-24,25 dihydrofusidate) given sequentially over a 2 day period. A euglycemic clamp was performed with a Biostator (Ames) that infused dextrose to keep the subject's blood glucose at his fasting level. Analysis of dextrose infusion curves for the low and high doses of IN insulin revealed an onset of action of 9.4 +/- 0.4 and 10.5 +/- 0.3 minutes, time to peak action of 20.6 +/- 5.6 and 23.7 +/- 4.4 minutes and duration of action of 82.1 +/- 5.2 and 95 +/- 5.7 minutes respectively. Both the onset of action and time to peak action were slightly longer (P less than .05) for the high as compared with the low dose IN insulin, although this should not represent a clinically significant difference. The total dextrose requirement was 21.9 +/- 2.3 g for the low dose IN insulin and 34.1 +/- 3.3 g for the high dose IN insulin, the latter value being significantly greater (P less than .01) than the former.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histamine decreases left ventricular contractility in normal human subjects.

To determine whether histamine alters human left ventricular contractility we measured heart rate, calibrated carotid arterial pressure, and left ventricular dimensions (echocardiogram) in nine healthy volunteers. We assessed baseline contractility using the end-systolic pressure-dimension relationship and the end-systolic meridional wall stress-rate-corrected velocity of circumferential fiber shortening relationship determined over a wide range of afterloads using phenylephrine and nitroprusside infusions. We then infused histamine for 3-5 min at a dose predetermined to decrease mean arterial pressure by 20%, both before and after H1 receptor antagonist pretreatment (diphenhydramine 50 mg i.v.). Histamine decreased end-systolic pressure but, unlike an equally hypotensive infusion of nitroprusside, did not decrease end-systolic dimension or increase fractional shortening. Histamine also decreased velocity of circumferential fiber shortening at the same end-systolic meridional wall stress as controls (P < 0.05). These effects of histamine were inhibited by H1 antagonist pretreatment. We conclude that the dominant effect of histamine on the human heart is to decrease left ventricular contractility and that this decrease in contractility is dependent, at least partially, on H1-receptor activation.     

Levels of peripheral blood cell DNA damage in insulin dependent diabetes mellitus human subjects

Increased production of reactive oxygen species (ROS) in vivo can lead to cellular biomolecule damage. Such damage has been suggested to contribute to the pathogenesis of insulin dependent diabetes mellitus (IDDM). In this study, we used the alkaline comet assay to measure DNA damage (single-stranded DNA breaks and alkali-labile sites) in freshly isolated whole blood, lymphocytes, monocytes, and neutrophils from 23 subjects with IDDM and 32 age- and sex-matched controls. Analysis of the results showed elevated levels of DNA damage (expressed as % comet tail DNA) in the lymphocyte (4.10+/-0. 47, 3.22+/-0.22), monocyte (4.28+/-0.47, 3.49+/-0.18), and whole blood (4.93+/-0.51, 4.51+/-0.23) fractions from IDDM subjects compared to controls, respectively, but the increases observed were not statistically significant. However, we found significantly elevated basal levels of DNA damage in the neutrophil fraction (8. 38+/-0.64, 4.07+/-0.23; p<0.001, Mann-Whitney U test) in IDDM subjects compared to controls. Given these novel neutrophil findings, we extended the study to include a total of 50 IDDM subjects and 50 age- and sex-matched control subjects and determined basal levels of DNA damage in the neutrophils of all 100 subjects. We found significantly elevated mean levels of DNA damage (8.40+/-0.83, 4. 34+/-0.27; p<0.001, Mann-Whitney U test) in the neutrophils from the IDDM subjects when compared to controls. Our results show that even with acceptable glycaemic control there is a significantly elevated level of DNA damage within diabetic neutrophils in vivo.     

Alpha-amidated peptides derived from pro-opiomelanocortin in normal human pituitary.

Normal human pituitaries were extracted in boiling water and acetic acid, and the alpha-amidated peptide products of pro-opiomelanocortin (POMC), alpha-melanocyte-stimulating hormone (alpha MSH), gamma-melanocyte-stimulating hormone (gamma 1MSH), and amidated hinge peptide (HP-N), as well as their glycine-extended precursors, were characterized by sequence-specific radioimmunoassays, gel-chromatography, h.p.l.c. and amino acid sequencing. alpha MSH and gamma 1MSH constituted 0.27-1.32% and 0.10-5.10%, respectively, of the POMC-derived products [calculated as the sum of adrenocorticotropic hormone (ACTH)-(1-39), ACTH-(1-14) and alpha MSH immunoreactivity]. alpha MSH and ACTH-(1-14) were only present in non- or mono-acetylated forms. Only large forms of gamma 1MSH and gamma 2MSH were present in partly glycosylated states. The hinge peptides were amidated to an extent two to three orders of magnitude greater than alpha MSH and gamma 1MSH. Most (99%) of the HP-N was of low molecular mass and consisted mainly of HP-N-30. The remaining part was high-molecular-mass HP-N, probably HP-N-108, although the presence of HP-N-44 could not be completely excluded. These results show that all the possible amidated POMC-related peptides are present in normal human pituitary. It also shows that cleavage in vivo at all dibasic amino acids but one, takes place at the N-terminal POMC region; the exception is at the POMC-(49-50) N-terminal of the gamma MSH sequence. The pattern of peptides produced suggests that the generation of amidated peptides is mainly regulated at the endopeptidase level.     

Quantitation of pharyngeal motor function in normal human subjects.

A need exists for accurate pressure recording of pharyngeal motor events. Results of this study indicate that accurate quantitation of pharyngeal motor activity is not possible using a water-filled catheter system, even when high infusion rates are used. An intraluminal strain gauge system, however, achieves high-fidelity recording. Quantitation of pharyngeal peristalsis using the intraluminal strain gauge system reveals peristaltic pressure amplitudes higher than those hitherto recorded. In normal subjects, peristaltic amplitude averages about 200 mmHg in the hypopharynx, complexes in one subject being as high as 600 mmHg. A zone of relatively low pressure exists in the oropharynx. Mean pharyngeal wave duration decreases progressively in an aboral direction, from 1.0 to 0.3 s, and peristaltic wave speeds range between 9 and 25 cm/s. Accurate quantitation of pharyngeal peristaltic variables provides the necessary basis for characterization and assessment of pharyngeal motor disorders.     

Recombinant human insulin: X simplification of folding of the biotechnological precursor of recombinant human insulin

The folding of biotechnological precursor of the human insulin precursor was carried out from solubilized inclusion bodies without a preliminary oxidizing or reducing its Cys residues. The inclusion bodies were dissolved in 8 M urea with the addition of 10 mM 2-mercaptoethanol. Hydrophobic cell components were removed from the solution by passing through a neutral weakly hydrophobic sorbent, the solution was five times diluted and refolded upon addition of 0.3 mM cystine for initiation of disulfide rearrangement. The presence of nucleic acids and cell protein impurities does not affect the folding efficiency. The resulting precursor of folded human insulin was purified by metal-chelate affinity chromatography and converted into insulin by two-stage enzymatic cleavage.     

Metformin increases insulin sensitivity and plasma beta-endorphin in human subjects.

Metformin has been widely used in clinical type 2 diabetes treatment and prevention. The present study was designed to explore the effect on people with a sedentary lifestyle at therapeutic doses. Twenty-two physically-inactive volunteers with normal glucose tolerance were studied. Escalating doses of metformin in low-dose (250 mg), intermediate-dose (500 mg), and high-dose (750 mg) treatment three times per day were administrated into each subject for a three-week treatment period. Fasting plasma glucose, A1C, HOMA-IR for insulin resistance, lipid profile, and plasma beta-endorphin-like immunoreactivity (BER) were measured before treatment and weekly at the end of each dosing period. Metformin significantly reduced fasting plasma glucose and HOMA-IR in healthy humans after receiving this treatment at therapeutic doses including low-dose (5 %, 17 %), intermediate-dose (6 %, 25 %) and high-dose treatment (6 %, 21 %). Plasma BER was also increased from 135.46 +/- 61.73 pg/ml to 137.52 +/- 66.11 pg/ml by low-dosing (p = 0.39), to 139.17 +/- 64.08 pg/ml by intermediate-dosing (p = 0.32), and to 149.59 +/- 63.32 pg/ml by high-dosing (p < 0.05). Also, serum cholesterol decreased significantly using metformin at therapeutic doses including low-dose (4 %), intermediate-dose (8 %) and high-dose treatment (7 %). However, metformin failed to modify levels of serum HDL-cholesterol and C-reactive protein (CRP) in healthy subjects. Also, the reduction of serum cholesterol by metformin did not correlate to the increase in insulin sensitivity. In conclusion, metformin causes a significant parallel increase in insulin sensitivity and plasma beta-endorphin level in human subjects.