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Biomedical subjects

G Seipke

Publications and source records attributed to G Seipke.

10 recordsLinked to original sources
PubMed2005

Effects of new insulin analogues HMR1964 (insulin glulisine) and HMR1423 on insulin receptors.

AIMS/HYPOTHESIS: New insulin analogues have been created by amino-acid exchange to provide an improved pharmacokinetic profile. However, safety issues have been raised regarding their use, as amino-acid exchange of insulin may induce altered metabolic and mitogenic effects. For example, the insulin analogue Asp(B10) causes breast cancer in rodents. The aim of this study was to compare two new insulin analogues HMR1964 (Lys[B3],Glu[B29]) (insulin glulisine) and HMR1423 (Gly[A21],His[B31],His[B32]) with regular insulin and the mitogenic analogue Asp(B10). MATERIALS AND METHODS: We analysed insulin receptor binding characteristics and dissociation kinetics, as well as insulin-induced receptor auto- and dephosphorylation kinetics, in rat-1 fibroblasts overexpressing the human insulin receptor isoform B. Mitogenic activity was tested in the non-malignant cell line MCF10. RESULTS: Regular insulin, HMR1964 and HMR1423 showed no significant differences in receptor association, dissociation and receptor binding affinity, while Asp(B10) displayed markedly increased insulin receptor affinity. All of the analogues induced rapid insulin receptor autophosphorylation, reaching a maximum 10 min after stimulation (10(-9) mmol/l insulin). In contrast, Asp(B10) induced a prolonged phosphorylation and dephosphorylation state of the 95 kDa insulin receptor beta-subunit. With respect to [3H]thymidine incorporation, the new analogues had similar (HMR1423) or even lower (HMR1964) effects than regular insulin in the mammary epithelial cell line MCF10, while Asp(B10) showed increased [3H]thymidine incorporation. CONCLUSIONS/INTERPRETATION: HMR1964 and HMR1423 displayed the same association, dissociation and insulin receptor affinity kinetics as regular insulin, and might therefore be useful for the treatment of diabetes.

Animals↗
PubMed2001

Effects of the long-acting insulin analog insulin glargine on cultured human skeletal muscle cells: comparisons to insulin and IGF-I.

The aim of this study was to determine whether the long-acting insulin analog, insulin glargine, behaves like human insulin for metabolic and mitogenic responses in differentiated cultured human skeletal muscle cells from nondiabetic and diabetic subjects. Human insulin and insulin glargine were equipotent in their ability to compete for [(125)I]insulin binding. Insulin glargine displaced [(125)I]IGF-I from the IGF-I-binding site with approximately 0.5% the potency of IGF-I. In nondiabetic muscle cells, all three ligands stimulated glucose uptake similarly, whereas the sensitivity of glucose uptake was greatest in response to IGF-I and lower and equal for human insulin and insulin glargine. In diabetic muscle cells, the final responsiveness of glucose uptake was greatest for IGF-I and equivalent for human insulin and insulin glargine; sensitivities were the same as those for nondiabetic cells. Thymidine uptake into DNA was stimulated foremost by IGF-I, whereas human insulin and insulin glargine showed equivalent, but greatly reduced, sensitivities and potencies (<1% IGF-I). Stimulation of Akt phosphorylation was slightly more responsive to IGF-I compared with human insulin and insulin glargine, with sensitivities similar to glucose uptake stimulation. We conclude that in human skeletal muscle cells, insulin glargine is equivalent to human insulin for metabolic responses and does not display augmented mitogenic effects.

Adult↗
PubMed1998

The long acting human insulin analog HOE 901: characteristics of insulin signalling in comparison to Asp(B10) and regular insulin.

HOE 901 is a new biosynthetic long-acting human insulin analog (GLY[A21]ARG[B31]ARG[B32]). We compared HOE 901 with normal human insulin and the insulin analog Asp(B10), which is known to have increased mitogenic activity at least partially mediated through the insulin receptor. We have analyzed receptor binding, insulin-induced receptor autophosphorylation and phosphorylation of receptor substrates in rat-1 fibroblasts overexpressing human insulin receptor isoform A (HIR A) or B (HIR B). In HIR A expressing cells, insulin and its analogs showed no significant differences in receptor association while clearly different dissociation kinetics were observed. In HIR B expressing cells, no significant differences in association and dissociation kinetics were observed. All insulins induced rapid autophosphorylation of the insulin receptor reaching a maximum after 10 min of stimulation. Asp(B10)insulin induced a prolonged phosphorylation state (over 60 minutes) of the 95 kDa receptor beta-subunit and of the substrates IRS-1/IRS-2 and Shc in contrast to normal human insulin and to HOE 901. In addition, we observed an increased and prolonged tyrosine phosphorylation of an unidentified protein with Asp(B10)insulin at about 60 kDa. Insulin-dependent dephosphorylation of the focal adhesion kinase (p125FAK) was equally induced by all these ligands. With respect to [3H]thymidine incorporation into DNA, HOE 901 had similar effects as normal human insulin, while Asp(B10)insulin showed increased [3H]thymidine incorporation. In summary, the data show that the increased mitogenic activity of Asp(B10)insulin may be explained with a prolonged kinetics of tyrosine phosphorylation of the insulin receptor and of insulin signalling elements together with the preferential phosphorylation of an yet unidentified 60 kDa protein. HOE 901 behaves with respect to insulin receptor binding, receptor autophosphorylation, phosphorylation of signalling elements and promotion of mitogenesis like regular human insulin.

Animals↗
PubMed1997

Growth promoting and metabolic activity of the human insulin analogue [GlyA21,ArgB31,ArgB32]insulin (HOE 901) in muscle cells.

[GlyA21,ArgB31,ArgB32]insulin (HOE 901) represents a biosynthetic human insulin analogue that, due to its isoelectric point, precipitates at neutral tissue pH leading to a retarded absorption rate and a corresponding longer duration of action. In the present investigation we have evaluated the growth promoting and metabolic activity of this analogue in muscle tissue using exponentially growing H9c2 cardiac myoblasts and adult rat ventricular cardiomyocytes. Equilibrium binding studies of 125I-labelled IGF-I (insulin-like growth factor I) to differentiating myoblasts revealed the presence of 7 x 10(3) IGF-I receptors per cell. In contrast, no specific binding of insulin could be detected. Competition binding experiments showed a slightly higher affinity of HOE 901 for the IGF-I receptor when compared to regular human insulin with IC50 (half-inhibitory concentration) values of 70 and 101 nM, respectively. However, the supermitogenic insulin analogue [AspB10]insulin competed significantly more efficiently for IGF-I binding (IC50: 44 nM). Maximum growth promoting activity of the peptides was then determined in serum-starved myoblasts by an incubation with the peptides (5 x 10(-7) M) for 16 h in the presence of [3H]thymidine. [Asp(B10)]Insulin produced a stimulation of DNA synthesis (about 3-fold) which was comparable to the effect of IGF-I and significantly (P < 0.005) higher than the effect of HOE 901 with the latter being essentially equipotent to native insulin. Comparable results were obtained at lower concentrations of the peptides (10(-9) to 10(-8) M). Metabolic activity of HOE 901 was determined by measuring the dose-dependent stimulation of 3-O-methylglucose transport in adult cardiomyocytes. Maximum transport stimulation was identical for insulin and HOE 901 with EC50 (half-effective concentration) values of 0.7 x 10(-10) and 1.9 x 10(-9) M, respectively. We concluded that the IGF-I receptor-mediated growth promoting activity of HOE 901 in muscle cells and the maximal metabolic activity of this analogue are not different from those of native human insulin. It is suggested that differential interaction with IGF-I receptors significantly contributes to the action profile of insulin analogues.

Animals↗
PubMed1990

[Recurrent hypoglycemia in the insulin autoimmune syndrome].

A 72-year-old man with recurrent hypoglycaemia was suspected of having an insulinoma. But several diagnostic methods (computed tomography; mesenteric and coeliac angiography; sella imaging) did not reveal any tumour. 72-hour hunger test did not precipitate any spontaneous hypoglycaemia. A falling insulin-glucose ratio spoke against an insulin-producing tumour. Reactive symptomatic hypoglycaemia occurred 4.5 hours after an oral glucose test. Measurement of insulin concentration demonstrated a slow but pronounced increase (3500 microU) over an already raised initial value (816 microU/ml), typical of prediabetic metabolic regulation. Demonstration of insulin autoantibodies confirmed the diagnosis of an insulin autoimmune syndrome, which has a good prognosis. The patient became free of symptoms on a regimen of frequent small, carbohydrate-poor but fat and bulk-rich meals. Hypoglycaemia recurred when the diet was not observed.

Aged↗
PubMed1988

Cellular processing of pro-atrial natriuretic factor (pro-ANF): studies using an antiserum that selectively binds ANF-(99-126) after its cleavage from pro-ANF.

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a 15-17K prohormone, but circulates in plasma as a 3K, carboxy (C)-terminal fragment of the prohormone. The tissue location at which the cleavage of pro-ANF to its hormonal form occurs is unknown. In the present study, an immunological approach was taken to address this question. A polyclonal antiserum was generated which recognizes the hormonal form of ANF [ANF-(99-126)] only after its cleavage from the prohormone. This was accomplished by immunizing rabbits with a synthetic peptide corresponding to the seven amino (N)-terminal residues of ANF-(99-126) coupled to carrier protein via a C-terminal cysteine. This antiserum, anti-ANF-(99-105), demonstrated high affinity for ANF-(99-126) (IC50 = 170 pM), but displayed 100-fold less affinity for recombinant pro-ANF [ANF-(2-126)]. The N-terminal specificity of anti-ANF-(99-105) was evident by its failure to bind ANF-(103-126) at concentrations up to 100 nM. The specificity of anti-ANF-(99-105) for the hormonal form of ANF was examined by using thrombin to cleave pro-ANF and testing for the generation of anti-ANF-(99-105) immunoreactivity. Cleavage of atrial pro-ANF or 35S biosynthetically-labeled pro-ANF resulted in the production of immunoreactive material from the prohormone, whereas pro-ANF itself demonstrated no cross-reactivity with anti-ANF-(99-105). Anti-ANF-(99-105) could also recognize ANF released from the isolated perfused rat heart. When anti-ANF-(99-105) was used in immunohistochemical studies of rat atrial myocardium, no staining was observed in unfixed frozen sections. This suggests that proteolytic processing of pro-ANF is not an intracardiocytic event.

Amino Acid Sequence↗
PubMed1987

Proatrial natriuretic factor is phosphorylated by rat cardiocytes in culture.

Proatrial natriuretic factor (proANF) is phosphorylated in primary cultures of neonatal rat cardiocytes. Rittenhouse et al. (Rittenhouse, J., Moberly, L., O'Donnell, M. E., Owen, N. E., and Marcus, F. (1986) J. Biol. Chem. 261, 7607-7610) observed that cyclic AMP-dependent protein kinase phosphorylated synthetic peptides related to atrial natriuretic factor (ANF) and that phosphorylated ANF peptides were more effective in stimulating Na/K/Cl cotransport in smooth muscle cells than nonphosphorylated forms. In our studies, rat cardiocytes in culture were incubated with [32P]orthophosphoric acid, and ANF-related peptides in cell extracts and culture media were isolated using antisera to ANF. Both atrial and ventricular cardiocytes contained and secreted phosphorylated proANF, a 126-amino acid precursor of ANF. Phosphorylated and nonphosphorylated isoforms of proANF were resolved by isoelectric focusing; approximately 35% of the proANF secreted by cardiocytes was phosphorylated. proANF is phosphorylated on a serine residue localized to a 42-amino acid tryptic fragment (proANF residues 26-67). We conclude that proANF is phosphorylated by rat cardiocytes but not within the portion of the molecule destined to become ANF (proANF residues 99-126). Phosphorylation may have a role in the cellular mechanisms of proANF storage and secretion or in the modulation of potential biological activities of the circulating amino-terminal portion of proANF.

Amino Acid Sequence↗
PubMed1977

Specificity of DNA basic polypeptide interactions. II+ Influence of aromatic amino acid residues investigated with agarose bound lysine copolypeptides.

Binding affinities towards DNA and base pair specificities of lysine copolymers, containing different amounts of Phe, Tyr or Trp residues, were estimated using a previously described chromatographic method. Incorporation of few aromatic residues into polylysine causes a decrease in the binding affinity, however, further raising the aromatic residue - lysine ratio results in a continous increase of affinity, which is most pronounced with the Tyr copolymers and not observed with polymers containing neutral aliphatic amino acid residues. AT-specificity increases concomitant with binding affinity in the case of the Tyr copolymers but not with the Phe copolymers. The interaction of DNA with the alternating Phe-Lys polymer is significantly stronger than with the random copolymer of equal residue composition. The molecular and conformational reasons determining specificity are discussed.

Base Sequence↗