Reductive methylation of insulin. Production of a biologically active tritiated insulin

Reductive methylation of the three amino groups of porcine insulin was accomplished by incubation with formaldehyde and sodium cyanoborohydride. The two amino termini and the epsilon amino group of B29 lysine were each dimethylated within 1 h of incubation. The fully methylated insulin bound more tightly to a reverse phase column than did native insulin, had a slightly more acid isoelectric point, and maintained approximately 50% biological activity when examined with an insulin sensitive cultured cell line. Reductive methylation with sodium cyanoboro [3H] hydride resulted in a [3H] methylated insulin with a specific activity of 6 Ci/mmol.     

Adenylate cyclase activity in isolated rat islets of Langerhans Effects of agents which alter rates of insulin secretion

Adenylate cyclase activity was estimated inhomogenates of rat islets of Langerhans. by measurement of the conversion of [α-³²P]ATP to adenosine cyclic 3′,5′-[³²P]monophosphate. Islet cell adenyulate cyclase activity was stimulated by the addition to the homogenates of glucagon, fluoride, prostaglandins E₁ or E₂ GTP or CTP although not by UTP, TTP, GDP, or GMP. Adrenaline, noradrenaline and isoproterenol were each found to inhibit the activity, the order of potency at a concentration of 10^?4 M being adrenaline > noradrenaline > isoproterenol. The effects of these agents were not altered by β-blackade with propanolol but could be preventived by α-blockade with phenoxybenzamine. The following agents, present at concentrations previously shown to increase rates of insulin secretion from rat islets of Langerhans, were ineffective in altering adenylate cyclase activity when tested in the presence or absence of 0.1 mM GTP: glucose, glibenclamide, xylitol leucine, arginine, or potassium. These results suggest that the activity of adenylate cyclase in the B cells of rat islets of Langerhans may play an important role in mediating the direct effects of hormones and adrenergic agents on insulin release, although the short term effects of substrates such as glucose or amino acids on the release process do not appear to be mediated through alterations in the activity of this enzyme.     

The preparation of stereospecific tritium-labeled reduced nicotinamide adenine dinucleotide phosphate

A method has been developed for the preparation of a high specific activity stereospecifically labeled tritiated NADPH. In this procedure, tritium is enzymatically transferred from d-isocitric acid-2-³H (8 Ci/mmole) to the A face of a pyridine nucleotide during its stereospecific reduction, resulting in the formation of NADPH-4A-³H (2 Ci/mmole).     

Possible modulatory effect of endogenous islet catecholamines on insulin secretion

Background

The possible participation of endogenous islet catecholamines (CAs) in the control of insulin secretion was tested.

Methods

Glucose-induced insulin secretion was measured in the presence of 3-Iodo-L-Tyrosine (MIT), a specific inhibitor of tyrosine-hydroxylase activity, in fresh and precultured islets isolated from normal rats. Incubated islets were also used to measure CAs release in the presence of low and high glucose, and the effect of α2-(yohimbine [Y] and idazoxan [I]) and α1-adrenergic antagonists (prazosin [P] and terazosin [T]) upon insulin secretion elicited by high glucose.

Results

Fresh islets incubated with 16.7 mM glucose released significantly more insulin in the presence of 1 μM MIT (6.66 ± 0.39 vs 5.01 ± 0.43 ng/islet/h, p < 0.02), but did not affect significantly the insulin response to low glucose. A similar enhancing effect of MIT upon insulin secretion was obtained using precultured islets devoid of neural cells, but absolute values were lower than those from fresh islets, suggesting that MIT inhibits islet rather than neural tyrosine hydroxylase. CAs concentration in the incubation media of fresh isolated islets was significantly higher in the presence of 16.7 than 3.3 mM glucose: dopamine 1.67 ± 0.13 vs 0.69 ± 0.13 pg/islet/h, p < 0.001, and noradrenaline 1.25 ± 0.17 vs 0.49 ± 0.04 pg/islet/h, p < 0.02. Y and I enhanced the release of insulin elicited by 16.7 mM glucose while P and T decreased such secretion.

Conclusion

Our results suggest that islet-originated CAs directly modulate insulin release in a paracrine manner.     

New development in the tritium labelling of peptides and proteins using solid catalytic isotopic exchange with spillover-tritium

Summary.  The mechanism of the reaction of high temperature solid state catalytic isotope exchange (HSCIE) of hydrogen in peptides with spillover-tritium at 140–180°C was analyzed. This reaction was used for preparing [³H]enkephalins such as [³H]DALG with specific activity of 138 Ci/mmol and [³H]LENK with specific activity of 120 Ci/mmol at 180°C. The analogues of [³H]ACTG_4–10 with specific activity of 80 Ci/mmol, [³H]zervamicin IIB with specific activity of 70 Ci/mmol and [³H]conotoxin G1 with specific activity 35 Ci/mmol were produced. The obtained preparations completely retained their biological activity. [³H]Peptide analysis using ³H NMR spectroscopy on a Varian UNITY-600 spectrometer at 640 MHz was carried out. The reaction ability of amino fragments in HSCIE was shown to depend both of their structures and on the availability and the mobility of the peptide chain. The reaction of HSCIE with the β-galactosidase from Termoanaerobacter ethanolicus was studied. The selected HSCIE conditions allow to prepare [³H] β-galactosidase with specific activity of 1440 Ci/mmol and completely retained its the enzymatic activity. Received November 30, 2001 Accepted January 31, 2002 Published online December 18, 2002 Acknowledgments The work was supported by the Russian Foundation for Basic Research, grant 01-04-48519a. Authors' address: Dr. Yurii A. Zolotarev, Institute of Molecular Genetics, Russian Academy of Sciences, pl. Kurchatova 2, 123182, Moscow, Russia, Fax: +7 (095) 196-0221, E-mail: zolya@img.ras.ru Abbreviations: HSCIE, the reaction of high temperature solid state catalytic isotope exchange; HS, hydrogen spillover; ³H NMR, tritium nuclear magnetic spectroscopy; CtxG1, conotoxin G1; AchR, acetylcholine receptor; HF, Hartree-Fock ab initio quantum-chemical calculation method     

Synthesis of luteinizing hormone-releasing hormone containing tritium-labelled pyroglutamic acid

A method of preparing luteinizing hormone-releasing hormone (LH-RH) pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂, by a combination of solid-phase and classical reactions was employed to conveniently synthesize a tritium-labelled hormone by incorporation of 4-[³H]-pyroglutamic acid into position I of the peptide chain. The tritiated LH-RH possessed a specific radioactivity of 18.3 Ci/mmole and a maximal biological potency.     

Insulin and insulin mutants stimulate glucose uptake in rat adipocytes

A simple method to determine thein vitro biological activity of insulin by measuring glucose uptake in the rat adipocytes is presented here. In the presence of insulin, the glucose uptake is 5–6 times more than the basal control. And the uptake of D-[3-³H]-glucose is linear as the logarithm of insulin concentration from 0.2 ώg/L to 1.0 ώg/L. Glucose and 3-O-methyl-glucose inhibit D-[3-³H]-glucose uptake into adipocytes. By this method, thein vitro biological activity of [B₂-Lys]-insulin and [B₃-Lys]-insulin was measured to be 61.6% and 154% respectively, relative to that of insulin.     

Glucose stimulates the biosynthesis of rat I and II insulin to an equal extent in isolated pancreatic islets

The effects of glucose on insulin biosynthesis were studied by measuring the incorporation of radiolabelled amino acids into proinsulin/insulin in isolated rat islets. The islets were pulse labelled for 15 min with [3H]leucine (present in rat insulin I and II) or [35S]methionine (unique to rat insulin II) and then incubated for a 165 min post-label (chase) period during which the majority of labelled proinsulin was converted to insulin but under conditions whereby greater than 95% of radiolabelled proinsulin or insulin was retained in the islets. The newly synthesized, labelled, insulin was analyzed by high performance liquid chromatography. Rat I and II insulin biosynthesis was stimulated by 16.7 mM glucose to the same extent.     

Stimulation of insulin biosynthesis in isolated mouse islets by L-leucine, 2-aminonorbornane-2-carboxylic acid and α-ketoisocaproic acid

An immune binding technique was used for measuring the effects of certain amino acids on the rate of insulin biosynthesis. [³H]phenylalanine served as the radioactive precursor for insulin synthesized by isolated mouse pancreatic islets. L-Leucine was found to stimulate the insulin biosynthesis and this effect was observed already at a physiologic concentration in contrast to the much higher concentrations needed to stimulate insulin secretion in vitro. Furthermore, it was found that 2-aminonorbornane-2-carboxylic acid and α-ketoisocaproic acid shared with glucose and L-leucine the ability to stimulate insulin biosynthesis. In contrast, L-alanine, L-arginine and D-leucine had no stimulatory effect in the absence of glucose, while in the presence of 5 mM glucose L-arginine decreased and L-alanine increased the incorporation rate of tritiated phenylalanine. The fact that many of those compounds which stimulated insulin biosynthesis have also been shown elsewhere to be metabolized by the B-cells supports the view that the rate of insulin biosynthesis may be substrate dependent.     

Fibroblast procollagen production rates in vitro based on [H]hydroxyproline production and procollagen hydroxyproline specific activity

In vitro procollagen production rates can be determined by culturing cells in the presence of [³H]proline and measuring the subsequent formation of [³H]hydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [³H]proline, and [³H]hydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [¹⁴C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. Biochem. 96, 45–55]. Procollagen production rates were readily determined by this method using 5 to 20 μCi [³H]proline and approximately 10⁶ cells. It was found that ³H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/μg DNA · h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro.     

Convenient methods to determine the specific radioactivity of (gamma-32P)ATP of high specific activity.

A satisfactory method for the determination of the specific activity of highly labeled [γ-³²P]ATP has not been reported previously. Yields of high specific activity ³²P labeled material usually are too small to be detected by ultraviolet spectrophotometry or phosphate analysis. Recent reports describing the assay of ATP by enzyme catalyzed phosphate transfer to ³H labeled glucose (1) or galactose (2) are not suitable for use with highly labeled ³²P material since the crossover into the ³H channel will greatly exceed the radioactivity of the ³H labeled phosphate acceptor. Recently Schendel and Wells reported the preparation of essentially carrier free [γ-³²P]ATP. They indicated, however, that the specific activity of the labeled product could not be determined by conventional methods (3). We have developed and now routinely use an expedient method for the determination of the specific activity of picomole quantities of highly labeled [γ-³²P]ATP. This procedure measures the phosphate transfer from [γ-³²P]ATP to oligothymidylic acid [dT(pT)₁₀] catalyzed by bacteriophage T4 induced polynucleotide kinase. The specific activity is determined by measuring the radioactivity present in d-³²pT(pT)₁₀, and can be verified by an isotope dilution method employing the same assay. Specific activities as high as 240 Ci/mmole have been determined.     

Stimulation of insulin biosynthesis in isolated mouse islets by L-leucine, 2-aminonorbornane-2-carboxylic acid and α-ketoisocaproic acid

An immune binding technique was used for measuring the effects of certain amino acids on the rate of insulin biosynthesis. [³H]phenylalanine served as the radioactive precursor for insulin synthesized by isolated mouse pancreatic islets. L-Leucine was found to stimulate the insulin biosynthesis and this effect was observed already at a physiologic concentration in contrast to the much higher concentrations needed to stimulate insulin secretion in vitro. Furthermore, it was found that 2-aminonorbornane-2-carboxylic acid and α-ketoisocaproic acid shared with glucose and L-leucine the ability to stimulate insulin biosynthesis. In contrast, L-alanine, L-arginine and D-leucine had no stimulatory effect in the absence of glucose, while in the presence of 5 mM glucose L-arginine decreased and L-alanine increased the incorporation rate of tritiated phenylalanine. The fact that many of those compounds which stimulated insulin biosynthesis have also been shown elsewhere to be metabolized by the B-cells supports the view that the rate of insulin biosynthesis may be substrate dependent.     

Effect of hexoses and mannoheptulose on cyclic AMP accumulation and insulin secretion in rat pancreatic islets

The effects of various sugars on the simultaneous release of insulin and accumulation of cyclic AMP were studied in collagenase isolated rat pancreatic islets. d-Glucose stimulated the formation of cyclic AMP at 3 and 60 min of incubation, whether measured by a label incorporation technique, or by the protein kinase binding assay of Gilman. Only d-glucose and d-mannose were able to stimulate insulin release and cyclic [³H]AMP accumulation in the absence of other substrate. d-fructose had a stimulatory effect in the presence of 3.3 mM d-glucose only at a high concentration (38.8 mM), and enhanced the effects of 8.3 mM glucose when added at the concentration of 8.3 mM. d-Galactose was effective only together with 8.3 mM d-glucose. The order of potency of these hexoses, both regarding insulin secretion and cyclic [³H]AMP accumulation, was glucose-mannose-fructose-galactose.l-Glucose and 3-O-methylglucose had no effects at 60 min when incubated together with 8.3 mM d-glucose, whereas at 3 min, 3-O-methylglucose induced a small stimulation of the cyclic [³H]AMP response.d-mannoheptulose and d-glucosamine inhibited the insulin and cyclic [³H]-AMP responses to 27.7 mM glucose. Mannoheptulose suppressed completely the glucose effect on cyclic nucleotide accumulation within 90 s.Although under all incubation conditions, the threshold stimulatory or inhibitory concentration of a given agent was identical for insulin release and cyclic [³H]AMP accumulation, these two variables showed quantitative differences in incubations of 60 min, the magnitude of the changes in insulin secretion being larger than that for the cyclic nucleotide. It is suggested that modulation of islet cyclic AMP level is an important step in the transmission of the effect of various sugars on insulin release; however, glucose and possibly other sugars may also enhance insulin release by additional mechanisms not involving the adenylate cyclase-cyclic AMP system of the β-cell.     

Effect of lithium and sodium ions on opiate-and dopamine-receptor binding

The effects of lithium and sodium were studied in the corpus striatum and cerebral cortex of rats. Lithium was inhibitory at low concentrations but at 20 mM it increased the binding of [G-³H]naloxone (specific activity 15.6 Ci/mmol). Sodium stimulated the high-affinity binding of this compound. Membranes obtained from the rats treated with lithium showed lower specific binding of both [³H]naloxone and [³H]DHM. Binding of [³H]d-alanine Leu-enkephalin was not changed in the brains of lithium-treated rats, but that of [³H]-spiroperidol was lowered. Cerebral cortex and striatum of lithium-treated rats had a decreased apparent dissociation constant and a lower receptor concentration of naloxone binding sites.     

Synthesis of tritiated abscisic acid of high specific activity

Stable abscisic acid (RS)-[³H] was synthesized at a specific activity of 21 Ci/mmol using a basic alumina catalyzed proton exchange of 1-hydroxy-4-keto-α-ionone with T₂O followed by a Wittig reaction. Abscisic acid -[³H] of specific activity 102 mCi/mmol was synthesized after carrying out a base catalyzed tritium exchange in solution.     

Mechanism of 3-phenylpyruvate-induced insulin release. Metabolic aspects.

1. The metabolism and metabolic effects of 3-phenylpyruvate were examined in rat pancreatic islets. 2. Islet homogenates catalysed transamination reactions between 3-phenylpyruvate and L-glutamate, L-leucine, L-norleucine or L-valine. 3-Phenylpyruvate failed to activate glutamate dehydrogenase. 3. 3-Phenylpyruvate rapidly entered into islet cells, was extensively converted into phenylalanine but slowly oxidized. 4. The conversion of phenylpyruvate into phenylalanine coincided with a fall in the content of several amino acids (especially glutamate and aspartate) in the islets and incubation medium, the accumulation of 2-oxoglutarate and a modest fall in the NH4+ production rate. 5. 3-Phenylpyruvate failed to affect 14CO2 output from islets prelabelled with [U-14C]palmitate, but augmented 14CO2 output from islets prelabelled or incubated with L-[U-14C]glutamine. 6. In the presence of L-glutamine, 3-phenylpyruvate augmented the ATP/ADP ratio and NAD(P)H islet content, and caused a rapid and sustained decrease in the outflow of radioactivity from islets prelabelled with [2-3H]adenosine. 7. These data support the view that the insulin-releasing capacity of 3-phenylpyruvate coincides with an increase in the catabolism of endogenous amino acids acting as 'partners' in transamination reactions leading to the conversion of 3-phenylpyruvate into phenylalanine.     

Insulin and insulin mutants stimulate glucose uptake in rat adipocytes

A simple method to determine the in vitro biological activity of insulin by measuring glucose uptake in the rat adipocytes is presented here. In the presence of insulin, the glucose uptake is 5-6 times more than the basal control. And the uptake of D-[3-3H]-glucose is linear as the logarithm of insulin concentration from 0.2 μg/L to 1.0 μg/L. Glucose and 3-O-methyl-glucose inhibit D-[3-3H]-glucose uptake into adipocytes. By this method, the in vitro biological activity of [B2-Lys]-insulin and [B3-Lys]-insulin was measured to be 61.6% and 154% respectively, relative to that of insulin.     

Cyclic AMP metabolism and insulin release in pancreatic islets of the rat Effects of agents which alter microtubular function

In order to investigate the relationship between microtubular function, insulin release and islet cyclic AMP metabolism, the effects of ²H₂O, colchicine and vincristine were studied in rat islets prelabeled with [³H]adenine. Glucose-induced insulin secretion and efflux of cyclic [³H]AMP was markedly inhibited by 8–50% ²H₂O. At a higher concentration (75%), deuterated water still suppressed insulin release, while the inhibition of nucleotide release was abolished. Glucose-induced intra-islet cyclic [³H]AMP accumulation was augmented by ²H₂O progressively with time. With 75% ²H₂O, although efflux of cyclic AMP was no more inhibited, intra-islet accumulation of the nucleotide was still enhanced. The cyclic AMP efflux induced by cholera toxin, or a high concentration of 3-isobutyl-1-methylxanthine was suppressed and the intra-islet nucleotide accumulation was enhanced by ²H₂O. The latter effect tended to be less pronounced than when glucose was the stimulator. All the effects of ²H₂O on glocuse-stimulated islets were mimicked by incubating the tissue in H₂O at 28°C.Colchicine and vincristine had no significant effect on glucose-induced insulin release, and did not enhance the intra-islet cyclic [³H]AMP response; efflux of the nucleotide was, however, significantly inhibited. This pattern of response was shared with probenecid. Preincubation of islets with colchicine did not influence the subsequent effects of ²H₂O on insulin release and cyclic AMP metabolism.It is concluded that: (1) enhancement of intra-islet cyclic AMP accumulation by ²H₂O is not due to inhibition of the nucleotide efflux; (2) the effects on cyclic AMP metabolism described here are not exclusive for microtubular affecting agents and do not seem to be related to the microtubular system of the islet.     

EFFECT OF TRITIATED THYMIDINE ON THE KINETICS AND VIABILITY OF 9L CELLS IN VITRO

The colony-forming efficiency of 9L rat gliosarcoma cells was unaffected by treatment with 0.1 μCi/ml of [³H]TdR. However, when cells were treated with 1 or 10 μCi/ml of [³H]Tdr, cell growth was reduced and cell survival decreased. When monolayer 9L cells were treated with 1 μCi/ml of [³H]TdR for up to 72 hr, approximately 5% survived, which is closely related to the percentage of non-cycling cells in this system. When cells were treated with 10 μCi/ml of [³H]TdR for 72 hr, less survival was observed. the additional cell kill observed may be induced by [³H]TdR released from doomed cells into petri dishes during the incubation period of the colony-formation assay.     

Amino acid dependent and independent insulin stimulation of cartilage metabolism

The effects of insulin on embryonic chicken cartilage in organ culture and the dependence of these effects on essential amino acids have been studied. In the presence of all essential amino acids, insulin: (1) increases 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake; (2) increases [5(-3H] uridine flux into uridine metabolites and the intracellular UTP pool; (3) expands the size of the intracellular UTP pool; (4) does not change the specific activity of the UTP pool; and (5) stimulates RNA, proteoglycan, and total protein synthesis. In lysine (or other essential amino acid)-deficient medium, the effects of insulin are different. While insulin stimulates incorporation of [5(-3)H] uridine into RNA, it does so by increasing the specific activity of the UTP pool without increasing RNA synthesis. Insulin stimulates 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake but no longer stimulates proteoglycan, total protein, or RNA synthesis or expands the size of the UTP pool. These data indicate that there are amino acid dependent and independent effects of insulin on cartilage. Transport processes are amino acid independent, while synthetic processes are amino acid dependent.