Showing papers on "Proinsulin published in 1983"

Formation of proinsulin by immobilized Bacillus subtilis

Abstract: There has been an increasing interest in the use of immobilized cells for the production of pharmaceuticals as well as for products such as high fructose syrup or ethanol1. Some of these compounds are now produced on an industrial scale2 whereby the cells are used in a resting or growing state or in a nonviable form as natural carriers of the enzyme(s) involved in the synthesis. The advantages of immobilized cell technology should also apply to microorganisms modified by recombinant DNA techniques to produce a variety of eukaryotic proteins such as hormones. We describe here the properties of immobilized Bacillus subtilis cells carrying plasmids encoding rat proinsulin. Cell proliferation normally coupled to DNA replication is undesirable in immobilized cell systems as ‘clogging’ of the system occurs due to cells growing outside the beads. Therefore, different ways were investigated to inhibit cell division while allowing continued protein synthesis. We found that the addition of certain antibiotics in the growth medium, such as novobiocin which inhibits DNA replication3, fulfills these requirements, allowing proinsulin synthesis and excretion to take place over a period of several days.

Effect of Poly(ADP-ribose) Synthetase Inhibitor Administration to Rats Before and After Injection of Alloxan and Streptozotocin on Islet Proinsulin Synthesis

Abstract: Nicotinamide (10 mmol/kg) and 3-aminobenzamide (1.25 mmol/kg), poly(ADP-ribose) synthetase inhibitors, were injected intravenously to rats either 30 min before the intravenous administration of 12 mg/kg alloxan or 50 mg/kg streptozotocin ("pretreatment") or 5 min after the administration ("posttreatment"). Fifteen minutes after the injection of the diabetogenic agents, pancreatic islets were isolated from the rats and proinsulin synthesis was determined. Proinsulin synthesis was decreased in islets from rats treated with alloxan or streptozotocin. Pretreatment with poly(ADP-ribose) synthetase inhibitors was found to protect against alloxan- or streptozotocin-induced decrease in proinsulin synthesis. By posttreatment with poly(ADP-ribose) synthetase inhibitors, streptozotocin-induced decrease in proinsulin synthesis was also significantly reversed, whereas the decrease induced by alloxan was not.

Insulin binding sites in various segments of the rabbit nephron.

Abstract: Insulin binds specifically to basolateral renal cortical membranes and modifies tubular electrolyte transport, but the target sites of this hormone in the nephron have not been identified. Using a microassay that permits measurement of hormone binding in discrete tubule segments we have determined the binding sites of /sup 125/I-insulin along the rabbit nephron. Assays were performed under conditions that minimize insulin degradation, and specific binding was measured as the difference between /sup 125/I-insulin bound in the presence or absence of excess (10(-5) M) unlabeled hormone. Insulin monoiodinated in position A14 was used in all assays. Specific insulin binding (attomol . cm-1 +/- SE) was highest in the distal convoluted tubule (180.5 +/- 15.0) and medullary thick ascending limb of Henle's loop (132.9 +/- 14.6), followed by the proximal convoluted and straight tubule. When expressed per milligram protein, insulin binding capacity was highest along the entire thick ascending limb (medullary and cortical portions) and the distal convoluted tubule, i.e., the ''diluting segment'' (congruent to 10(-13) mol . mg protein-1), and was lower (congruent to 4 X 10(-14) mol . mg protein-1), and remarkably similar, in all other nephron segments. Binding specificity was verified in competition studies with unlabeled insulin, insulin analoguesmore » (proinsulin and desoctapeptide insulin), and unrelated hormones (glucagon, 1-34 parathyroid hormone, prolactin, follicle-stimulating hormone). In addition, serum containing antiinsulin receptor antibody from two patients with type B insulin resistance syndrome markedly inhibited insulin binding to isolated tubules. Whether calculated per unit tubule length or protein content, insulin binding is highest in the thick ascending limb and the distal convoluted tubule, the same nephron sites where a regulatory role in sodium transport has been postulated for this hormone.« less

Insulin growth factor and epidermal growth factor trigger mitosis in lenses cultured in a serum-free medium.

Abstract: The mitogenicity of insulin, insulin growth factor (IGF), and epidermal growth factor (EGF) was evaluated on rabbit lenses cultured in medium KEI-4. IGF, the most highly purified of the insulin-like growth factors was a potent mitogen for mammalian lens epithelia cells. IGF and EGF triggered cell proliferation throughout the normally amitotic central and pre-equatorial region of the epithelium. The mitotic response elicited by IGF and EGF was dose dependent, was preceded by DNA synthesis, exceeded that engendered by equimolar insulin, and exhibited a chronology identical to that brought about by crystalline insulin. Lenses cultured in KEI-4 alone or in KEI-4 supplemented with growth hormone, proinsulin, the A and/or B chain of insulin, or MSA, another of the insulin-like growth factors belonging to the somatomedin family, did not show a mitotic response. Simultaneous exposure of the lens to IGF and EGF resulted in an increase in the total number of mitotic figures over that obtained with equimolar concentrations of IGF and EGF. Our results suggest that IGF or other insulin-like growth factors may be capable of regulating cell division in the mammalian lens in vivo. That the lens epithelium responded to IGF and EGF may indicate that lens epithelial cells are subject to multiple hormonal interaction. Since growth factors appear to be cell type specific, information obtained from the rabbit lens epithelium should be useful in delineating the factors and conditions required for the growth of cultured human lens cells.

Discrepancy Between Plasma C-Peptide and Insulin Response to Oral and Intravenous Glucose

Abstract: Plasma insulin, proinsulin, and C-peptide responses to 25 g glucose orally and intravenously administered were measured in 10 healthy males. Plasma insulin response was higher during the oral load in accordance with the “incretin” concept. However, the actual amountof insulin secreted, as measured by the plasma C-peptide response, was similar during the two glucose loads. The higher plasma insulin response after oral glucose was not due to crossreactivity with proinsulin in the insulin assay. These results suggest that the higher plasma insulin response during an oral glucose load is due at least partially to a lower hepatic extraction of insulin.

Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism.

Abstract: The direct addition of insulin to highly purified nuclear envelopes prepared from the livers of diabetic rats resulted in a decrease in the incorporation of 32P into trichloroacetic acid-precipitable proteins. Autoradiography of 32P-labeled envelopes, solubilized in sodium dodecyl sulfate and subjected to electrophoresis, revealed that insulin decreased the phosphorylation of all major protein bands. Insulin produced detectable effects at concentrations between 0.1 and 1 pM, maximal effects at 10 pM, and progressively diminished effects at higher concentrations. Two insulin analogs, desdipeptide proinsulin and desoctapeptide insulin, had approximately 10% and 1%, respectively, the activity of native insulin. When nuclear envelopes were first phosphorylated with [gamma-32P]ATP and insulin was then added with an excess of unlabeled ATP, dephosphorylation was enhanced, suggesting that insulin was regulating nuclear envelope phosphatase activity. The direct addition of insulin to isolated rat liver nuclei in the presence of ATP stimulated the release of previously 14C-labeled trichloroacetic acid-precipitable mRNA-like material, and the direct addition of insulin to nuclear envelopes stimulated the activity of nucleoside triphosphatase, the enzyme that participates in mRNA nucleocytoplasmic transport. Moreover, the dose-response curves for these functions mirrored insulin's inhibition of nuclear envelope phosphorylation. These data suggest, therefore, a mechanism whereby insulin directly inhibits the phosphorylation of the nuclear envelope, leading in turn to the regulation of mRNA metabolism.

Effects of Species of Origin, Purification Levels, and Formulation on Insulin Immunogenicity

Abstract: The immunogenicity of purified pork insulins (PPI) with and without (groups 1 and 2, respectively) trace contamination of beef insulin was contrasted with mixed beef pork insulin of lower purity (MBP, group 3) in 137 patients who had not previously been treated with insulin. Patients and physicians were blinded with regard to the species source of insulin and studies were conducted for a minimum of 1 yr. Antibody development to insulin was assessed by species-specific binding of 125I-insulin by acid charcoal extracted sera, as well as by measurement of insulin prebound to immunoglobulins by a polyethylene glycol precipitation method. NPH- and lente-treated individuals had equivalent antibody responses with regard to the rate of development of antibodies, and maximum immune responses to insulin. In all patient groups, antibody bound insulin as well as species-specific binding of 125I-insulin increased significantly over time (all P less than 0.01 for specific binding of pork and beef insulins SBP and SBB, as well as bound insulin). Maximum bound insulin and SBB as well as bound insulin and SBB over the entire course of the study were significantly greater in group 1 than in group 2 patients (both P less than 0.05). The rate of development and magnitude of antibodies' responses in both PPI-treated groups were significantly less than that seen in the MBP group (all P less than 0.01). New formation of antibeef proinsulin antibodies was seen in one patient from groups 1 and 3, but not in group 2. In all groups, insulin dose per day and fasting serum glucose concentrations increased by about 5 U/day and 10 mg/dl over 1 yr, but groups did not differ. MBP insulin used in these studies proved to be significantly less immunogenic than previously available Argentine pure beef insulin, purified by gel filtration. PPI containing even trace contamination of beef insulin was more immunogenic than PPI alone.

The Production and Characterization of Monoclonal Antibodies Specific for Human Proinsulin Using a Sensitive Microdot Assay Procedure

Abstract: The development of a simple microscale solid phase screening RIA and improved methods for cell cloning has lead to the establishment of 2 prohormone- and species-specific mouse monoclonal antibodies against human proinsulin (HPI). These antibodies react to determinant(s) only expressed on the HPI molecule. In addition, 11 rat monoclonal antibodies were generated which react with both human C-peptide (HCP) and HPI. All 11 rat antibodies recognize a very similar antigenic determinant in the C-peptide that appears to be made up of residues 40–45 and 57–63, which are probably brought into close proximity by a β-turn near the center of the connecting segment. The identical behavior of both HPI-specific mouse antibodies in competition experiments indicates that the antigenic structure recognized in proinsulin might be the same for both antibodies. This structure could not be regenerated by mixing equimolar amounts of human insulin and C-peptide, including the chemically synthesized complete proinsulin connectin...

Insulin antibody determination: Theoretical and practical considerations

Abstract: The immunogenicity of insulin preparations is of both academic and clinical interest. The links between insulin antibodies and insulin allergy, some forms of insulin resistance and injection site lipoatrophy are well-established, but other more subtle metabolic effects require further examination. Contamination with impurities (e.g. proinsulin) has been a major factor in the immunogenicity of conventional bovine insulin preparations but the less frequent, although still detectable, immunogenicity of highly purified porcine and human preparations remains enigmatic. Further work is required to analyse the physico-chemical factors involved, while the genetic control of the immune response to insulin is of fundamental interest.

Identification of a 31,500 molecular weight islet cell protease as cathepsin B.

Abstract: A method for the preparation of a radioisotopically labeled active-site directed reagent for proteases (125I-Tyr-Ala-Lys-ArgCH2Cl) is described, and an example of its use as a sensitive method for identifying trypsin-like proteases is provided. This high specific activity reagent was then used in an attempt to identify proteases in rat islets of Langerhans involved in the conversion of proinsulin to insulin. Previous studies have indicated that the endoprotease involved in proinsulin conversion is a cysteine proteinase and that 125I-Tyr-Ala-Lys-ArgCH2Cl affinity labels an islet crude granule fraction protein having a molecular weight of 31,500. Here we demonstrate, using a probe of higher specific activity, that the major affinity-labeled proteins of the islet crude granule fraction, when displayed by sodium dodecyl sulfate gel electrophoresis, have molecular weights of approximately 39,000 (5%), 31,500 (53%), and 5,000-6,000 (37%), with several other minor proteins (less than 5%) also labeled. The two predominant labeled proteins were mainly soluble rather than membrane bound, and they exhibited patterns of competition with various inhibitors that were similar to the pattern shown by the conversion of proinsulin to insulin in vitro. A rabbit antibody to rat liver cathepsin B immunoprecipitated both affinity-labeled 31,500 and 5,000-6,000 molecular weight proteins, and on the basis of this and structural considerations the 31,500 molecular weight cysteine protease is identified as cathepsin B. The 5,000-6,000 molecular weight peptide is an NH2-terminal, active site cysteine-containing, proteolytic fragment of the 31,500 molecular weight protein. Because cathepsin B is not per se a candidate for the proinsulin convertase because of its excessively broad substrate specificity, these studies suggest that a similar enzyme or a modified form of this enzyme is active within the secretory progranules, whereas the more typical cathepsin B may be largely confined to lysosomal contaminants in our granule preparations.

Binding of Insulin by Monkey and Pig Hypothalamus

Abstract: Membrane preparations from monkey and pig hypothalami bound [ 125 ]insulin specifically. The binding appeared to be greater by preparations from anterior than posterior portions of the pig hypothalamus. Binding was time dependent, and its dissociation was first order with a half-time at 22°C of 14 min. Desalanine insulin was as effective as native insulin in inhibiting the binding of [ 125 I]insulin, while proinsulin was less effective and desoctapeptide insulin still less effective in accord with their biologic activities. Binding by membranes from cortex and thalamus appeared to be less than from hypothalamus. [ 125 I]lnsulin was infused into an arterial split monkey brain preparation to determine if insulin that was blood borne bound specifically to the primate hypothalamus. Half the brain was perfused with [ 125 I]insulin alone and the other half with [ 125 I]insulin plus an excess of unlabeled insulin. Radioautography showed specific binding of insulin localized to the median eminence, infundibular nucleus, and microvessels. Thus, the monkey and pig hypothalami bind insulin with characteristics similar to those reported for known target tissues for insulin. Furthermore, insulin from the blood stream binds to specific anatomical structures in the hypothalamus of the monkey.

Insulin in the Central Nervous System

Abstract: Publisher Summary This chapter provides an overview of insulin in the central nervous system (CNS). Immunohistochemical studies have been employed to visualize the localization of peptide hormones in the brain and other tissues. Before hypothesizing synthesis of insulin in nonpancreatic tissues, one must determine with some accuracy the insulin concentrations in tissues such as the brain of various species or in IM-9 lymphocytes, or of non-guinea pig insulin in guinea pig tissues. If the concentrations are no more than a few percentage points of the levels initially reported by the NIH laboratory, then some explanation should be given for the erroneously high concentrations that were earlier reported. The absence of mechanisms in nonendocrine cells for the complex processing of insulin precursors to the 6000-dalton peptide and the absence of proinsulin in the extracts of the variety of tissues reported from the NIH laboratory suggest that the insulin found in these extracts was ultimately derived from pancreatic insulin.

Elevated proinsulin biosynthesis in vitro from a rat model of non-insulin-dependent diabetes mellitus

Abstract: Rats were injected with streptozotocin at 2 days of age. After 10-14 wk, the injected rats showed normal body weight with only modestly elevated fed plasma glucose (controls: 118 mg/dl, diabetic rats: 209 mg/dl). No significant difference between control and diabetic rats was found for overnight fasting plasma insulin levels. The diabetic rats had a retarded rate of disposal of intravenously injected glucose, with only 13% of the incremental increase in plasma insulin seen in control rats 2 min after glucose injection. The immunoreactive insulin content of islets isolated from the diabetic rats was 14% of controls. However, these islets had 74% of the B-cell composition of controls, as estimated by quantitative morphometry. Insulin release (30 min) was lower from islets from diabetic rats than from controls, but was not lowered to the same extent as islet insulin content. Proinsulin biosynthesis was estimated by measuring the incorporation of [3H] leucine or of [3H] phenylalanine into immunoprecipitable material. Proinsulin biosynthesis was strikingly elevated in islets from diabetic rats. Although incorporation of labeled amino acids into total islet protein was also elevated for islets from diabetic rats, the elevation was less pronounced than for incorporation into immunoprecipitable material. The diabetic state of these animals is thus associated with glucose intolerance in vivo, and an increased rate of insulin turnover in isolated islets studied in vitro.

Insulin Gene Expression in the Developing Rat Pancreas

Abstract: In the developing rat pancreas from about day 15 until birth there is a marked increase in the concentration of insulin. The present experiments were designed to determine whether enhanced insulin biosynthesis is accompanied by a coordinate increment in proinsulin mRNA. Using a sensitive RNA filter hybridization technique and a cloned rat proinsulin 32 P-cDNA as a hybridizing probe, the concentration of proinsulin mRNA was measured. A greater than 900-fold accumulation of proinsulin mRNA was found, which closely paralleled that of immunoreactive insulin. Proinsulin mRNA and immunoreactive insulin were highest 1–2 days after birth, more than eightfold higher than that in adult pancreas. The patterns of digestion by a number of restriction endonucleases of the two nonallelic insulin genes were identical in adult and fetal DNA and in DNA from an insulinoma. We found no evidence of major gene rearrangement during embryogenesis as a prerequisite to insulin gene expression. Glucose stimulated insulin biosynthesis at the earliest time examined (day 15 of gestation). Insulin biosynthesis was enhanced almost fourfold while proinsulin mRNA differed by less than 20% during a 2-h incubation. This suggested that the short-term effect of glucose on insulin biosynthesis in the developing rat pancreas occurs at the level of translation of existing mRNA, similar to that noted in adult pancreatic islets. These data taken together suggest that the same controls for insulin biosynthesis in the adult exist at these early times in pancreatic development and that the controls for insulin biosynthesis appear coordinately with the appearance of increased insulin during endocrine pancreatic differentiation.

Serum levels of true insulin, C-peptide and proinsulin in peripheral blood of patients with cirrhosis

Abstract: The levels of proinsulin, immunoreactive insulin, true insulin (calculated from the difference, namely immunoreactive insulin-proinsulin) and C-peptide were determined in the fasting state and during a 3-h oral glucose tolerance test after administration of 100 g of glucose in 12 patients with cirrhosis with normal oral glucose tolerance test (50 g) and in 12 healthy subjects serving as controls. In the patients with cirrhosis the serum levels of proinsulin and immunoreactive insulin were significantly higher in the fasting state and after glucose loading than in the healthy subjects. The serum level of true insulin was also higher in the patients with cirrhosis, but the difference was less pronounced and only significant at a few of the time points. The serum level of C-peptide was very similar in both groups. These results emphasize that cirrhosis is a condition in which the serum proinsulin level is raised and that this hyperproinsulinaemia contributes greatly to the increased immunoreactive insulin levels observed in patients with this disease.

The Independence of Insulin Release and Ambient Insulin In Vitro

Abstract: The effect of insulin on its own secretion was tested in three independent experimental models using insulin concentrations that approached physiologic values. The collected secretions from glucose-stimulated islet tissue had no effect on insulin release from other islets. Perifused insulin had no effect on the release of endogenous insulin even when the assay was completely controlled for dilutional effects. Perifused insulin had no effect on the release of prelabeled insulin from glucose-stimulated islets. Similarly, proinsulin did not affect insulin release. Porcine insulin did not affect the function of porcine or canine islets. These studies strongly support the independence of glucose-stimulated insulin secretion and ambient insulin.

DNA strand breaks and poly(ADP-ribose) synthetase activation in pancreatic islets--a new aspect to development of insulin-dependent diabetes and pancreatic B-cell tumors.

Abstract: Alloxan and streptozotocin, which produce diabetes mellitus in experimental animals, have been known to inhibit various functions of pancreatic islets including proinsulin synthesis. However, little is known about the mechanisms underlying the action of these agents in pancreatic islets. Our recent in vivo and in vitro study using rats and isolated islets showed that one of the primary targets of the diabetogenic agents is the DNA of pancreatic islets. The first step is the generation of hydroxyl radical by alloxan which attacks DNA to produce strand breaks. In the case of streptozotocin, the alkylating activity of this compound may be causally related to its ability to induce DNA strand breaks. Subsequently, the fragmented DNA activates poly(ADP-ribose) synthetase which depletes cellular NAD. Since NAD is the most abundant of cellular coenzymes and participates in many biological reactions in mammalian cells, the reduction in intracellular NAD to such a nonphysiological level may severely affect islet cell functions including proinsulin synthesis. These results, in turn, raise the possibility that insulin-dependent diabetes may be preventable by inhibiting the occurrence of DNA strand breaks or the poly(ADP-ribose) synthetase. In fact, by poly(ADP-ribose) synthetase inhibitors such as nicotinamide and picolinamide, alloxan- and streptozotocin-induced NAD depletion was completely prevented, and B-cell functions including proinsulin synthesis proceeded normally. However, poly(ADP-ribose) synthetase inhibitors did not prevent the DNA strand breaks at all. Therefore, B-cells may survive with the residual DNA damage within their genome. About one year after the combined administration to rats of alloxan or streptozotocin with poly(ADP-ribose) synthetase inhibitors, diabetes did not develop but islet B-cell tumors were found frequently. This suggests that insulin-dependent diabetes and B-cell tumors are closely related with respect to their developmental processes. In other words, DNA breaks initiate two kinds of pathological state in B-cells, one is degeneratively and the other is oncogenically expressed.

In vitro lymphocyte proliferation response to therapeutic insulin components. Evidence for genetic control by the human major histocompatibility complex.

Abstract: Genes in the major histocompatibility complex of mice and guinea pigs control immunologic responsiveness to insulins from other animal species. In order to determine if similar genetic control exists in man, we have examined lymphocyte proliferation responses to components of therapeutic insulins by employing lymphocytes from diabetic patients that receive insulin. Distinct groups of individuals demonstrated positive lymphocyte proliferative responses to beef insulin, beef and pork insulin, beef proinsulin, pork proinsulin, and protamine. Lymphocytes from the patient population were typed for the HLA-A, B, C, and DR antigens. An increased frequency of certain HLA antigens was found in those individuals that responded to the following therapeutic insulin components: beef, HLA-DR4; beef and pork, HLA-DR3; beef proinsulin, HLA-BW4, CW2, CW5, DR2, and DR5; protamine, HLA-CW3, CW5, and DR7. The results demonstrate that the human immune system recognized the structural differences between human and beef and/or pork insulin. These differences are two amino acids in the A chain, alpha loop, of beef insulin and the single terminal amino acid, alanine, which is common to pork and beef insulins. Positive responses to both beef proinsulin and pork proinsulin demonstrated the capability of restricted recognition of more complex proteins represented by the C-peptide in these insulin preparations. Lymphocyte proliferative responses to protamine were also restricted, which suggests a genetic control to this antigen. The association of these responses with HLA alloantigens strongly suggests that genes within the human major histocompatibility complex control recognition and lymphocyte response to therapeutic insulin components.

The C-peptide of proinsulin: Its diagnostic use and a possible physiological role.

Abstract: A radioimmunoassay was developed and validated for human C-peptide in unextracted plasma, using a synthetic 31 amino acid human C-peptide for immunogen, standard and tracer The sensitivity of the assay (10 pg/tube) enabled the measurement of both fasting and stimulated circulating C-peptide levels Normal ranges were established in lean healthy volunteers after (a) fasting (b) stimulation of insulin secretion using oral and intravenous stimuli (c) suppression of endogenous insulin secretion using exogenous insulin Human C-peptide measurements were used to investigate patients presenting with hypoglycaemia due to a number of clinical conditions and were found to be of especial use in the differencial diagnosis of the factitious hypoglycaemia of insulin abuse A rat C-peptide radioimmunoassay was developed and validated to investigate the possibility that C-peptide, as well as insulin, inhibits fat stimulated GIP release Both exogenous and endogenous C-peptide were shown to inhibit fat stimulated GIP release in rat fed normal laboratory food However, neither insulin or C-peptide were effective in inhibiting fat stimulated GIP release in rats maintained on shortterm high fat diets Studies were, therefore, extended to investigate the feed-back inhibition of exogenous insulin on GIP release in humans maintained on low and high fat dietary regimens Exogenous insulin was found to be ineffective in inhibiting fat stimulated GIP secretion in subjects maintained on a high fat diet The control of GIP secretion with its consequent effect on insulin secretion via the enteroinsular axis therefore appears to be affected by dietary fat intake

Insulin-Stimulated Methylaminoisobutyric Acid Uptake in 3T3-L1 Fibroblasts and Fat Cells

Abstract: 3T3-L1 fibroblasts and fat cells have been extensively used to study the development of insulin-stimulated glucose and lipid metabolism during adipocyte differentiation in vitro. In this paper we explore the ability of insulin to stimulate amino acid uptake in 3T3-L1 cells using the nonmetabolizable amino acid analog methylaminoisobutyric acid (MAIB). In differentiated 3T3-L1 fat cells, a 12-h preincubation with insulin was required for maximal stimulation of MAIB uptake. In contrast, in the undifferentiated fibroblasts, insulin stimulation peaked between 6 and 8 h and then declined significantly. Maximal stimulation of MAIB uptake in the differentiated fat cell exceeded that in the fibroblast phenotype. This increased ability of insulin to stimulate MAIB uptake in fat cells appeared within the first day after removal of the differentiation medium. 3T3-L1 fat cells were 30 times more sensitive to the effects of insulin on MAIB transport than the undifferentiated fibroblasts. These findings are consistent with previous data on insulin-stimulated deoxyglucose uptake, in that the increased sensitivity to insulin with differentiation is more than can be accounted for by the increase in receptor number. The activity of porcine proinsulin indicates that this stimulation reflects the known characteristics of the insulin receptor. The stimulation of MAIB uptake by insulin in 3T3-L1 fat cells was blocked by inhibitors of protein synthesis (cycloheximide and puromycin) and mRNA synthesis (actinomycin D). Colchicine, an inhibitor of microtubule function, showed little inhibition of insulin-stimulated MAIB uptake. Insulin stimulation of MAIB uptake was greater when 3T3-L1 cells were preincubated with insulin in the absence of essential amino acids. Basal transport in 3T3-L1 cells was not influenced by the presence or absence of amino acids. Thus, amino acid deprivation appears specifically to enhance the ability of insulin to stimulate amino acid transport in cultured adipocytes.

Adaptors, and synthesis and cloning of proinsulin genes

Abstract: A human-like proinsulin gene and its analogs, have been synthesized by a combination of chemical and enzymatic methods. A number of different human-like proinsulin gene analogs with altered C-chains have also been designed and can be readily constructed as described. As a part of the strategy, an adaptor for trimming DNA has been designed, synthesized and used to recover the A-chain insulin gene with the desired sequence from a hybrid plasmid; a related adaptor for trimming DNA has been designed to shorten the C-chain gene or any gene. The synthetic proinsulin gene has been joined to a replicable cloning vehicle and the hybrid DNA transferred to a host cell. The transformed host cell has been shown to contain the desired human-like proinsulin gene.