Insulin-like Biological Activity Research Articles

Expression of the Biologically Active Insulin Analog SCI-57 in Nicotiana Benthamiana.

Diabetes mellitus is a growing problem worldwide; however, only 23% of low-income countries have access to insulin, and ironically it costs higher in such countries than high-income ones. Therefore, new strategies for insulin and insulin analogs production are urgently required to improve low-cost access to therapeutic products, so as to contain the diabetes epidemic. SCI-57 is an insulin analog with a greater affinity for the insulin receptor and lower thermal degradation than native insulin. It also shows native mitogenicity and insulin-like biological activity. In this work, SCI-57 was transiently expressed in the Nicotiana benthamiana (Nb) plant, and we also evaluated some of its relevant biological effects. An expression plasmid was engineered to translate an N-terminal ubiquitin and C-terminal endoplasmic reticulum-targeting signal KDEL, in order to increase protein expression and stability. Likewise, the effect of co-expression of influenza M2 ion channel (M2) on the expression of insulin analog SCI-57 (SCI-57/M2) was evaluated. Although using M2 increases yield, it tends to alter the SCI-57 amino acid sequence, possibly promoting the formation of oligomers. Purification of SCI-57 was achieved by FPLC cation exchange and ultrafiltration of N. benthamiana leaf extract (NLE). SCI-57 exerts its anti-diabetic properties by stimulating glucose uptake in adipocytes, without affecting the lipid accumulation process. Expression of the insulin analog in agroinfiltrated plants was confirmed by SDS-PAGE, RP-HPLC, and MS. Proteome changes related to the expression of heterologous proteins on N. benthamiana were not observed; up-regulated proteins were related to the agroinfiltration process. Our results demonstrate the potential for producing a biologically active insulin analog, SCI-57, by transient expression in Nb.

Isolation and characterization of polyphenol type-A polymers from cinnamon with insulin-like biological activity.

The causes and control of type 2 diabetes mellitus are not clear, but there is strong evidence that dietary factors are involved in its regulation and prevention. We have shown that extracts from cinnamon enhance the activity of insulin. The objective of this study was to isolate and characterize insulin-enhancing complexes from cinnamon that may be involved in the alleviation or possible prevention and control of glucose intolerance and diabetes. Water-soluble polyphenol polymers from cinnamon that increase insulin-dependent in vitro glucose metabolism roughly 20-fold and display antioxidant activity were isolated and characterized by nuclear magnetic resonance and mass spectroscopy. The polymers were composed of monomeric units with a molecular mass of 288. Two trimers with a molecular mass of 864 and a tetramer with a mass of 1152 were isolated. Their protonated molecular masses indicated that they are A type doubly linked procyanidin oligomers of the catechins and/or epicatechins. These polyphenolic polymers found in cinnamon may function as antioxidants, potentiate insulin action, and may be beneficial in the control of glucose intolerance and diabetes.

Insulin-like biological activity of culinary and medicinal plant aqueous extracts in vitro.

To evaluate the possible effects on insulin function, 49 herb, spice, and medicinal plant extracts were tested in the insulin-dependent utilization of glucose using a rat epididymal adipocyte assay. Cinnamon was the most bioactive product followed by witch hazel, green and black teas, allspice, bay leaves, nutmeg, cloves, mushrooms, and brewer's yeast. The glucose oxidation enhancing bioactivity was lost from cinnamon, tea, witch hazel, cloves, bay leaf and allspice by poly(vinylpyrrolidone) (PVP) treatment, indicating that the active phytochemicals are likely to be phenolic in nature. The activity of sage, mushrooms, and brewers's yeast was not removed by PVP. Some products such as Korean ginseng, flaxseed meal, and basil have been reported to be effective antidiabetic agents; however, they were only marginally active in our assay. Our technique measures direct stimulation of cellular glucose metabolism, so it may be that the active phytochemicals in these plants improve glucose metabolism via other mechanisms or that this in vitro screening is not a reliable predictor of hypoglycemic effects in vivo for some products. In summary, the positive effects of specific plant extracts on insulin activity suggest a possible role of these plants in improving glucose and insulin metabolism.

Determinants of growth-promoting activity reside in the A-domain of insulin-like growth factor I.

A two-chain, disulfide linked, insulin-like compound embodying the A-domain of insulin-like growth factor I (IGF-I) and the B-chain of insulin has been synthesized and characterized with respect to insulin-like biological activity and growth-promoting potency. The compound displays a potency of ca. 41% relative to insulin in assays for insulin-like activity (e.g., lipogenesis) but significantly higher activity than insulin, ca. 730% relative to insulin, in growth factor assays (e.g., thymidine incorporation). The compound is, however, a less potent growth factor than IGF-I itself, ca. 26.5% relative to IGF-I, and is not recognized by IGF carrier proteins. We conclude that structural features contained in the A-domain of IGF-I are primarily responsible for the growth-promoting ability displayed by IGF-I, while features in the B-domain are responsible for recognition by IGF carrier proteins.

Isolation and Partial Characterization of Insulin of the Honeybee (Apis Mellifica)

In the honeybee (Apis mellifica), insulin-like material was partially purified with acid ethanol extractions by a classic method for recovering insulin and following gel filtration on a Sephadex G-50 column. The preparations were characterized by their ability to cross-react with porcine insulin antibodies. Insulin-like biological activity was demonstrated using the insulin bioassay. Stimulation of glucose oxidation or lipogenesis was measured by isolated rat adipocytes. Insulin seems to be more widespread in invertebrates than was previously assumed.

Insulin-like activity of chromium-binding fractions from brewer's yeast

51CrCl 3 was added to the incubation medium of Saccharomyces cerevisiae for up to 48 hr. After repeated freezing and thawing, lysing in 9 M urea with 1% NP-40 detergent, and dialysis against water, the lower molecular weight (Mr < 3500) dialysate was retained on a SE53 cationic exchange column, eluted with 0.25 M NH 4OH and fractionated on a Bio-gel P-2 column. The insulinlike biological activity of the fractions was measured by the 14C-glucose oxidation in isolated rat adipocytes. The biological activity that was found in two of nine fractions did not correspond to their chromium content. Moreover, identical findings were obtained when chromium was added not to the live yeast but to the yeast extract, which showed that its binding was a chemical process not requiring cellular activity. No fraction demonstrated insulin-potentiating activity on rat adipocytes.

Identification of an insulin fragment produced by an insulin degrading enzyme, neutral thiopeptidase

Previous studies have shown that neutral thiopeptidase (E.C.3.4.22.11, insulinase) degrades (processes) insulin with a high affinity ( K m = 30 × 10 −9 M). In the current studies, insulin was subjected to digestion with a highly purified rat liver neutral thiopeptidase and the peptides generated were separated by HPLC using a C 8 column. With the use of structural analysis (which included the determination of amino terminal residues and amino acid composition), the major product was identified as a peptide containing portions of both chains of insulin, A 1 to A 13 and B 1 to B 9 having two disulfide bonds, an interchain disulfide bond and presumably the intra-A chain disulfide bond as well. Examination of insulin-like biological activity using a primary cultured hepatocyte test system showed that the fragment promoted neither short-term (α-aminoisobutyric acid uptake) nor long-term (glycogen synthesis) bioactivities of insulin.

Insulin-like growth factors/somatomedins: structure, secretion, biological actions and physiological role.

Insulin-like growth factors (IGFs, somatomedins) are structural homologues of insulin with insulin-like biological activity. They are mainly synthesized and secreted by the liver, but may also be produced by extrahepatic tissues. In their native from in blood they are bound to specific carrier protein(s). This determines essentially their biological actions. The complexed factors stimulate growth indices in vitro and in vivo, but, in contrast to the free factors, do not exert acute effects on insulin target tissues.

Specific binding sites for insulin in the rat pituitary gland

Studies were carried out in order to characterize specific insulin binding sites in the rat pituitary gland. Binding of labeled insulin by pituitary microsomes reached equilibrium after 4 h at 4° C and remained stable over 16 h; at 25°C the plateau was reached in 20 min. Equilibrium binding data analysis of competitive displacement of bound 125I-iodo insulin by unlabeled insulin yielded a non-linear Scatchard plot. At 25°C the K d for the high affinity component was 2.8 ± 0.1 × 10 −9 M and the receptor concentration was 260 ± 80 fmol/mg of microsomal protein. A K d value of 4.6 ± 0.4 × 10 −8 M and a binding capacity of 800 ± 200 fmol/mg microsomal protein were obtained for the low affinity sites. Insulin binding to microsomes was enhanced 2.7 times by increasing the ionic strength of the incubation medium with 2 M NaCl, and was abolished when the microsome preparation was preincubated with trypsin prior to binding measurements. Other hormones, such as bovine thyrotropin, ovine follitropin, human somatotropin and ovine prolactin did not interact with the insulin receptor. Proinsulin displaced the labeled hormone in direct proportion to its insulin-like biological activity.

Insulin binds to specific receptors and stimulates 2-deoxy-D-glucose uptake in cultured glial cells from rat brain.

The kinetics of 125I-insulin binding and physiological activity of insulin on glial cells cultured from brains of 1-day-old rats have been studied. Binding of 125I-insulin to cultured glial cells was specific, reversible, and time-dependent. Porcine and chicken insulin competed equally for 125I-insulin binding while other hormones or insulin analogs competed in proportion to their insulin-like biological activity. Incubation of glial cultures with insulin resulted in a time- and dose-dependent stimulation of 2-deoxy-D-glucose uptake. Maximal stimulation (190% of control) was observed with 18 nM insulin, and 0.1 nM insulin caused half-maximum effect. The stimulatory effect of insulin on 2-deoxy-D-glucose uptake was due to its effect on Vmax without affecting the Km. These observations suggest that insulin stimulates glucose uptake in glial cells cultured from rat brain, the effect mediated by insulin specific receptors.

Insulin-like peptides in the lobster Homarus americanus II. Insulin-like biological activity

The in vitro incorporation of [ 14C]glucose into glycogen in lobster muscle was used to measure insulin-like biological activity. Glycogenesis was significantly increased by the same hepatopancreas eluate which was previously found to have the greatest insulin immunoreactivity. Hemolymph but not gut extract also increased the rate of glycogenesis.

Insulin-ricin B chain conjugate has enhanced biological activity in insulin-insensitive cells.

Insulin-ricin B chain conjugate, a hybrid molecule consisting of insulin covalently linked to the binding chain of ricin, was tested for insulin-like biological activity in HTC and H35 rat hepatoma cells, rat adipocytes, rat thymocytes, and human fibroblasts. In H35 cells and adipocytes, cells that have abundant insulin receptors and are very sensitive to insulin (ED50 of 30 pM and 50 pM, respectively), the conjugate had 5% the biological activity of native insulin (ED50 of 500 pM and 1000 pM, respectively). Since the insulin portion of the conjugate has 5% the potency of native insulin in binding to the insulin receptor, these observations suggested that (in these cells) the conjugate was acting via the insulin receptor. Moreover lactose and galactose, potent inhibitors of ricin binding to its receptor, had no effect on the action of the conjugate on H35 cells. In contrast, in thymocytes, HTC cells, and fibroblasts cells that have relatively few insulin receptors and require high concentrations of insulin to elicit biological actions (ED50 of 10 nM, 20 nM, and 1 nM, respectively), the conjugate had more biological activity than was predicted on the basis of its ability to bind to the insulin receptor. In addition, in these three insulin-insensitive cell types, the activity of the conjugate was inhibited by either lactose or galactose. Thus, these observations indicate that in cells which are relatively insensitive to insulin, the biological effects of the conjugate require the interaction of the ricin B chain moiety with the ricin receptor. In addition, in fibroblasts, the activity of the conjugate was inhibited by the addition of a monoclonal antibody to the insulin receptor which inhibits the response of fibroblasts to insulin. These data suggest, therefore, that in insulin-insensitive cells the binding of the ricin B chain moiety to its receptor enhances the interaction of the insulin portion of the conjugate with the insulin receptor.

Insulin-cholera toxin binding unit conjugate: a hybrid molecule with insulin biological activity and cholera toxin binding specificity.

The polypeptide hormone insulin and the binding unit of cholera toxin (CTB) were coupled via a disulfide bond. This hybrid molecule had 1/30 the ability of native insulin to bind to the insulin receptor and 1/30 the biological activity of native insulin in H35 rat hepatoma cells and rat adipocytes. Thus, in these two cell types that are very sensitive to insulin, the biological activity of the hybrid molecule was as predicted on the basis of the ability of the molecule to interact with the insulin receptor. In contrast, in HTC rat hepatoma cells and rat thymocytes, two poorly responsive cell types, the insulin-CTB conjugate had 1/3 the biological activity of native insulin, a value 10 times greater than its insulin receptor binding potency. This increased activity of the conjugate did not appear to be due to cholera toxin in the preparation, since a control of uncoupled CTB had no biological activity. Furthermore, native cholera toxin increased intracellular levels of cAMP by 20-fold, whereas the conjugate had no effect on cAMP levels. The CTB moiety did, however, contribute to the biological activity of the conjugate, since the activity of the hybrid molecule, like cholera toxin, was inhibited by gangliosides, whereas the activity of native insulin was not. Finally, the binding to thymocytes of insulin-CTB conjugate, but not insulin, was inhibited by gangliosides. Thus, a hybrid hormone molecule has been constructed which has insulin-like biological activity with the receptor specificity of cholera toxin in poorly responsive cells.

Isolation of material displaying insulin-like immunological biological activity from the brain of the blowfly Calliphora vomitoria.

An insulin-like material from the brain of the blowfly Calliphora vomitoria was partially purified by acid alcohol extraction, gel filtration and ion-exchange cellulose chromatography. In addition, the RF value on polyacrylamide-gel electrophoresis was determined. The material was characterized by its ability to cross-react with bovine insulin antibody and by displaying diminished immunoreactivity on dilution. It displaced specifically bound 125I-labelled insulin from rat liver plasma membrane insulin receptors and displayed insulin-like biological activity on the isolated rat fat-cell. Within 30 min of injection into Calliphora, made hypertrehalocaemic and hyperglucaemic as a result of median neurosecretory cell removal, it caused the concentrations of both sugars to return to normal. The hypothesis is put forward that the median neurosecretory cells are the source of the material.

Interaction of Concanavalin A and Wheat Germ Agglutinin with the Insulin Receptor of Fat Cells and Liver

Abstract Wheat germ agglutinin enhances the specific binding of insulin to isolated fat cells and to liver cell membranes at a concentration of about 1 µg per ml. Wheat germ agglutinin increases insulin binding by increasing the rate of insulin-receptor complex formation; the protein does not alter the rate of dissociation of the insulin-membrane complex or the total number of binding sites for insulin. Higher concentrations of wheat germ agglutinin, as well as concanavalin A, block the binding of insulin to fat cells when the plant lectins are added to the cells before insulin. If the insulin-receptor complex is formed before addition of the plant lectin, only an increase in insulin binding with wheat germ agglutinin and virtually no effect of concanavalin A are demonstrable. Studies using combinations of both plant lectins suggest that these proteins are binding to different regions of the insulin receptor and that some lectin molecules can bind to the cell in a way which is without effect on insulin binding unless the other lectin is also present. Wheat germ agglutinin and concanavalin A modify insulin binding in membrane preparations in a manner similar to that described for intact fat cells. Similar alterations of insulin binding occur with soluble preparations of the insulin receptor of liver and fat cell membranes. These results indicate that the changes in the binding of insulin occur by direct binding of the plant lectins to the insulin receptor macromolecule. All of the effects of the plant lectins are reversed rapidly by adding simple sugars having selective specificity for the proteins, N-acetyl-d-glucosamine (for wheat germ agglutinin) or α-methyl-d-mannopyranoside (for concanavalin A). Digestion of fat cells with trypsin, which results in a marked fall in the affinity of the membrane receptor for insulin, similarly reduces the apparent affinity of wheat germ agglutinin for the membrane sites with which the lectin interacts to enhance glucose oxidation in the fat cells. These studies suggest that the insulin-like biological activity of wheat germ agglutinin may result from direct interactions with the insulin receptor in a way which closely resembles the interaction with insulin. If concanavalin A stimulates glucose transport by interacting with the insulin receptor, such an interaction must be somewhat dissimilar from that of insulin since tryptic digestion of cells does not markedly affect the biological response to this protein. These studies indicate that the insulin-binding macromolecules of liver and fat cell membranes are proteins of complex carbohydrate composition which have several chemically distinct sites capable of binding plant lectins in a manner which perturbs the insulin-receptor interaction.