An alternative to allosteric interactions as causes of sigmoid dose versus response curves. Application to glucose-induced secretion of insulin.

It is shown that a sigmoid dose vs. response curve will occur in any biological system in which the stimulant compound must diffuse significant distances to reach responding cells, even when the cells themselves respond according to Michaelis-Menten kinetics. Isolated pancreatic islets releasing insulin in response to glucose stimulation have been used as a specific example. Since diffusion and/or other physical processes can produce global effects which could account for the sigmoidal nature of a dose vs. response curve, the existence of complex molecular mechanisms of hormone-receptor interaction can not be inferred solely from the character of a dose vs. response relation.     

Biphasic response of insulin secretion to arginine stimulation: daily changes

8 healthy subjects were infused with arginine at h. 08 and h. 16 of different days in order to observe if some changes occurred in the biphasic insulin response according with the different times of the day. A statistical difference between the two responses could be observed only for the first phase of the insulin response (time 0' - 10'), that appeared greater in the morning. When the insulin areas were plotted against the percentage of Ideal Body Weight an highly statistically significant correlation was found.     

Overexpression of protein-tyrosine phosphatase PTP sigma is linked to impaired glucose-induced insulin secretion in hereditary diabetic Goto-Kakizaki rats

The impaired glucose-induced insulin release in type 2 diabetes mellitus may be accounted for by reduced B-cell ATP/ADP ratio or decreased phosphorylation of proteins regulating exocytosis of insulin. This, in turn, could be due to enhanced phosphatase activity. Using in situ hybridization techniques to assess the expression of 11 different phosphotyrosine phosphatases (PTPases), known to be present in the B-cells, overexpression by approximately 60% of PTP sigma (also known as LAR-PTP2 or PTP NE-3) was demonstrated in pancreatic islets and liver of spontaneously type 2 diabetic Goto-Kakizaki (GK) rats. In agreement with these findings Western blot of islet lysates, using a polyclonal PTP sigma antiserum, showed increased amounts of the protein in GK relative to control rat islets. Exposure of isolated islets for 20 h to 5 muM antisense to PTP sigma, composed of an antisense PNA sequence of 15 bases linked to the cell penetrating peptide transportan, increased glucose-induced insulin secretion from GK rat islets, but not from control islets. In parallel, the amounts of the phosphatase decreased. In conclusion, increased expression of PTP sigma may be of pathogenetic significance for the defective insulin secretion in GK rat islets.     

Overexpression of the ped/pea-15 gene causes diabetes by impairing glucose-stimulated insulin secretion in addition to insulin action

Overexpression of the ped/pea-15 gene is a common feature of type 2 diabetes. In the present work, we show that transgenic mice ubiquitously overexpressing ped/pea-15 exhibited mildly elevated random-fed blood glucose levels and decreased glucose tolerance. Treatment with a 60% fat diet led ped/pea-15 transgenic mice to develop diabetes. Consistent with insulin resistance in these mice, insulin administration reduced glucose levels by only 35% after 45 min, compared to 70% in control mice. In vivo, insulin-stimulated glucose uptake was decreased by almost 50% in fat and muscle tissues of the ped/pea-15 transgenic mice, accompanied by protein kinase Calpha activation and block of insulin induction of protein kinase Czeta. These changes persisted in isolated adipocytes from the transgenic mice and were rescued by the protein kinase C inhibitor bisindolylmaleimide. In addition to insulin resistance, ped/pea-15 transgenic mice showed a 70% reduction in insulin response to glucose loading. Stable overexpression of ped/pea-15 in the glucose-responsive MIN6 beta-cell line also caused protein kinase Calpha activation and a marked decline in glucose-stimulated insulin secretion. Antisense block of endogenous ped/pea-15 increased glucose sensitivity by 2.5-fold in these cells. Thus, in vivo, overexpression of ped/pea-15 may lead to diabetes by impairing insulin secretion in addition to insulin action.     

The stimulus-secretion coupling of glucose-induced insulin release. LIII. Calcium-dependency of the cyclic AMP response to nutrient secretagogues

Above a threshold value in excess of 5.6 mM, D-glucose increases the amount of cyclic AMP measured by radioimmunoassay in pancreatic rat islets and their surrounding incubation medium. As judged from the cyclic AMP content of islets exposed to isobutylmethylxanthine (1.0 mM), the glucose-induced increment in the rate of cyclic AMP generation represents a rapid and sustained phenomenon. The stimulant action of glucose on cyclic AMP accumulation is mimicked by L-leucine, and L-glutamine, these amino acids acting synergistically of one another. Trifluoperazine slightly decreases but fails to abolish the effect of glucose. In the absence of extracellular Ca2+, however, the cyclic AMP response to D-glucose, L-leucine and/or L-glutamine is severely impaired. These findings are compatible with the view that an increase in the generation rate of cyclic AMP participates in the process of nutrient-stimulated insulin release. This increase could be secondary to the nutrient-induced accumulation of Ca2+ in the islet cells leading to activation of adenylate cyclase by calmodulin.     

Activation of protein kinase C is essential for sustained insulin secretion in response to cholinergic stimulation

Insulin secretion from isolated rat islets of Langerhans is enhanced by cholinergic agonists, such as carbachol (CCh), in the presence of a stimulatory concentration of glucose. Depletion of islet protein kinase C activity by prolonged exposure to a tumour-promoting phorbol ester did not prevent the initial secretory response to CCh, but markedly reduced the duration of CCh-induced elevated secretory rates. These results suggest that the major action of PKC is in maintaining rather than initiating the insulin secretory response to cholinergic agonists.     

An imidoester spin probe of membrane protein interactions: application to cytochrome c

The synthesis of an imidoester spin label, whose advantages relative to other spin labels include its water solubility, lysine specificity, and retention of positive charge at the reaction site is described. Cytochrome c is spin labeled and shown to exhibit spectral changes upon interacting with lipid vesicles and lipid-rich cytochrome oxidase preparations. Spin labeled cytochrome c in buffer or in the presence of mitochondria at high ionic strength had a correlation time of τ = 0.91 ± 10^?9 s; at low ionic strength the mitochondrial signal was more immobilized, τ = 2.27 ± 0.13 × 10^?9 s; and further immobilization was observed when cytochrome c was bound to the high-affinity site of purified oxidase containing 37% phospholipid (τ = 2.71 ± 0.22 × 10^?9). Cytochrome c-oxidase electron transfer rates were unaltered by spin labeling. The results suggest that this imidoester spin label will be useful for studies of protein-protein and protein-lipid interactions.     

An endotoxin induced serum factor that causes enhancement of antibody response to heterologous erythrocytes.

The sera obtained from blood of the mice, which had been intravenously injected with LPS several hours in advance, contained some active substance capable of enhancing anti-sheep red blood cell (SRBC) antibody responses in mice. Activity of the sera was still retained after passage through a rabbit anti-LPS antibody-coated Sepharose 4B column, but greatly reduced by passage through a rabbit anti-mouse thymocyte antibody-coated Sepharose 4B column. The active substance in the sera was eluted through a Sephadex G-200 column at the same position as the serum albumin. The addition of this substance to B cell rich spleen cell cultures in vitro in the presence of SRBC generated tremendous numbers of antibody forming cells 4 days after the incubation, suggesting that this substance was able to take over the helper function of T cells in thymus dependent antibody responses. However, this substance was not capable of stimulating 3H-thymidine-uptake into cultured spleen cells. The possible role of this substance in the adjuvant effect of LPS is discussed.     

LONGTMR3IGF-I as a more potent alternative to insulin in serum-free culture of HEK293 cells

LONG^TMR³IGF-I, an analogue of insulin-like growth factor (IGF)-I, was specifically engineered for use in biopharmaceutical protein production in mammalian cells. LONG^TMR³IGF-I is capable of supporting the growth and survival of Chinese hamster ovary cells in serum-free media at concentrations at least 200-fold lower than required for insulin. LONG^TMR³IGF-I also acts as a more potent growth and survival factor than either insulin or native IGF-I in SF culture of human embryonic kidney (HEK293) cells. To investigate the basis of the enhanced potency of LONG^TMR³IGF-I we have examined the mechanism of action of these mitogens in HEK293 cells. All mitogens tested were found to activate the TypeI IGF receptor (IGF-IR) and insulin receptor (IR) in a dose-responsive manner. However, the level of activation of both receptors after stimulation with LONG^TMR³IGF-I, at lower concentrations, was greater than with either insulin or IGF-I. The greater potency of LONG^TMR³IGF-I in activating the IR, despite having a low affinity for IRs, suggests the presence of heterotetrameric IGF-IR/IR dimers. Interestingly, the decrease in IGF-IR activation at higher concentrations of LONG^TMR³IGF-I suggests that the dose-response curve may be bell-shaped.     

Effect of octanoic acid on the insulin secretion in response to glucose in vitro (author's transl)

The effect of octanoic acid (1.5 mM) on insulin secretion in 4.4 and 16.7 mM glucose stimulation has been studied in rat's isolated and perfused pancreas. The absence of octanoic acid does not produce any significant insulin secretion increase in response to 4.4 mM glucose infusion, whereas its presence produces a significant insulinic response of a monophasic nature. Both in the presence and absence of octanoic acid, the 16.7 mM glucose-stimulation produces a biphasic insulin secretion. The octanoic acid enhances both the first and the second phase of insulin secretion. The present results show that octanoic acid clearly potentiates the insulin secretion in response to 4.4 mM and 16.7 mM glucose.     

iWRAP: An interface threading approach with application to prediction of cancer-related protein-protein interactions

Current homology modeling methods for predicting protein-protein interactions (PPIs) have difficulty in the “twilight zone” (< 40%) of sequence identities. Threading methods extend coverage further into the twilight zone by aligning primary sequences for a pair of proteins to a best-fit template complex to predict an entire three-dimensional structure. We introduce a threading approach, iWRAP, which focuses only on the protein interface. Our approach combines a novel linear programming formulation for interface alignment with a boosting classifier for interaction prediction. We demonstrate its efficacy on SCOPPI, a classification of PPIs in the Protein Databank, and on the entire yeast genome. iWRAP provides significantly improved prediction of PPIs and their interfaces in stringent cross-validation on SCOPPI. Furthermore, by combining our predictions with a full-complex threader, we achieve a coverage of 13% for the yeast PPIs, which is close to a 50% increase over previous methods at a higher sensitivity. As an application, we effectively combine iWRAP with genomic data to identify novel cancer-related genes involved in chromatin remodeling, nucleosome organization, and ribonuclear complex assembly. iWRAP is available at http://iwrap.csail.mit.edu.     

Identification of RhoGAP22 as an Akt-dependent regulator of cell motility in response to insulin

Insulin exerts many of its metabolic actions via the canonical phosphatidylinositide 3 kinase (PI3K)/Akt pathway, leading to phosphorylation and 14-3-3 binding of key metabolic targets. We previously identified a GTPase-activating protein (GAP) for Rac1 called RhoGAP22 as an insulin-responsive 14-3-3 binding protein. Insulin increased 14-3-3 binding to RhoGAP22 fourfold, and this effect was PI3K dependent. We identified two insulin-responsive 14-3-3 binding sites (pSer(16) and pSer(395)) within RhoGAP22, and mutagenesis studies revealed a complex interplay between the phosphorylation at these two sites. Mutating Ser(16) to alanine blocked 14-3-3 binding to RhoGAP22 in vivo, and phosphorylation at Ser(16) was mediated by the kinase Akt. Overexpression of a mutant RhoGAP22 that was unable to bind 14-3-3 reduced cell motility in NIH-3T3 fibroblasts, and this effect was dependent on a functional GAP domain. Mutation of the catalytic arginine of the GAP domain of RhoGAP22 potentiated growth factor-stimulated Rac1 GTP loading. We propose that insulin and possibly growth factors such as platelet-derived growth factor may play a novel role in regulating cell migration and motility via the Akt-dependent phosphorylation of RhoGAP22, leading to modulation of Rac1 activity.