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During feeding, increases in circulating pancreatic insulin inhibit hepatic glucose output through the activation of the Ser/Thr kinase AKT and subsequent phosphorylation of the forkhead transcription factor FOXO1 (refs 1-3). [...]

Acylated and unacylated ghrelin (AG and UAG) are gut hormones that exert pleiotropic actions, including regulation of insulin secretion and glucose metabolism. In this study, we investigated whether AG and UAG differentially regulate portal and systemic insulin levels after a glucose load. We studied the effects of the administration of AG (30 nmol/kg), UAG (3 and 30 nmol/kg), the ghrelin receptor antagonist [D-[Lys.sup.3]]GHRP-6 (1 [micro]mol/kg), or various combinations of these compounds on portal and systemic levels of glucose and insulin after an intravenous glucose tolerance test (IVGTT, o-glucose 1 g/kg) in anesthetized fasted Wistar rats. UAG administration potently and dose-dependently enhanced the rise of insulin concentration induced by IVGTT in the portal and, to a lesser extent, the systemic circulation. This UAG-induced effect was completely blocked by the coadministration of exogenous AG at equimolar concentrations. Similarly to UAG, [o-[Lys.sup.3]]GHRP-6, alone or in combination with AG and UAG, strongly enhanced the portal insulin response to IVGTT, whereas exogenous AG alone did not exert any further effect. Our data demonstrate that, in glucose-stimulated conditions, exogenous UAG acts as a potent insulin secretagogue, whereas endogenous AG exerts a maximal tonic inhibition on glucose-induced insulin release. acylated ghrelin; insulin secretion; portal vein; rat

We have previously demonstrated that long-term exposure of bovine tracheal smooth muscle (BTSM) strips to insulin induces a functional hypercontractile phenotype. To elucidate molecular mechanisms by which insulin might induce maturation of contractile phenotype airway smooth muscle (ASM) cells, we investigated effects of insulin stimulation in serum-free primary BTSM cell cultures on protein accumulation of specific contractile phenotypic markers and on the abundance and stability of mRNA encoding these markers. In addition, we used microscopy to assess insulin effects on ASM cell morphology, phenotype, and induction of phosphatidylinositol (PI) 3-kinase signaling. It was demonstrated that protein and mRNA levels of smooth muscle-specific contractile phenotypic markers, including sm-myosin, are significantly increased after stimulation of cultured BTSM cells with insulin (1 [micro]M) for 8 days compared with cells treated with serum-free media, whereas mRNA stability was unaffected. In addition, insulin treatment promoted the formation of large, elongate ASM cells, characterized by dramatic accumulation of contractile phenotype marker proteins and phosphorylated [p70.sup.S6K] (downstream target of PI 3-kinase associated with ASM maturation). Insulin effects on protein accumulation and cell morphology were abrogated by combined pretreatment with the Rho kinase inhibitor Y-27632 (1 [micro]M) or the PI 3-kinase inhibitor LY-294002 (10 [micro]M), indicating that insulin increases the expression of contractile phenotypic markers in BTSM in a Rho kinase- and PI 3-kinase-dependent fashion. In conclusion, insulin increases transcription and protein expression of contractile phenotypic markers in ASM. This could have important implications for the use of recently approved aerosolized insulin formulations in diabetes mellitus. airway smooth muscle; hypercontractile phenotype; insulin; phosphatidylinositol 3-kinase; Rho kinase doi:10.1152.ajpcell.00502.2006

The mechanisms responsible for controlling growth rates and size assessment in insects are discussed. Researchers have found that the insulin-dependent growth of the prothoracic gland is involved in assessing when critical weight is reached and mutations in DHR4, a repressor of ecdysone signaling, reduce critical weight and adult size.

The redox chemistry of insulin was investigated at glassy carbon (GC) electrodes that were coated with films of chitosan (CHIT) and multiwalled carbon nanotubes (CNT). While bare electrodes deactivated quickly during insulin oxidation, the GC electrodes coated with CHIT and CHIT-CNT films generated stable insulin currents. The GC/ CHIT-CNT electrodes were used for investigating the electrooxidation process of insulin and amperometric determination of insulin. The mass spectrometric, electron paramagnetic resonance, and separation studies of electrolyzed insulin solutions suggested that the loss of 4 mass units upon insulin oxidation at CNT could be accounted for by the formation of two dityrosine cross-links intramolecularly. At a potential of 0.700 V and physiological pH 7.40, the GC/CHIT-CNT electrodes displayed a detection limit of ~30 nM insulin (S/N = 3), sensitivity of 135 mA [M.sup.-1] [cm.sup.-2], linear dynamic range from 0.10 to 3.0 [micro]M ([R.sup.2] = 0.995), and superior operational and long-term stability. The CNT-based electrodes are promising new insulin detectors for diabetes-related studies such as fast chromatographic analysis of therapeutic insulin formulations or evaluation of quality of pancreatic islets prior to their transplantation.

We report a novel enzyme-based microcantilever sensor by using layer-by-layer nanoassembly modification. A glucose oxidase (GOx) functionalized microcantilever underwent bending when it was exposed to glucose solutions. The magnitudes of bending were proportional to the concentrations of glucose. The cantilever bending was specific toward glucose, but not to other sugars such as mannose, fructose, or galactose. The flow rate effect on the cantilever bending response is also discussed. The bending mechanism was investigated, and the kinetic and thermodynamic analysis and experimental results showed that the conformational change of GOx and gluconic formation were the origin of cantilever deflection.

Voltage-gated [K.sup.+] channel activities are involved in regulating growth factor-stimulated cell proliferation in a variety of cell types. Here we report that suppression of a voltage-gated [K.sup.+] channel with 4-aminopyridine (4-AP), barium, and tetraethylammonium inhibited both EGF- and insulin-stimulated myeloblastic leukemia ML-1 cell proliferation in a concentration-dependent manner. Both MAPK/ERK and Akt pathways are known to mediate cell proliferative signals of a variety of growth factors including insulin. In serum-starved ML-1 cells, insulin rapidly stimulated phosphorylation of ERK1/2 and Akt, and the phosphorylation levels peaked ~30 min after treatment. Pretreatment of ML-1 cells with 4-AP potently and dose-dependently prevented phosphorylation of ERK1/2 and Akt. However, insulin-induced activation of the Akt pathway also played a role in promoting ML-1 cell proliferation. Flow cytometry analysis revealed that although ML-1 cells were primarily arrested at [G.sub.1] phase by serum starvation for 36 h, they reentered the cell cycle after treatment with serum or insulin for 24 h. However, concomitant 4-AP treatment was able to attenuate cell cycle progression in synchronized ML-1 cells stimulated with growth factors. Our results strongly suggest that a 4-AP-sensitive [K.sup.+] channel activity plays an important role in controlling proliferation of ML-1 cells by affecting the activation of multiple signal transduction processes induced by insulin. growth factors; myeloblastic cells; signaling; ion channel blocker

Fat tissue produces a variety of secreted proteins (adipocytokines) with important roles in metabolism. We isolated a newly identified adipocytokine, visfatin, that is highly enriched in the visceral fat of both humans and mice and whose expression level in plasma increases during the development of obesity. Visfatin corresponds to a protein identified previously as pre-B cell colony-enhancing factor (PBEF), a 52-kilodalton cytokine expressed in lymphocytes. Visfatin exerted insulin-mimetic effects in cultured cells and lowered plasma glucose levels in mice. Mice heterozygous for a targeted mutation in the visfatin gene had modestly higher levels of plasma glucose relative to wild-type littermates. Surprisingly, visfatin binds to and activates the insulin receptor. Further study of visfatin's physiological role may lead to new insights into glucose homeostasis and/or new therapies for metabolic disorders such as diabetes., Recent work in obesity research has revealed that adipose tissue functions as an endocrine organ, producing a variety of secreted factors including leptin (1), adiponectin/ACRP30/ AdipoQ (2-4), tumor necrosis factor-α [...]

In pancreatic [beta]-cells, insulin selectively up-regulates the transcription of its own gene and that of the glucokinase gene by signaling through the two isoforms of the insulin receptor, i.e., A-type (Ex11-) and B-type (Ex11+), using different signaling pathways. However, the molecular mechanism(s) that allows the discrete activation of signaling cascades via the two receptor isoforms remains unclear. Here we show that activation of the insulin promoter via A-type and of the glucokinase promoter via B-type insulin receptor is not dependent on receptor isoform-specific differences in internalization but on the different localization of the receptor types in the plasma membrane. Our data demonstrate that localization and function of the two receptor types depend on the 12-amino acid string encoded by exon 11, which acts as a sorting signal rather than as a physical spacer. Moreover, our data suggest that selective activation of the insulin and glucokinase promoters occurs by signaling from noncaveolae lipid rafts that are differently sensitive toward cholesterol depletion.

Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P < 0.01) i.v. infused with insulin (1.5 milliunits/kg of fat-free mass per min) while clamping glucose, amino acids, glucagon, and growth hormone. Increased ATP production occurred in association with increased mRNA levels from both mitochondrial (NADH dehydrogenase subunit IV) and nuclear [cytochrome c oxidase (COX) subunit IV] genes (164-180%) encoding mitochondrial proteins (P < 0.05). In addition, muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (P < 0.05). Further studies demonstrated no effect of low to high insulin levels on muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16-26% (P < 0.02) when four different substrate combinations were used. In conclusion, insulin stimulates mitochondrial oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels. cytochrome c oxidase | NADH dehydrogenase subunit IV | amino acids | citrate synthase

Dietary restriction has been shown to have several health benefits including increased insulin sensitivity, stress resistance, reduced morbidity, and increased life span. The mechanism remains unknown, but the need for a long-term reduction in caloric intake to achieve these benefits has been assumed. We report that when C57BL/6 mice are maintained on an intermittent fasting (alternate-day fasting) dietary-restriction regimen their overall food intake is not decreased and their body weight is maintained. Nevertheless, intermittent fasting resulted in beneficial effects that met or exceeded those of caloric restriction including reduced serum glucose and insulin levels and increased resistance of neurons in the brain to excitotoxic stress. Intermittent fasting therefore has beneficial effects on glucose regulation and neuronal resistance to injury in these mice that are independent of caloric intake. caloric restriction | hippocampus | insulin | [beta]-hydroxybutyrate | ketosis

Mutational analysis indicate that insulin possessing a tertiary fold can express the biological activity provided the molecule is flexible and has the propensity to form the secondary structure as seen in the mutant insulin. This suggests that native insulin's compact secondary structure is not required for biological activity.

Results indicate that the insulin B-chain central helix can accommodate beta-branched aliphatic amino acids, such as valine, at the B12 position by virtue of their receptor binding potency and their ability to form dimers. Data reveal that valine A3 and B12 exhibit similar structure-activity relationships and insulin-receptor interaction.

Research demonstrates that segmental unfolding of insulin's alpha-helix span A1-A8 is brought about by untethering of the disulfide bridge and it is not influenced by the polarity of the serine side chain.

Results show that cadmium binding of the copper containing insulin crystals changes EPR as well as combined EPR and Electron spin-echo envelope modulation spectral characteristics of the insulin. Data indicate that the changes are due to alteration in the insulin structure.

A new method is described for direct monitoring of the conformational stability of proteins that are physically adsorbed from solution onto a solid substrate. The adsorption-induced conformational changes of insulin are studied using a combination of hydrogen/deuterium (H/D) exchange and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The effect of the surface hydrophobicity on the adsorption-induced conformational changes in the insulin structure is probed by adsorbing insulin on a hydrophilic silica and a hydrophobic methylated silica surface before subjecting the insulin molecules to the isotopic exchange process. The present study describes the experimental procedure of this new application of MALDI. Results show that insulin is more highly and more irreversibly adsorbed to a hydrophobic methylated silica surface than to a hydrophilic silica surface. Hydrogen-exchange experiments clearly demonstrate that the strong interaction of insulin with the hydrophobic surface is accompanied by a strong increase in the H/D-exchange rates and thus in a reduction in the insulin native structural stability. In contrast, H/D-exchange rates of insulin are somewhat reduced upon adsorption on silica from solution.

Several phosphoinositolglycan (PIG) compounds were synthesized composed of the complete or shortened/mutated glycan moiety derived from the structure of the glycosylphosphatidylinositol (GPI) anchor of the GPI-anchored protein, Gce1p, from Saccharomyces cerevisiae. Findings indicate the strength and specificity of the interaction of PIG molecules with putative signal components in adipose and muscle cells which finally result to insulin-mimetic metabolic activity even in insulin-resistant states.

An examination of the effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae reveals that glycogen accumulation and glycogen synthase activity was enhanced by 40% to 60% by human insulin. Under two conditions of growth limitation, it was found that insulin causes glycogen phosphorylase and glycogen synthase to be more sensitive to inactivation and activation, respectively. The effects are observed to be specific and selectively act on storage carbohydrate metabolism.

Exploration of the effects of heterotropic interactions between insulin hexamer binding sites found that applying the Seydoux, Malhotra, and Bernhard (SMB) model provides for the agreement between ligand binding behavior and X-ray crystal structure data. Experimental data and computer simulation support a three-state SMB model with two positive homotropic cooperative transitions connected by a negative homotropic interaction. An equation describes the effects of transition parameters on the ligand binding properties of insulin hexamers.

Quantitative studies of the allosteric binding 2,6- and 2,7-DHN to both the Co(II)-substituted wild type and the GluB13-Gln mutant insulin hexamers show the sensitivity of ligand binding to changes in the side chains that constitute phenolic binding pockets. Results show that ligand affinity and specificity and negative cooperative subunit interactions come from the differences in the binding sites of T3degreeR3degree and R6 resulting from the symmetry properties of these oligomers. Data indicate that pairwise pseudosymmetry is also an important property of insulin.

The insulin family of peptides and their receptors influence cellular growth in very early preimplantation embryos. In this study their expression and role in renal organogenesis was investigated. By immunofluorescence microscopy and in situ hybridization, insulin receptor (IR) expression was seen in the ureteric bud branches and early nephron precursors in mouse metanephroi harvested at day 13 of gestation. The expression gradually decreased in successive stages of gestation, and it was confined mainly to renal tubules in 1-week-old mice. Similar developmental regulation of the IR and insulin was observed by reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. Addition of insulin into the culture medium at low concentrations, ranging from 40 to 400 ng/ml, induced trophic changes and increased [3H]thymidine incorporation in the embryonic renal explants, and inclusion of IR [Beta]-subunit-specific antisense oligodeoxynucleotide caused marked dysmorphogenesis and growth retardation of the metanephroi. Specificity of the antisense effect was reflected by immunoprecipitation experiments in which translational blockade of the [Beta] subunit of the IR was observed. RT-PCR analyses revealed that the a subunit of the IR was unaffected by the antisense treatment of metanephric explants. Concomitantly, de novo synthesis of morphogenetic regulatory extracellular matrix proteins, especially the proteoglycans, was decreased. Gel-shift analyses indicated a failure in the activation of c-fos promoter region binding protein(s) by insulin in the antisense oligodeoxynucleotide-treated explants. These studies suggest that insulin and its putative receptor are developmentally regulated in the murine embryonic metanephros, and they play a role in renal organogenesis, possibly by affecting other modulators of morphogenesis - i.e., extracellular matrix proteins and protooncogenes.

The energy expenditure of three isocaloric oral loads composed of glucose, fat or both fat and glucose were evaluated in ten normal weight subjects. Results showed that the energetic cost of glucose and fat utilization and/or storage is the same while the main source of energy after each load corresponds to the substrate ingested. Plasma insulin and free fatty acids (FFA) were also found to influence the substrate oxidative patterns. Results also suggest that alimentary fat may contribute to fat oxidation by maintaining elevated plasma FFA levels.

The crystal structure of desheptapeptide (B24-B30) insulin (DHPI), a virtually inactive analog of insulin, was determined at 1.6 [Angstrom] resolution. In the refined structure model, DHPI retains three [Alpha]-helices (A1-A8, A12-A18, and B9-B19) as its structural framework, while great conformational changes occur in the N and C termini of B-chain. The [Beta]-turn, which lies in B20-B30 in insulin and insulin analogs with high potency, no longer exists in DHPI. Relative motion is observed among the three [Alpha]-helices, each as a rigid functional group. In contrast, a region covering B5-B6 and A6-A11 exhibits a relatively stable conformation. We interpret our results as identifying: (i) the importance of [Beta]-turn in determining the receptor-binding potency of insulin and (ii) a leading role of [Phe.sup.B24] in maintaining the [Beta]-turn structure.

Fluorescence spectroscopy was done to characterize the conformational states of recombinant forms of insulin holoreceptor in which insulin binding and ligand-induced autophosphorylation are normal. The results confirmed the applicability of the fluorescence spectroscopic technique in monitoring the real time changes in insulin receptor conformation. The method was also found useful for elucidating the mechanism of insulin action in terms of receptor autophosphorylation and activation.

The effect of T-tubule disruption on glucose transport was examined with an enhanced immunogold labeling procedure for plastic-embedded thin sections and a hypertonic shock technique. Transgenic mice overexpressing either Glut1 or Glut4 in skeletal muscle were also used. Data demonstrate that Glut1 and Glut4 are targeted to different plasma membrane domains in skeletal muscle. Basal transport was also found to occur via Glut1 localized at the sarcolemma and insulin-induced transport occurs primarily via Glut4 located at the T-tubule. Data also indicate that insulin induces conformational changes in skeletal muscle Glut4.

A study of the different features of the folding constancy revealed by the insulin monomer shows specific areas whose conformation is altered by site-directed mutagenesis. Hydrogen bonds are produced to increase the constancy of the insulin fold. The N- and C-caps of alpha-helices are also changed to increase the constancy. The insulin molecule reveals increased biological activity due to certain mutations which contribute to the powerful constancy of the A-chain NH2-terminal helix and the B-chain helix at the core.

The mediation of acute insulin action on glucose transporter translocation by activated, GTP-bound Ras protein was studied. 3T3-L1 fibroblasts were subjected to 2-deoxyglucose uptake measurement, immunoprecipitate characterization, subcellular membrane fractionation and glucose transporter protein immunobblot analysis. The intermediation of Ras proteins in the insulin signaling pathway was established.

A study was conducted on the spatial requirements of the B12 region in insulin and the biological properties of synthesized insulin analogues in which other hydrophobic amino acid residues replaced the B11 and B12 residues. The analogues that resulted from alpha-aminoisobutyric acid, D-Ala and Phe substitution of B12 Val were found to exhibit biological activities of 8.5%, 2% and 0.2%, respectively compared to natural insulin, while the analogue formed by inverting the B11-B12 sequence, had 3.3% activity. Results indicate that the B12 position's Val residue plays a role in the special side-chain packing requirements that contributes to insulin's structural stability.

Ovariectomized rats yield hepatocytes which reveal the influences of female sex hormones on the binding process of insulin and disintegration of insulin, which is controlled by a receptor. This process is affected by perinatal and peripubertal androgenization. The cell surface insulin receptor number is augmented to enhance insulin binding and insulin degeneration, controlled by a receptor, in estradiol treatment. The events of binding and disintegration are directly proportional and equally high under the influence of progesterone. A rise in binding and fall in disintegration are caused by perinatal exposure to testosterone, and a total prevention of enhancements controlled by progesterone is seen.

A method for quantitative determination of amide proton exchange rates of the order of reciprocal hours is presented. The method, which makes use of the 2D nuclear magnetic resonance spectroscopy for recording of the exchange processes, is applied in the study of the 50-residue des-(Phe(B25)) mutant of human insulin. Results show that the exchange rates of the mutant insulins' amide protons have been successfully measured and experimental conditions exert an influence on the rate of proton exchanges.

Research determined the structure, solubility and stability of such hydrophobic ion pairing (HIP) phenomenon as the sodium dodecyl sulfate-insulin complex which resulted from the addition of anionic detergent SDS to acqueous insulin solutions using circular dichroism spectroscopy. Results show that SDS decreased the solubility of insulin in water while increasing its solubility in organic matter. These findings support the effectivenes of the HIP tecchnique in obtaining peptide and protein solutions.

Insulin receptor gene mutants were studied with respect to the regulation of insulin action in transfected NIH-3T3 fibroblasts. Unlike wild-type human insulin receptors, the mutants were found to hinder insulin-stimulated c-jun expression and thymidine incorporation. The Val382 mutant, which is defective in receptor autophosphorylation, exhibited the most severe impairment. However, it is concluded that the ability to undergo normal autophosphorylation does not prevent mutations from impairing mediating activity of insulin receptors.

The nuclear magnetic resonance (NMR) structure of des-pentapeptide-(B26-B30) insulin (DPI), a monomeric insulin analog was determined. The NMR structure of DPI in solution was found to be similar to its secondary- and teriary-structure crystal forms. Two-dimensional nuclear Overhauser enhancement (NOESY) spectroscopy was used toanalyse the crystal and NMR structures in order to predict the physical realityof the NMR ensemble. Results suggest that the compact, folded state of DPI is an intermediate form rather than the native form of the protein, while the molten state is the biologically active state of the protein.

This article describes the health aspects of dietary fiber and its genetic aspects. Topics include insulin secretion, disease prevention, and biosynthesis.

The results from (super 15)N relaxation measurements carried out in both the laboratory and frames for the insulin-like growth factors binding proteins (IGFBPs) are presented. The results strongly suggest possible conformation switching or population shifting between pre-existing conformations in C-terminal domain of IGFBP-6 upon binding to insulin-like growth factors (IGF)-II.

Results show that dissociation and transformation of insulin hexamers into dimers and partially unfolded dimers and monomers occurs with varying concentrations of guanidine hydrochloride, with high concentrations yielding mostly unfolded monomers with some residual structure. Further, low concentration of guanidine hydrochloride significantly accelerates fibrillation conforming to the partially folded state of insulin monomers.