Structure-based stabilization of insulin as a therapeutic protein assembly via enhanced aromatic-aromatic interactions

Key contributions to protein structure and stability are provided by weakly polar interactions, which arise from asymmetric electronic distributions within amino acids and peptide bonds. Of particular interest are aromatic side chains whose directional π-systems commonly stabilize protein interiors and interfaces. Here, we consider aromatic-aromatic interactions within a model protein assembly: the dimer interface of insulin. Semi-classical simulations of aromatic-aromatic interactions at this interface suggested that substitution of residue TyrB26 by Trp would preserve native structure while enhancing dimerization (and hence hexamer stability). The crystal structure of a [TrpB26]insulin analog (determined as a T3Rf3 zinc hexamer at a resolution of 2.25 Å) was observed to be essentially identical to that of WT insulin. Remarkably and yet in general accordance with theoretical expectations, spectroscopic studies demonstrated a 150-fold increase in the in vitro lifetime of the variant hexamer, a critical pharmacokinetic parameter influencing design of long-acting formulations. Functional studies in diabetic rats indeed revealed prolonged action following subcutaneous injection. The potency of the TrpB26-modified analog was equal to or greater than an unmodified control. Thus, exploiting a general quantum-chemical feature of protein structure and stability, our results exemplify a mechanism-based approach to the optimization of a therapeutic protein assembly

miR-375 gene dosage in pancreatic β-cells: implications for regulation of β-cell mass and biomarker development

MicroRNAs play a crucial role in the regulation of cell growth and differentiation. Mice with genetic deletion of miR-375 exhibit impaired glycemic control due to decreased β-cell and increased α-cell mass and function. The relative importance of these processes for the overall phenotype of miR-375KO mice is unknown. Here, we show that mice overexpressing miR-375 exhibit normal β-cell mass and function. Selective re-expression of miR-375 in β-cells of miR-375KO mice normalizes both, α- and β-cell phenotypes as well as glucose metabolism. Using this model, we also analyzed the contribution of β-cells to the total plasma miR-375 levels. Only a small proportion (≈1 %) of circulating miR-375 originates from β-cells. Furthermore, acute and profound β-cell destruction is sufficient to detect elevations of miR-375 levels in the blood. These findings are supported by higher miR-375 levels in the circulation of type 1 diabetes (T1D) subjects but not mature onset diabetes of the young (MODY) and type 2 diabetes (T2D) patients. Together, our data support an essential role for miR-375 in the maintenance of β-cell mass and provide in vivo evidence for release of miRNAs from pancreatic β-cells. The small contribution of β-cells to total plasma miR-375 levels make this miRNA an unlikely biomarker for β-cell function but suggests a utility for the detection of acute β-cell death for autoimmune diabetes

Global Stability and Periodicity in a Glucose-Insulin Regulation Model with a Single Delay

A two-dimensional system of differential equations with delay modelling the glucose-insulin interaction processes in the human body is considered. Sufficient conditions are derived for the unique positive equilibrium in the system to be globally asymptotically stable. They are given in terms of the global attractivity of the fixed point in a limiting interval map. The existence of slowly oscillating periodic solutions is shown in the case when the equilibrium is unstable. The mathematical results are supported by extensive numerical simulations. It is shown that typical behaviour in the system is the convergence to either a stable periodic solution or to the unique stable equilibrium. The coexistence of several periodic solutions together with the stable equilibrium is demonstrated as a possibility.Comment: Accepted to Communications in Nonlinear Science and Numerical Simulatio

Mathematical studies of the glucose-insulin regulatory system models.

Three dynamic models are proposed to study the mechanism of glucose-insulin regulatory system and the possible causes of diabetes mellitus. The progression of diabetes comes along with the apoptosis of pancreatic beta-cells. A dynamical system model is formulated based on physiology and studied by geometric singular perturbation theory. The analytical studies reveal rich analytical features, such as persistence of solutions, Hopf bifurcation and backward bifurcation, while numerical studies successfully fit available longitudinal T2DM data of Pima Indian tribe. These studies together not only validate our model, but also point out key intrinsic factors leading to the development of T2DM. We found that the intermittent rests of beta-cells in insulin secretion are essential for the cells to survive through the observation of the existence of a limit cycle. A delay differential equation model for IVGTT is also studied thoroughly to determine the range of time delay and the globally asymptotic stability by Liapunov function. The third kinetic model aims to investigate the scaling effect of local insulin in islet on proliferation and apoptosis of beta-cells. It is revealed that the local concentration of monomeric insulin within the islet is in the biologist defined picomolar ‘sweet spot’ range of insulin doses, which activate the insulin receptors and have the most potent effects on beta-cells in vitro

Bayesian Analysis of ODE's: solver optimal accuracy and Bayes factors

In most relevant cases in the Bayesian analysis of ODE inverse problems, a numerical solver needs to be used. Therefore, we cannot work with the exact theoretical posterior distribution but only with an approximate posterior deriving from the error in the numerical solver. To compare a numerical and the theoretical posterior distributions we propose to use Bayes Factors (BF), considering both of them as models for the data at hand. We prove that the theoretical vs a numerical posterior BF tends to 1, in the same order (of the step size used) as the numerical forward map solver does. For higher order solvers (eg. Runge-Kutta) the Bayes Factor is already nearly 1 for step sizes that would take far less computational effort. Considerable CPU time may be saved by using coarser solvers that nevertheless produce practically error free posteriors. Two examples are presented where nearly 90% CPU time is saved while all inference results are identical to using a solver with a much finer time step.Comment: 28 pages, 6 figure

Mathematical investigation of diabetically impaired ultradian oscillations in the glucose-insulin regulation

We study the effect of diabetic deficiencies on the production of an oscillatory ultradian regime using a deterministic nonlinear model which incorporates two physiological delays. It is shown that insulin resistance impairs the production of oscillations by dampening the ultradian cycles. Four strategies for restoring healthy regulation are explored. Through the introduction of an instantaneous glucose-dependent insulin response, explicit conditions for the existence of periodic solutions in the linearised model are formulated, significantly reducing the complexity of identifying an oscillatory regime. The model is thus shown to be suitable for representing the effect of diabetes on the oscillatory regulation and for investigating pathways to reinstating a physiological healthy regime

RNA-seq reveals post-transcriptional regulation of Drosophila insulin-like peptide dilp8 and the neuropeptide-like precursor Nplp2 by the exoribonuclease Pacman/XRN1

Ribonucleases are critically important in many cellular and developmental processes and defects in their expression are associated with human disease. Pacman/XRN1 is a highly conserved cytoplasmic exoribonuclease which degrades RNAs in a 5' - 3' direction. In Drosophila, null mutations in pacman result in small imaginal discs, a delay in onset of pupariation and lethality during the early pupal stage. In this paper, we have used RNA-seq in a genome-wide search for mRNAs misregulated in pacman null wing imaginal discs. Only 4.2% of genes are misregulated ±>2-fold in pacman null mutants compared to controls, in line with previous work showing that Pacman has specificity for particular mRNAs. Further analysis of the most upregulated mRNAs showed that Pacman post-transcriptionally regulates the expression of the secreted insulin-like peptide Dilp8. Dilp8 is related to human IGF-1, and has been shown to co-ordinate tissue growth with developmental timing in Drosophila. The increased expression of Dilp8 is consistent with the developmental delay seen in pacman null mutants. Our analysis, together with our previous results, show that the normal role of this exoribonuclease in imaginal discs is to suppress the expression of transcripts that are crucial in apoptosis and growth control during normal development