Novel Role for Matricellular Proteins in the Regulation of Islet β Cell Survival: THE EFFECT OF SPARC ON SURVIVAL,PROLIFERATION, AND SIGNALING*

Understanding the mechanisms regulating islet growth and survival is critical for developing novel approaches to increasing or sustaining β cell mass in both type 1 and type 2 diabetes patients. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that is important for the regulation of cell growth and adhesion. Increased SPARC can be detected in the serum of type 2 diabetes patients. The aim of this study was to investigate the role of SPARC in the regulation of β cell growth and survival. We show using immunohistochemistry that SPARC is expressed by stromal cells within islets and can be detected in primary mouse islets by Western blot. SPARC is secreted at high levels by pancreatic stellate cells and is regulated by metabolic parameters in these cells, but SPARC expression was not detectable in β cells. In islets, SPARC expression is highest in young mice, and is also elevated in the islets of non-obese diabetic (NOD) mice compared with controls. Purified SPARC inhibits growth factor-induced signaling in both INS-1 β cells and primary mouse islets, and inhibits IGF-1-induced proliferation of INS-1 β cells. Similarly, exogenous SPARC prevents IGF-1-induced survival of primary mouse islet cells. This study identifies the stromal-derived matricellular protein SPARC as a novel regulator of islet survival and β cell growth.     

The early adaptive evolution of calmodulin

Interaction between gene duplication and natural selection in molecular evolution was investigated utilizing a phylogenetic tree constructed by the parsimony procedure from amino acid sequences of 50 calmodulin- family protein members. The 50 sequences, belonging to seven protein lineages related by gene duplication (calmodulin itself, troponin-C, alkali and regulatory light chains of myosin, parvalbumin, intestinal calcium-binding protein, and glial S-100 phenylalanine-rich protein), came from a wide range of eukaryotic taxa and yielded a denser tree (more branch points within each lineage) than in earlier studies. Evidence obtained from the reconstructed pattern of base substitutions and deletions in these ancestral loci suggests that, during the early history of the family, selection acted as a transforming force on expressed genes among the duplicates to encode molecular sites with new or modified functions. In later stages of descent, however, selection was a conserving force that preserved the structures of many coadapted functional sites. Each branch of the family was found to have a unique average tempo of evolutionary change, apparently regulated through functional constraints. Proteins whose functions dictate multiple interaction with several other macromolecules evolved more slowly than those which display fewer protein-protein and protein-ion interactions, e.g., calmodulin and next troponin-C evolved at the slowest average rates, whereas parvalbumin evolved at the fastest. The history of all lineages, however, appears to be characterized by rapid rates of evolutionary change in earlier periods, followed by slower rates in more recent periods. A particularly sharp contrast between such fast and slow rates is found in the evolution of calmodulin, whose rate of change in earlier eukaryotes was manyfold faster than the average rate over the past 1 billion years. In fact, the amino acid replacements in the nascent calmodulin lineage occurred at residue positions that in extant metazoans are largely invariable, lending further support to the Darwinian hypothesis that natural selection is both a creative and a conserving force in molecular evolution.      

Release of growth hormone from ox pituitary slices after pronase treatment

Proteolytic enzymes have been used both to modify properties of the cell membrane and to dissociate cells from many tissues including pituitary (4, 5, 12). Exposure of secretory tissues to pronase can alter their secretory response. Thus incubation of pancreatic islets of Langerhans in the presence of low concentrations of pronase increased the subsequent release of insulin in the presence of stimulatory and nonstimulatory glucose concentrations (7). The purpose of the present investigation was to determine whether low concentrations of pronase have the same stimulatory effect on the release of a pituitary hormone, growth hormone. Such an effect on hormone release could be of some importance in view of the development of dissociated cell systems as models for the study of the control of hormone release (4, 5).     

Fractal properties of human muscle sympathetic nerve activity

Muscle sympathetic nerve activity (MSNA) in resting humans is characterized by cardiac-related bursts of variable amplitude that occur sporadically or in clusters. The present study was designed to characterize the fluctuations in the number of MSNA bursts, interburst interval, and burst amplitude recorded from the peroneal nerve of 15 awake, healthy human subjects. For this purpose, we used the Allan and Fano factor analysis and dispersional analysis to test whether the fluctuations were time-scale invariant (i.e., fractal) or random in occurrence. Specifically, we measured the slopes of the power laws in the Allan factor, Fano factor, and dispersional analysis curves. In addition, the Hurst exponent was calculated from the slope of the power law in the Allan factor curve. Whether the original time series contained fractal fluctuations was decided on the basis of a comparison of the values of these parameters with those for surrogate data blocks. The results can be summarized as follows. Fluctuations in the number of MSNA bursts and interburst interval were fractal in each of the subjects, and fluctuations in burst amplitude were fractal in four of the subjects. We also found that fluctuations in the number of heartbeats and heart period (R-R interval) were fractal in each of the subjects. These results demonstrate for the first time that apparently random fluctuations in human MSNA are, in fact, dictated by a time-scale-invariant process that imparts "long-term memory" to the sequence of cardiac-related bursts. Whether sympathetic outflow to the heart also is fractal and contributes to the fractal component of heart rate variability remains an open question.     

Structural destabilization of tropomyosin induced by the cardiomyopathy‐linked mutation R21H

The missense mutation R21H in striated muscle tropomyosin is associated with hypertrophic cardiomyopathy, a genetic cardiac disease and a leading cause of sudden cardiac death in young people. Tropomyosin adopts conformation of a coiled coil which is critical for regulation of muscle contraction. In this study, we investigated the effects of the R21H mutation on the coiled‐coil structure of tropomyosin and its interactions with its binding partners, tropomodulin and leiomodin. Using circular dichroism and isothermal titration calorimetry, we found that the mutation profoundly destabilized the structural integrity of αTM1a_1‐28Zip, a chimeric peptide containing the first 28 residues of tropomyosin. The mutated αTM1a_1‐28Zip was still able to interact with tropomodulin and leiomodin. However, the mutation resulted in a ～30‐fold decrease of αTM1a_1‐28Zip's binding affinity to leiomodin. We used a crystal structure of αTM1a_1‐28Zip that we solved at 1.5 Å resolution to study the mutation's effect in silico by means of molecular dynamics simulation. The simulation data indicated that while the mutation disrupted αTM1a_1‐28Zip's coiled‐coil structure, most notably from residue Ala18 to residue His31, it may not affect the N‐terminal end of tropomyosin. The drastic decrease of αTM1a_1‐28Zip's affinity to leiomodin caused by the mutation may lead to changes in the dynamics at the pointed end of thin filaments. Therefore, the R21H mutation is likely interfering with the regulation of the normal thin filament length essential for proper muscle contraction.     

Increases in intramuscular pressure raise arterial blood pressure during dynamic exercise.

This investigation was designed to determine the role of intramuscular pressure-sensitive mechanoreceptors and chemically sensitive metaboreceptors in affecting the blood pressure response to dynamic exercise in humans. Sixteen subjects performed incremental (20 W/min) cycle exercise to fatigue under four conditions: control, exercise with thigh cuff occlusion of 90 Torr (Cuff occlusion), exercise with lower body positive pressure (LBPP) of 45 Torr, and a combination of thigh cuff occlusion and LBPP (combination). Indexes of central command (heart rate, oxygen uptake, ratings of perceived exertion, and electromyographic activity), cardiac output, stroke volume, and total peripheral resistance were not significantly different between the four conditions. Mechanical stimulation during LBPP and combination conditions resulted in significant elevations in intramuscular pressure and mean arterial pressure from control at rest and throughout the incremental exercise protocol (P < 0.05). Conversely, there existed no significant changes in mean arterial pressure when the metaboreflex was stimulated by cuff occlusion. These findings suggest that under normal conditions the mechanoreflex is tonically active and is the primary mediator of exercise pressor reflex-induced alterations in arterial blood pressure during submaximal dynamic exercise in humans.