Control of Precursor Maturation and Disposal Is an Early Regulative Mechanism in the Normal Insulin Production of Pancreatic β-Cells

The essential folding and maturation process of proinsulin in β-cells is largely uncharacterized. To analyze this process, we improved approaches to immunoblotting, metabolic labeling, and data analysis used to determine the proportion of monomers and non-monomers and changes in composition of proinsulin in cells. We found the natural occurrence of a large proportion of proinsulin in various non-monomer states, i.e., aggregates, in normal mouse and human β-cells and a striking increase in the proportion of proinsulin non-monomers in Ins2+/Akita mice in response to a mutation (C96Y) in the insulin 2 (Ins2) gene. Proinsulin emerges in monomer and abundant dual-fate non-monomer states during nascent protein synthesis and shows heavy and preferential ATP/redox-sensitive disposal among secretory proteins during early post-translational processes. These findings support the preservation of proinsulin's aggregation-prone nature and low relative folding rate that permits the plentiful production of non-monomer forms with incomplete folding. Thus, in normal mouse/human β-cells, proinsulin's integrated maturation and degradation processes maintain a balance of natively and non-natively folded states, i.e., proinsulin homeostasis (PIHO). Further analysis discovered the high susceptibility of PIHO to cellular energy and calcium changes, endoplasmic reticulum (ER) and reductive/oxidative stress, and insults by thiol reagent and cytokine. These results expose a direct correlation between various extra-/intracellular influences and (a)typical integrations of proinsulin maturation and disposal processes. Overall, our findings demonstrated that the control of precursor maturation and disposal acts as an early regulative mechanism in normal insulin production, and its disorder is crucially linked to β-cell failure and diabetes pathogenesis

Intracellular Ca2+ signals in human-derived pancreatic somatostatin-secreting cells (QGP-1N)

Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1-100 μM) on the intracellular free calcium ion concentration (Ca2+i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in Ca2+i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of Ca2+i transients. Overall, the pattern of response was concentration related. In general, 0.1-10 μM acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca2+, whilst at higher concentrations the responses consisted of a rapid rise in Ca2+i followed by a smaller more sustained increase. Without external Ca2+, 100 μM acetylcholine caused only a transient rise in Ca2+i, whereas lower concentrations of the agonist were able to initiate, but not maintain, Ca2+i oscillations. Acetylcholine-evoked Ca2+ signals were abolished by atropine (1-10 μM), verapamil (100 μM) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in Ca2+i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in Ca2+i. Ryanodine (50-500 nM) and caffeine (1-20 mM) did not increase basal Ca2+ levels, but the Ca2+-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated Ca2+i levels. These data demonstrate for the first time cytosolic Ca2+ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca2+ influx and Ca2+ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events. © 1994 Springer-Verlag

Coastal eutrophication as a driver of salt marsh loss

Salt marshes are highly productive coastal wetlands that provide important ecosystem services such as storm protection for coastal cities, nutrient removal and carbon sequestration. Despite protective measures, however, worldwide losses of these ecosystems have accelerated in recent decades1. Here we present data from a nine-year whole-ecosystem nutrient-enrichment experiment. Our study demonstrates that nutrient enrichment, a global problem for coastal ecosystems2,3,4, can be a driver of salt marsh loss. We show that nutrient levels commonly associated with coastal eutrophication increased above-ground leaf biomass, decreased the dense, below-ground biomass of bank-stabilizing roots, and increased microbial decomposition of organic matter. Alterations in these key ecosystem properties reduced geomorphic stability, resulting in creek-bank collapse with significant areas of creek-bank marsh converted to unvegetated mud. This pattern of marsh loss parallels observations for anthropogenically nutrient-enriched marshes worldwide, with creek-edge and bay-edge marsh evolving into mudflats and wider creeks5,6,7. Our work suggests that current nutrient loading rates to many coastal ecosystems have overwhelmed the capacity of marshes to remove nitrogen without deleterious effects. Projected increases in nitrogen flux to the coast, related to increased fertilizer use required to feed an expanding human population, may rapidly result in a coastal landscape with less marsh, which would reduce the capacity of coastal regions to provide important ecological and economic services

Mitochondrial function in normal and diabetic beta-cells

The aetiology of type 2, or non-insulin-dependent, diabetes mellitus has been characterized in only a limited number of cases. Among these, mitochondrial diabetes, a rare subform of the disease, is the consequence of pancreatic beta-cell dysfunction caused by mutations in mitochondrial DNA, which is distinct from the nuclear genome. The impact of such mutations on beta-cell function reflects the importance of mitochondria in the control of insulin secretion. The beta-cell mitochondria serve as fuel sensors, generating factors that couple nutrient metabolism to the exocytosis of insulin-containing vesicles. The latter process requires an increase in cytosolic Ca2+, which depends on ATP synthesized by the mitochondria. This organelle also generates other factors, of which glutamate has been proposed as a potential intracellular messenger