Matrix alkalinization: a novel mitochondrial signal for sustained pancreatic β‐cell activation

Nutrient secretagogues activate mitochondria of the pancreatic β‐cell through the provision of substrate, hyperpolarisation of the inner mitochondrial membrane and mitochondrial calcium rises. We report that mitochondrial matrix pH, a parameter not previously studied in the β‐cell, also exerts an important control function in mitochondrial metabolism. During nutrient stimulation matrix pH alkalinises, monitored by the mitochondrial targeted fluorescent pH‐sensitive protein mtAlpHi or ³¹P‐NMR inorganic phosphate chemical shifts following saturation transfer. Compared with other cell types, the resting mitochondrial pH was surprisingly low, rising from pH 7.25 to 7.7 during nutrient stimulation of rat β‐cells. As cytosolic alkalinisation to the nutrient was of much smaller amplitude, the matrix alkalinisation was accompanied by a pronounced increase of the ΔpH across the inner mitochondrial membrane. Furthermore, matrix alkalinisation closely correlates with the cytosolic ATP net increase, which is also associated with elevated ATP synthesis rates in mitochondria. Preventing ΔpH increases in permeabilised cells abrogated substrate‐driven ATP synthesis. We propose that the mitochondrial pH and ΔpH are key determinants of mitochondrial energy metabolism and metabolite transport important for cell activation.     

Combination of microwell structures and direct oxygenation enables efficient and size‐regulated aggregate formation of an insulin‐secreting pancreatic β‐cell line

Spherical three‐dimensional (3D) cellular aggregates are valuable for various applications such as regenerative medicine or cell‐based assays due to their stable and high functionality. However, previous methods to form aggregates have shown drawbacks, being labor‐intensive, showing low productivity per unit area or volume and difficulty to form homogeneous aggregates. We proposed a novel strategy based on oxygen‐permeable polydimethylsiloxane (PDMS) honeycomb microwell sheets, which can theoretically supply about 80 times as much oxygen as conventional polystyrene culture dishes, to produce recoverable aggregates in controllable sizes using mouse insulinoma cells (MIN6‐m9). In 48 hours of culture, the PDMS sheets produced aggregates whose diameters were strictly controlled (?32, 60, 90, 150 and 280 mm) even at an inoculum density eight times higher (8.0×105 cells/cm²) than that of normal confluent monolayers (1.0×105 cells/cm²). Measurement of the oxygen tension near the cell layer and glucose/lactate analysis clearly showed that cells exhibit aerobic respiration on the PDMS‐based culture system. Glucose‐responsive insulin secretion of the recovered aggregates showed that the aggregates around 90 mm in diameter secreted the largest amounts of insulin. This confirmed the advantages of 3D cellular organization and the existence of a suitable aggregate size, above which excess organization leads to a decreased metabolic response. These results demonstrated that this microwell‐based PDMS culture system provides a promising method to form size‐regulated and better functioning 3D cellular aggregates of various kinds of cells with a high yield per surface area. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:178–187, 2014     

Unconventional protein secretion: an evolving mechanism

The process by which proteins are secreted without entering the classical endoplasmic reticulum (ER)–Golgi complex pathway, in eukaryotic cells, is conveniently called unconventional protein secretion. Recent studies on one such protein called Acb1 have revealed a number of components involved in its secretion. Interestingly, conditions that promote the secretion of Acb1 trigger the biogenesis of a new compartment called CUPS (Compartment for Unconventional Protein Secretion). CUPS form near the ER exit site but lack ER‐specific proteins. Other proteins that share some of the features common with the secretion of Acb1 are interleukin‐1β and tissue transglutaminase. Here I will review recent advances made in the field and propose a new model for unconventional protein secretion.     

Posttranslational modification impact on the mechanism by which amyloid‐β induces synaptic dysfunction

Oligomeric amyloid‐β (Aβ) 1‐42 disrupts synaptic function at an early stage of Alzheimer's disease (AD). Multiple posttranslational modifications of Aβ have been identified, among which N‐terminally truncated forms are the most abundant. It is not clear, however, whether modified species can induce synaptic dysfunction on their own and how altered biochemical properties can contribute to the synaptotoxic mechanisms. Here, we show that a prominent isoform, pyroglutamated Aβ3(pE)‐42, induces synaptic dysfunction to a similar extent like Aβ1‐42 but by clearly different mechanisms. In contrast to Aβ1‐42, Aβ3(pE)‐42 does not directly associate with synaptic membranes or the prion protein but is instead taken up by astrocytes and potently induces glial release of the proinflammatory cytokine TNFα. Moreover, Aβ3(pE)‐42‐induced synaptic dysfunction is not related to NMDAR signalling and Aβ3(pE)‐42‐induced impairment of synaptic plasticity cannot be rescued by D1‐agonists. Collectively, the data point to a scenario where neuroinflammatory processes together with direct synaptotoxic effects are caused by posttranslational modification of soluble oligomeric Aβ and contribute synergistically to the onset of synaptic dysfunction in AD.