Inhibition of Protein Fibrillation by Hydrogen Sulfide<xref rid="fn1" ref-type="fn">¹</xref>

Amyloid fibrils are misfolded proteins, which are often associated with various neurodegenerative diseases such as Alzheimer’s. The amount of hydrogen sulfide (H2S) is known to be reduced in the brain tissue of people diagnosed with Alzheimer’s disease relative to that of healthy individuals. Hen Egg-White Lysozyme (HEWL) forms typical β-sheet-rich fibrils during 70 minutes at low pH and high temperatures. These results are consistent with the ThT findings that β-sheets structure is also present in myoglobin (Mb), and hemoglobin (Hb) in the presence of 45% TFE. The addition of H2S in the process completely inhibits the formation of amyloid fibrils in HEWL, Mb, and Hb as revealed by several spectroscopic techniques. Non-resonance Raman bands corresponding to disulfide (RSSR) vibrational modes in the 550-500 cm-1 spectral range decreases in intensity and is accompanied by the appearance of a new 490 cm-1 band assigned to the trisulfide group (RSSSR). Intrinsic tryptophan fluorescence shows a partial denaturation of HEWL containing trisulfide bonds. Overall, the Mb and Hb result ties excellent with the HEWL data showing that the presence of H2S during these proteins fibrillation processes protects the α-helical protein structures, preventing the formation of amyloids in these different proteins moieties

A Novel High-Throughput Assay for Islet Respiration Reveals Uncoupling of Rodent and Human Islets

The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets.The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets.The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors

Objective: Islets from the same pancreas show remarkable variability in glucose sensitivity. While mitochondrial respiration is essential for glucose-stimulated insulin secretion, little is known regarding heterogeneity in mitochondrial function at the individual islet level. This is due in part to a lack of high-throughput and non-invasive methods for detecting single islet function. Methods: We have developed a novel non-invasive, high-throughput methodology capable of assessing mitochondrial respiration in large-sized individual islets using the XF96 analyzer (Agilent Technologies). Results: By increasing measurement sensitivity, we have reduced the minimal size of mouse and human islets needed to assess mitochondrial respiration to single large islets of >35,000 μm2 area (∼210 μm diameter). In addition, we have measured heterogeneous glucose-stimulated mitochondrial respiration among individual human and mouse islets from the same pancreas, allowing population analyses of islet mitochondrial function for the first time. Conclusions: We have developed a novel methodology capable of analyzing mitochondrial function in large-sized individual islets. By highlighting islet functional heterogeneity, we hope this methodology can significantly advance islet research. Keywords: Islets, Mitochondria, Respirometry, Glucos

Deletion of ABCB10 in beta-cells protects from high-fat diet induced insulin resistance

ObjectiveThe contribution of beta-cell dysfunction to type 2 diabetes (T2D) is not restricted to insulinopenia in the late stages of the disease. Elevated fasting insulinemia in normoglycemic humans is a major factor predicting the onset of insulin resistance and T2D, demonstrating an early alteration of beta-cell function in T2D. Moreover, an early and chronic increase in fasting insulinemia contributes to insulin resistance in high-fat diet (HFD)-fed mice. However, whether there are genetic factors that promote beta-cell-initiated insulin resistance remains undefined. Human variants of the mitochondrial transporter ABCB10, which regulates redox by increasing bilirubin synthesis, have been associated with an elevated risk of T2D. The effects of T2D ABCB10 variants on ABCB10 expression and the actions of ABCB10 in beta-cells are unknown.MethodsThe expression of beta-cell ABCB10 was analyzed in published transcriptome datasets from human beta-cells carrying the T2D-risk ABCB10 variant. Insulin sensitivity, beta-cell proliferation, and secretory function were measured in beta-cell-specific ABCB10 KO mice (Ins1Cre-Abcb10flox/flox). The short-term role of beta-cell ABCB10 activity on glucose-stimulated insulin secretion (GSIS) was determined in isolated islets.ResultsCarrying the T2Drisk allele G of ABCB10 rs348330 variant was associated with increased ABCB10 expression in human beta-cells. Constitutive deletion of Abcb10 in beta-cells protected mice from hyperinsulinemia and insulin resistance by limiting HFD-induced beta-cell expansion. An early limitation in GSIS and H2O2-mediated signaling caused by elevated ABCB10 activity can initiate an over-compensatory expansion of beta-cell mass in response to HFD. Accordingly, increasing ABCB10 expression was sufficient to limit GSIS capacity. In health, ABCB10 protein was decreased during islet maturation, with maturation restricting beta-cell proliferation and elevating GSIS. Finally, ex-vivo and short-term deletion of ABCB10 in islets isolated from HFD-fed mice increased H2O2 and GSIS, which was reversed by bilirubin treatments.ConclusionsBeta-cell ABCB10 is required for HFD to induce insulin resistance in mice by amplifying beta-cell mass expansion to maladaptive levels that cause fasting hyperinsulinemia

Measuring islet oxygen consumption in a high throughput format.

<p>A) Islet plate development. The XF24 measures oxygen consumption from monolayers of adherent cells in a 24 well cell culture plate format. Oxygen consumption is measured with probe heads coated with oxygen sensitive fluorophores that are optically read (marked white and red). During the “measure” mode a volume of only a few micro-liters is formed. In this minute volume oxygen tension rapidly drops which enables OCR calculations. In “mix” mode, the probe head moves up and down, reoxygenating the cells and exposing them to injected compounds. The wells with flat bottoms, originally designed for cell monolayers, proved inadequate for islets as they gathered in the well periphery, too far from the probe head (left panel). To address this problem, an islet plate with a central depression covered by a screen was developed. This trapped the islets while still maintaining sufficient media access (right panel). The screen consists of a polycarbonate ring attached to a nylon net with 50 µm pore size. B) Image of islet plate well. After experiments each well of the 24-well plate was imaged and the images were used for calculating OCR per islet and to measure islet diameter. The islet images were then used to obtain an exact islet count per well and to measure islet diameter. C) Flat bottom (V7) plates are not suitable for islets. 70 islets were seeded per well. Traces recorded from separate wells in a plate are shown. Note the large data variation and that subsequent to 20 mM glucose injection the OCR only increase in one well out of four. D) Islet plate reduces data variability. As islet seeding could vary and thus the absolute OCR, data from each well was often normalized to its initial steady state values before any compound was injected. Traces from islets in 3 mM and 20 mM glucose are shown with absolute (left) and normalized rates (right); note the stability over 2.5 hours. Subsequent to 20 mM glucose injection (after 1^st data point) the OCR increased in all the wells. Islets that stay in 3 mM glucose do not display any major change in OCR. E) Initial measurements are unstable. OCR in the islet plate showed an initial drift during the first 1–2 measurements (dashed square), therefore these data points where always omitted. F) Experimental set up – bioenergetic principles. Respiration recorded may be deciphered by using drugs acting on the mitochondrial inner membrane/complexes. Oligomycin blocks complex V and remaining respiration represents the proton leak. Rotenone/Myxothiazol blocks complexes I/III and remaining respiration is non-mitochondrial. FCCP stimulation shows maximal respiratory capacity. Nutrient stimulation (glucose) may be used in addition to these drugs to study the effect of nutrient metabolism.</p

Islets are highly uncoupled; mice islets more than human.

<p>A) Uncoupling of wild type mouse islets, INS-1 cells, C2C12 myotubes and human islets. The graph presents a summary of multiple experiments where OCR under oligomycin (defined as uncoupling) was measured until stable. Note that uncoupled OCR is significantly higher in the mouse islets and that the C2C12 myotubes show the lowest uncoupled OCR. N = 29 (INS-1), 20 (C2C12), 35 (C57Bl6/J islets), 14 (FVB/N islets), 8 (Human islets). **indicates p<0.01. B) Comparison of total ATP levels in INS-1 cells vs. C2C12 myotubes.</p