Glucose and fatty acids synergize to promote B-cell apoptosis through activation of glycogen synthase kinase 3β independent of JNK activation

Background

The combination of elevated glucose and free-fatty acids (FFA), prevalent in diabetes, has been suggested to be a major contributor to pancreatic β-cell death. This study examines the synergistic effects of glucose and FFA on β-cell apoptosis and the molecular mechanisms involved. Mouse insulinoma cells and primary islets were treated with palmitate at increasing glucose and effects on apoptosis, endoplasmic reticulum (ER) stress and insulin receptor substrate (IRS) signaling were examined.

Principal Findings

Increasing glucose (5–25 mM) with palmitate (400 µM) had synergistic effects on apoptosis. Jun NH2-terminal kinase (JNK) activation peaked at the lowest glucose concentration, in contrast to a progressive reduction in IRS2 protein and impairment of insulin receptor substrate signaling. A synergistic effect was observed on activation of ER stress markers, along with recruitment of SREBP1 to the nucleus. These findings were confirmed in primary islets. The above effects associated with an increase in glycogen synthase kinase 3β (Gsk3β) activity and were reversed along with apoptosis by an adenovirus expressing a kinase dead Gsk3β.

Conclusions/Significance

Glucose in the presence of FFA results in synergistic effects on ER stress, impaired insulin receptor substrate signaling and Gsk3β activation. The data support the importance of controlling both hyperglycemia and hyperlipidemia in the management of Type 2 diabetes, and identify pancreatic islet β-cell Gsk3β as a potential therapeutic target.     

Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM₆), octasaccharide (HM₈), and decasaccharide (HM₁₀), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM₈ and HM₁₀ are significantly more potent than HM₆ in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM₈ relative to HM₆ in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM₈ to bind and dimerize FGF2. Notably FGF2·HM₈ exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.     

Selective reduction of AMPA currents onto hippocampal interneurons impairs network oscillatory activity

Reduction of excitatory currents onto GABAergic interneurons in the forebrain results in impaired spatial working memory and altered oscillatory network patterns in the hippocampus. Whether this phenotype is caused by an alteration in hippocampal interneurons is not known because most studies employed genetic manipulations affecting several brain regions. Here we performed viral injections in genetically modified mice to ablate the GluA4 subunit of the AMPA receptor in the hippocampus (GluA4(HC-/-) mice), thereby selectively reducing AMPA receptor-mediated currents onto a subgroup of hippocampal interneurons expressing GluA4. This regionally selective manipulation led to a strong spatial working memory deficit while leaving reference memory unaffected. Ripples (125-250 Hz) in the CA1 region of GluA4(HC-/-) mice had larger amplitude, slower frequency and reduced rate of occurrence. These changes were associated with an increased firing rate of pyramidal cells during ripples. The spatial selectivity of hippocampal pyramidal cells was comparable to that of controls in many respects when assessed during open field exploration and zigzag maze running. However, GluA4 ablation caused altered modulation of firing rate by theta oscillations in both interneurons and pyramidal cells. Moreover, the correlation between the theta firing phase of pyramidal cells and position was weaker in GluA4(HC-/-) mice. These results establish the involvement of AMPA receptor-mediated currents onto hippocampal interneurons for ripples and theta oscillations, and highlight potential cellular and network alterations that could account for the altered working memory performance.     

mTORC1 signaling and regulation of pancreatic β-cell mass

The capacity of β cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of β cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for β-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of β-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in β cells. This review will discuss recent advances in the understanding of how this pathway regulates β-cell mass and present data on the role of TSC1 in modulation of β-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic β cells results in improved glucose tolerance, hyperinsulinemia and expansion of β-cell mass that persists with aging.     

Mitochondrial NAD+ Controls Nuclear ARTD1-Induced ADP-Ribosylation

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Structure activity relationship studies on rhodanines and derived enethiol inhibitors of metallo-β-lactamases

Metallo-β-lactamases (MBLs) enable bacterial resistance to almost all classes of β-lactam antibiotics. We report studies on enethiol containing MBL inhibitors, which were prepared by rhodanine hydrolysis. The enethiols inhibit MBLs from different subclasses. Crystallographic analyses reveal that the enethiol sulphur displaces the di-Zn(II) ion bridging ‘hydrolytic’ water. In some, but not all, cases biophysical analyses provide evidence that rhodanine/enethiol inhibition involves formation of a ternary MBL enethiol rhodanine complex. The results demonstrate how low molecular weight active site Zn(II) chelating compounds can inhibit a range of clinically relevant MBLs and provide additional evidence for the potential of rhodanines to be hydrolysed to potent inhibitors of MBL protein fold and, maybe, other metallo-enzymes, perhaps contributing to the complex biological effects of rhodanines. The results imply that any medicinal chemistry studies employing rhodanines (and related scaffolds) as inhibitors should as a matter of course include testing of their hydrolysis products.     

Parasitism may alter functional response comparisons: a case study on the killer shrimp <Emphasis Type="Italic">Dikerogammarus villosus</Emphasis> and two non-invasive gammarids

The Ponto-Caspian freshwater amphipod Dikerogammarus villosus has colonized most of the water bodies of continental Europe where it causes strong structural alterations in recipient communities that can lead to changes in ecosystem-level processes, mainly because of a strong predatory behaviour. Most of the D. villosus populations from the invaded range have been found infected with the co-introduced microsporidian parasite Cucumispora dikerogammari, known to decrease the predation rate of its host. Infection might thus mitigate the ecological impact of D. villosus and we wanted to test this assumption using the comparative functional response approach. We compared the relationship between resource use and resource availability (i.e. the functional response, FR) of D. villosus, either with infected individuals or not, to that of two non-invasive gammarids: Gammarus pulex and Echinogammarus berilloni. With infected individuals included, D. villosus displayed a higher FR than the two non-invasive gammarids. Although this effect was not significant, C. dikerogammari infection tended to alter the FR of D. villosus with a slight decrease in attack rate and handling time, resulting in a less steep initial slope and a higher asymptote, respectively. Removing infected D. villosus from the dataset did not affect the FR comparison with G. pulex but suppressed the difference in FR with E. berilloni. Although we cannot exclude the role of sample size reduction in this effect, this suggests that C. dikerogammari infection might increase the predation pressure on local prey populations in case of species replacement between D. villosus and E. berilloni. From a more general perspective, our study illustrates how parasites may alter our capacity to predict invasive species impacts from FR comparisons.     

Is non‐host pollen suitable for generalist bumblebees?

Current evidence suggests that pollen is both chemically and structurally protected. Despite increasing interest in studying bee–flower networks, the constraints for bee development related to pollen nutritional content, toxicity and digestibility as well as their role in the shaping of bee–flower interactions have been poorly studied. In this study we combined bioassays of the generalist bee Bombus terrestris on pollen of Cirsium, Trifolium, Salix, and Cistus genera with an assessment of nutritional content, toxicity, and digestibility of pollen. Microcolonies showed significant differences in their development, non‐host pollen of Cirsium being the most unfavorable. This pollen was characterized by the presence of quite rare δ7‐sterols and a low digestibility. Cirsium consumption seemed increase syrup collection, which is probably related to a detoxification mixing behavior. These results strongly suggest that pollen traits may act as drivers of plant selection by bees and partly explain why Asteraceae pollen is rare in bee generalist diet.     

Setup and characterization of a multiphoton FLIM instrument for protein-protein interaction measurements in living cells.

BACKGROUND: Fluorescence lifetime microscopy (FLIM) is currently one of the best techniques to perform accurate measurements of interactions in living cells. It is independent of the fluorophore concentration, thus avoiding several common artifacts found in F?rster Resonance Energy Transfer (FRET) imaging. However, for FLIM to achieve high performance, a rigorous instrumental setup and characterization is needed. METHODS: We use known fluorophores to perform characterization experiments in our instrumental setup. This allows us to verify the accuracy of the fluorescence lifetime determination, and test the linearity of the instrument by fluorescence quenching. RESULTS: We develop and validate here a protocol for rigorous characterization of time-domain FLIM instruments. Following this protocol, we show that our system provides accurate and reproducible measurements. We also used HeLa cells expressing proteins fused to Green Fluorescent Proteins variants (CFP and YFP) to confirm its ability to detect interactions in living cells by FRET. CONCLUSIONS: We report a well-designed protocol in which precise and reproducible lifetime measurements can be performed. It is usable for all confocal-based FLIM instruments and is a useful tool for anyone who wants to perform quantitative lifetime measurements, especially when studying interactions in living cells using FRET.