The precision of line position measurements of unresolved quasar absorption lines and its influence on the search for variations of fundamental constants

Optical quasar spectra can be used to trace variations of the fine-structure constant alpha. Controversial results that have been published in last years suggest that in addition to to wavelength calibration problems systematic errors might arise because of insufficient spectral resolution. The aim of this work is to estimate the impact of incorrect line decompositions in fitting procedures due to asymmetric line profiles. Methods are developed to distinguish between different sources of line position shifts and thus to minimize error sources in future work. To simulate asymmetric line profiles, two different methods were used. At first the profile was created as an unresolved blend of narrow lines and then, the profile was created using a macroscopic velocity field of the absorbing medium. The simulated spectra were analysed with standard methods to search for apparent shifts of line positions that would mimic a variation of fundamental constants. Differences between position shifts due to an incorrect line decomposition and a real variation of constants were probed using methods that have been newly developed or adapted for this kind of analysis. The results were then applied to real data. Apparent relative velocity shifts of several hundred meters per second are found in the analysis of simulated spectra with asymmetric line profiles. It was found that each system has to be analysed in detail to distinguish between different sources of line position shifts. A set of 16 FeII systems in seven quasar spectra was analysed. With the methods developed, the mean alpha variation that appeared in these systems was reduced from the original Dalpha/alpha=(2.1+/-2.0)x10^-5 to Dalpha/alpha=(0.1+/-0.8)x10^-5. We thus conclude that incorrect line decompositions can be partly responsible for the conflicting results published so far

JNK1 Deficient Insulin-Producing Cells Are Protected against Interleukin-1 beta-Induced Apoptosis Associated with Abrogated Myc Expression

The relative contributions of the JNK subtypes in inflammatory β-cell failure and apoptosis are unclear. The JNK protein family consists of JNK1, JNK2, and JNK3 subtypes, encompassing many different isoforms. INS-1 cells express JNK1α1, JNK1α2, JNK1β1, JNK1β2, JNK2α1, JNK2α2, JNK3α1, and JNK3α2 mRNA isoform transcripts translating into 46 and 54 kDa isoform JNK proteins. Utilizing Lentiviral mediated expression of shRNAs against JNK1, JNK2, or JNK3 in insulin-producing INS-1 cells, we investigated the role of individual JNK subtypes in IL-1β-induced β-cell apoptosis. JNK1 knockdown prevented IL-1β-induced INS-1 cell apoptosis associated with decreased 46 kDa isoform JNK protein phosphorylation and attenuated Myc expression. Transient knockdown of Myc also prevented IL-1β-induced apoptosis as well as caspase 3 cleavage. JNK2 shRNA potentiated IL-1β-induced apoptosis and caspase 3 cleavage, whereas JNK3 shRNA did not affect IL-1β-induced β-cell death compared to nonsense shRNA expressing INS-1 cells. In conclusion, JNK1 mediates INS-1 cell death associated with increased Myc expression. These findings underline the importance of differentiated targeting of JNK subtypes in the development of inflammatory β-cell failure and destruction

JNK1 protects against glucolipotoxicity-mediated beta-cell apoptosis

Pancreatic β-cell dysfunction is central to type 2 diabetes pathogenesis. Prolonged elevated levels of circulating free-fatty acids and hyperglycemia, also termed glucolipotoxicity, mediate β-cell dysfunction and apoptosis associated with increased c-Jun N-terminal Kinase (JNK) activity. Endoplasmic reticulum (ER) and oxidative stress are elicited by palmitate and high glucose concentrations further potentiating JNK activity. Our aim was to determine the role of the JNK subtypes JNK1, JNK2 and JNK3 in palmitate and high glucose-induced β-cell apoptosis. We established insulin-producing INS1 cell lines stably expressing JNK subtype specific shRNAs to understand the differential roles of the individual JNK isoforms. JNK activity was increased after 3 h of palmitate and high glucose exposure associated with increased expression of ER and mitochondrial stress markers. JNK1 shRNA expressing INS1 cells showed increased apoptosis and cleaved caspase 9 and 3 compared to non-sense shRNA expressing control INS1 cells when exposed to palmitate and high glucose associated with increased CHOP expression, ROS formation and Puma mRNA expression. JNK2 shRNA expressing INS1 cells did not affect palmitate and high glucose induced apoptosis or ER stress markers, but increased Puma mRNA expression compared to non-sense shRNA expressing INS1 cells. Finally, JNK3 shRNA expressing INS1 cells did not induce apoptosis compared to non-sense shRNA expressing INS1 cells when exposed to palmitate and high glucose but showed increased caspase 9 and 3 cleavage associated with increased DP5 and Puma mRNA expression. These data suggest that JNK1 protects against palmitate and high glucose-induced β-cell apoptosis associated with reduced ER and mitochondrial stress

Butyrate Protects Pancreatic Beta Cells from Cytokine-Induced Dysfunction

Pancreatic beta cell dysfunction caused by metabolic and inflammatory stress contributes to the development of type 2 diabetes (T2D). Butyrate, produced by the gut microbiota, has shown beneficial effects on glucose metabolism in animals and humans and may directly affect beta cell function, but the mechanisms are poorly described. The aim of this study was to investigate the effect of butyrate on cytokine-induced beta cell dysfunction in vitro. Mouse islets, rat INS-1E, and human EndoC-βH1 beta cells were exposed long-term to non-cytotoxic concentrations of cytokines and/or butyrate to resemble the slow onset of inflammation in T2D. Beta cell function was assessed by glucose-stimulated insulin secretion (GSIS), gene expression by qPCR and RNA-sequencing, and proliferation by incorporation of EdU into newly synthesized DNA. Butyrate protected beta cells from cytokine-induced impairment of GSIS and insulin content in the three beta cell models. Beta cell proliferation was reduced by both cytokines and butyrate. Expressions of the beta cell specific genes Ins, MafA, and Ucn3 reduced by the cytokine IL-1β were not affected by butyrate. In contrast, butyrate upregulated the expression of secretion/transport-related genes and downregulated inflammatory genes induced by IL-1β in mouse islets. In summary, butyrate prevents pro-inflammatory cytokine-induced beta cell dysfunction

JNK knockdown increase <i>Puma</i> mRNA expression.

<p>INS1 cells stably expressing shRNA directed against JNK1, JNK2, JNK3 or the non-sense control were exposed to 0.5 mM palmitate and 25 mM glucose for 12 or16 h (+). Relative mRNA expression was measured using quantitative RT-PCR and normalized to <i>Hprt1</i>. Relative <i>DP5</i> mRNA expression at A: 12 h, B: 16 h. Relative <i>Puma</i> mRNA expression C: 12 h, D: 16 h. Data are shown with+SEM of five - six independent experiments. INS1 cells stably expressing shRNA directed against JNK1, JNK2, JNK3 or the non-sense control were exposed to 0.5 mM palmitate and 25 mM glucose (black bars) or vehicle (white bars) for 24 h. Relative <i>Ins1</i> mRNA expression E: 24 h, Relative <i>Ins2</i> mRNA expression, F: 24 h. G: Total insulin content (ng insulin/total DNA) after GSIS. Data are shown with+SEM of four independent experiments *P<0.05, **P<0.01, ***P<0.001.</p

Figure 2. JNK1 knockdown increases palmitate and high glucose-induced β-cell apoptosis.

<p>INS1 cell lines stably expressing shRNA for JNK1, JNK2, JNK3, non-sense shRNA (ns), empty vector controls (ev) or wildtype INS1 cells were exposed to 0.5 mM palmitate and 25 mM glucose (GLT) (+) or vehicle (−) for 24 h. A: JNK1, B: JNK2, C: JNK3 knockdown specificity and efficiency in JNK1, 2 and 3 shRNA expressing INS1 cell lines were assessed by Western blotting with actin as loading control. Blots are representative of knockdown efficiency in the shRNA expressing INS1 cell lines. D: The specificity of the JNK antibodies were verified against JNK1 (73 kDa with GST tag), JNK2 (72 kDa with GST tag) and JNK3 (61 kDa with GST tag) recombinant proteins. INS1 cell lines stably expressing shRNA for JNK1, JNK2, JNK3, non-sense shRNA (ns), empty vector controls (ev) or wildtype INS1 cells were exposed to 0.5 mM palmitate and 25 mM glucose (black bars) or vehicle (white bars) for 24 h. E: Apoptosis was measured as the relative levels of cytoplasmic nucleosomes in INS1 shRNA stable cell lines lysates compared to ns vehicle using the Roche Cell Death detection ELISA kit. Data are shown as means+SEM of five independent experiments. F, G: Cleaved caspase 9 or 3 was assessed by Western blotting and normalized to actin. Data are shown as means+SEM of five independent experiments; representative blots are shown. *P<0.05, ***P<0.001.</p

Primer sequence.

<p>Primer sequence.</p

JNK knockdown does not affect <i>sXbp-1</i>, P-eIF2α, <i>ATF4</i> and <i>ATF3</i> expression.

<p>INS1 cell lines stably expressing shRNA directed against JNK1, JNK2, JNK3 or the non-sense (ns) control were exposed to 0.5 mM palmitate and 25 mM glucose for 4–16 h (+). Relative mRNA expression was measured using quantitative RT-PCR and normalized to <i>Hprt1</i>. Relative <i>sXBP1</i>mRNA expression at A: 4 h, B: 12 h. C: Time-course analysis of P-eIF2α protein expression analyzed by Western blotting. Protein was isolated from INS1 cells exposed to 0.5 mM palmitate and 25 mM glucose for 0.5–24 h. Actin was used as loading control, representative blots are shown. Data are shown with+SEM of three independent experiments. D: Protein was isolated from INS1 cell lines stably expressing JNK1, JNK2 or JNK3 shRNA or the non-sense (ns) shRNA control after 16 h of palmitate and high glucose exposure, and p-eIF2α levels were analyzed by Western blotting. Actin was used as the loading control, representative blots are shown. Relative <i>ATF-4</i> mRNA expression at E: 12 h, F: 16 h. Relative <i>ATF-3</i> mRNA expression at G: 12 h, H: 16 h. Data are shown with+SEM of five independent experiments. *P<0.05, **P<0.01, ***P<0.001.</p

JNK1 knockdown increases CHOP expression and ROS formation.

<p>INS1 cells stably expressing shRNA directed against JNK1, JNK2, JNK3 or the non-sense (ns) control were exposed to 0.5 mM palmitate and 25 mM glucose for 12–24 h (+). Relative mRNA expression was measured using quantitative RT-PCR and normalized to <i>Hprt1</i>. Relative <i>CHOP</i> mRNA expression at A: 12 h, B: 16 h. C: Protein was isolated from JNK1, 2 or 3 shRNA expressing INS1 cells after 24 h of 0.5 mM palmitate and 25 mM glucose exposure. CHOP levels were analyzed by Western blotting. Actin was used as the loading control, representative blots are shown. Data are shown with ± SEM of five - six independent experiments D: ROS production was measured by a fluorophore probe as described in the methods section. JNK1, JNK2, JNK3 or non-sense (ns) shRNA expressing INS1 cells were exposed to palmitate and high glucose for 24 h and fluorescent signal was normalized to ns (100%). Data are shown+SEM of four independent experiments. *P<0.05, **P<0.01, ***P<0.001.</p