Inhibition of the Nuclear Factor-κB Pathway Prevents Beta Cell Failure and Diet Induced Diabetes in Psammomys obesus

BACKGROUND: High doses of anti-inflammatory drugs, such as aspirin and salicylates, improve glucose metabolism in insulin resistant and type 2 diabetic patients. It has also been shown that the glucose lowering effect is related to the unspecific ability of these drugs to inhibit inhibitor kinaseβ (IKKβ). In this study we have investigated the effect of a selective IKKβ-inhibitor on beta cell survival and the prevention of diet induced type 2 diabetes in the gerbil Psammomys obesus (P. obesus). METHODOLOGY/PRINCIPAL FINDINGS: P. obesus were fed a diabetes inducing high energy diet for one month in the absence or presence of the IKKβ-inhibitor. Body mass, blood glucose, HbA(1C), insulin production and pancreatic insulin stores were measured. The effects on beta cell survival were also studied in INS-1 cells and primary islets. The cells were exposed to IL-1β and subsequently reactive oxygen species, insulin release and cell death were measured in the absence or presence of the IKKβ-inhibitor. In primary islets and beta cells, IL-1β induced the production of reactive oxygen species, reduced insulin production and increased beta cell death, which were all reversed by pre-treatment with the IKKβ-inhibitor. In P. obesus the IKKβ-inhibitor prevented the development of hyperglycaemia and hyperinsulinaemia, and maintained pancreatic insulin stores with no effect on body weight. CONCLUSIONS/SIGNIFICANCE: Inhibition of IKKβ activity prevents diet-induced diabetes in P. obesus and inhibits IL-1β induced reactive oxygen species, loss of insulin production and beta cell death in vitro

Inhibition of nuclear factor-kappaB or Bax prevents endoplasmic reticulum stress- but not nitric oxide-mediated apoptosis in INS-1E cells.

Accumulating evidence suggests that endoplasmic reticulum (ER) stress by mechanisms that include ER Ca(2+) depletion via NO-dependent down-regulation of sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) contributes to beta-cell death in type 1 diabetes. To clarify whether the molecular pathways elicited by NO and ER Ca(2+) depletion differ, we here compare the direct effects of NO, in the form of the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP), with the effects of SERCA2 inhibitor thapsigargin (TG) on MAPK, nuclear factor kappaB (NFkappaB), Bcl-2 proteins, ER stress, and apoptosis. Exposure of INS-1E cells to TG or SNAP caused caspase-3 cleavage and apoptosis. Both TG and SNAP induced activation of the proapoptotic transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP). However, other classical ER stress-induced markers such as up-regulation of ER chaperone Bip and alternative splicing of the transcription factor Xbp-1 were exclusively activated by TG. TG exposure caused NFkappaB activation, as assessed by IkappaB degradation and NFkappaB DNA binding. Inhibition of NFkappaB or the Bcl-2 family member Bax pathways protected beta-cells against TG- but not SNAP-induced beta-cell death. These data suggest that NO generation and direct SERCA2 inhibition cause two quantitative and qualitative different forms of ER stress. In contrast to NO, direct ER stress induced by SERCA inhibition causes activation of ER stress signaling pathways and elicit proapoptotic signaling via NFkappaB and Bax.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Fluorescence response of H₂DCFDA to various reactive oxygen and reactive nitrogen species [28].

<p>Fluorescence response of H₂DCFDA to various reactive oxygen and reactive nitrogen species <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013341#pone.0013341-Setsukinai1" target="_blank">[28]</a>.</p

Prevention of IL-1 mediated ROS in INS-1 cells by an IKKβ-inhibitor.

<p><i>A</i>: ROS was measured in Krebs-Ringer bicarbonate buffer with 10 µM fluorescent probe CM-H2DCFDA (Molecular probes) following incubation of the cells with and without IL-1β for 4, 8, 16 and 24 hours. Results are shown as delta fluorescence (t 90 min – t45 min). <i>B</i>: ROS was measured after 24 hours incubation in the presence or absence of IL-1β and the IKKβ-inhibitor. <i>C</i>: Microscopy of INS-1 after 24 hours incubation in the presence or absence of IL-1β and the IKKβ-inhibitor (magnification x10). Phase contrast (upper photos) and fluorescence (lower photos) after incubation with 10 µM fluorescent probe (CM-H₂DCFDA). All data are shown as the mean ± S.E.M. (*p<0,05; **p<0,01; ***p<0,001).</p

IKKβ inhibition prevents diet induced diabetes in <i>P. obesus</i>.

<p>Diabetes-prone male and female <i>P. obesus</i>, age of 13–14 weeks, were transferred from <i>ad libitum</i> low energy diet to <i>ad libitum</i> high energy diet. At the same day (day 0) treatment was initiated with vehicle (open circles n = 12) or with the IKKβ-inhibitor (black circles n = 10). <i>A</i>: Body weight was measured daily. <i>B</i>: Non fasting blood glucose was measured twice weekly. <i>C</i>: HbA1c was measured once weekly. <i>D</i>: Insulin was measured at day 0, 11 and then twice a week through the rest of the study. All data are shown as the mean ± S.E.M. (*p<0,05; **p<0,01; ***p<0,001).</p

Islets from <i>P. obesus</i> are rescued from of IL-1β induced ROS production and diminished insulin production by an IKKβ-inhibitor.

<p><i>A</i>: Islets from healthy adult <i>P. obesus</i> were incubated in the presence and absence of L-1β and the IKKβ-inhibitor for 24 hours before ROS was measured. <i>B</i>: GSIS was performed after 6 days incubation in the present or absent of IL-1β and the IKKβ-inhibitor. All data are shown as the mean ± S.E.M. (*p<0,05; **p<0,01; ***p<0,001).</p

Prevention of IL-1 mediated beta cell toxicity in INS-1 cells by an IKKβ-inhibitor.

<p>Rat insulinoma (INS-1) cells were incubated in the presence and absence of IL-1β and the IKKβ-inhibitor for 48 hours. <i>A</i>: mitochoiondrial activity, a measurement of viability was determined by the MTT assay (Promega, USA). <i>B</i>: Cell death was measure as caspase 3/7 activity. <i>C</i>: iNOS mRNA expression was quantified using RT-PCR after cells had been incubation for 24 hours in the presence and absence of IL-1β and the IKKβ-inhibitor. <i>D</i>: Accumulated Insulin in the media was measured after 48 hours incubation in the presence or absence of IL-1β and the IKKβ-inhibitor. <i>E</i>: GSIS was performed after 48 hours incubation in the presence or absence of IL-1β and the IKKβ-inhibitor. All data are shown as the mean ± S.E.M. (*p<0,05; **p<0,01; ***p<0,001).</p

Prevention of IL-1β mediated ROS in newborn rat islets of Langerhans by an IKKβ-inhibitor correlates with transcription of iNOS.

<p><i>A</i>: In newborn rat islets ROS was measured after 24 hours incubation in the presence or absence of IL-1β and the IKKβ-inhibitor. <i>B</i>: iNOS mRNA expression was quantified using RT-PCR after the islets had been incubation for 24 hours in the presence and absence of IL-1β and the IKKβ-inhibitor. All data are shown as the mean ± S.E.M. (*p<0,05; **p<0,01; ***p<0,001).</p