Lipopolysaccharides Impair Insulin Gene Expression in Isolated Islets of Langerhans via Toll-Like Receptor-4 and NF-κB Signalling

BACKGROUND:Type 2 diabetes is characterized by pancreatic β-cell dysfunction and is associated with low-grade inflammation. Recent observations suggest that the signalling cascade activated by lipopolysaccharides (LPS) binding to Toll-Like Receptor 4 (TLR4) exerts deleterious effects on pancreatic β-cell function; however, the molecular mechanisms of these effects are incompletely understood. In this study, we tested the hypothesis that LPS alters insulin gene expression via TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in islets. METHODOLOGY/PRINCIPAL FINDINGS:A 24-h exposure of isolated human, rat and mouse islets of Langerhans to LPS dose-dependently reduced insulin gene expression. This was associated in mouse and rat islets with decreased mRNA expression of pancreas-duodenum homebox-1 (PDX-1) and mammalian homologue of avian MafA/l-Maf (MafA). Accordingly, LPS exposure also decreased glucose-induced insulin secretion. LPS repression of insulin, PDX-1 and MafA expression, as well as its inhibition of insulin secretion, were not observed in islets from TLR4-deficient mice. LPS inhibition of β-cell gene expression in rat islets was prevented by inhibition of the NF-κB pathway, but not the p38 mitogen-activated protein kinase (p38 MAPK) pathway. CONCLUSIONS/SIGNIFICANCE:Our findings demonstrate that LPS inhibit β-cell gene expression in a TLR4-dependent manner and via NF-κB signaling in pancreatic islets, suggesting a novel mechanism by which the gut microbiota might affect pancreatic β-cell function

Exposure to LPS dose-dependently represses insulin pre-mRNA expression in isolated rat and human islets.

<p>Insulin pre-mRNA levels in response to increasing doses of LPS in isolated rat (A) and human (B) islets. Pre-mRNA levels were measured by RT-PCR and normalized to β-actin mRNA levels. Data are mean ± S.E.M. of 2–6 independent experiments; *p<0.05 vs 0 pg/mL.</p

Potential mechanism by which LPS repress insulin gene expression in isolated islets.

<p>Exposure to LPS activates the NF-κB pathway in isolated islets and inhibits the expression of insulin, PDX-1 and MafA. The decrease in insulin expression might indirectly results from LPS inhibition of PDX-1 and MafA. NF-κB could also inhibit insulin gene expression by interacting with other proteins such as C/EBPβ and/or CREB, as observed in other cell types.</p

Inhibition of NF-κB, but not p38 MAPK, restores insulin, PDX-1, and MafA gene expression in islets exposed to LPS.

<p>(A) Insulin pre-mRNA, (B) PDX-1 mRNA and (C) MafA mRNA expression in isolated rat islets exposed for 24 h to 16.7 mM (16.7 G) glucose in the presence or absence of 10 ng/mL LPS or 0.5 ng/mL IL-1β with or without SB202190 (10 µM) and IKK-2 Inh IV (10 µM). Data are mean ± S.E.M. of 3–4 independent experiments; *p<0.05.</p

PDX-1 cellular localization is not altered in response to LPS.

<p>HIT-T15 cells were transfected with a construct encoding a PDX-1-GFP fusion protein. PDX-1 localization (green) (A–F) was visualized by GFP fluorescence using a laser-scanning confocal microscope in cells cultured in 0.1 and 5 mM glucose with or without 0.5 mM palmitate or increasing doses of LPS. 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used for nuclear staining (G–L). Images are representative of 3 replicate experiments.</p

PI3 kinases p110α and PI3K-C2β negatively regulate cAMP via PDE3/8 to control insulin secretion in mouse and human islets

Objectives: Phosphatidylinositol-3-OH kinase (PI3K) signalling in the endocrine pancreas contributes to glycaemic control. However, the mechanism by which PI3K modulates insulin secretion from the pancreatic beta cell is poorly understood. Thus, our objective was two-fold; to determine the signalling pathway by which acute PI3K inhibition enhances glucose-stimulated insulin secretion (GSIS) and to examine the role of this pathway in islets from type-2 diabetic (T2D) donors. Methods: Isolated islets from mice and non-diabetic or T2D human donors, or INS 832/13 cells, were treated with inhibitors of PI3K and/or phosphodiesterases (PDEs). The expression of PI3K-C2β was knocked down using siRNA. We measured insulin release, single-cell exocytosis, intracellular Ca2+ responses ([Ca2+]i) and Ca2+ channel currents, intracellular cAMP concentrations ([cAMP]i), and activation of cAMP-dependent protein kinase A (PKA) and protein kinase B (PKB/AKT). Results: The non-specific PI3K inhibitor wortmannin amplifies GSIS, raises [cAMP]i and activates PKA, but is without effect in T2D islets. Direct inhibition of specific PDE isoforms demonstrates a role for PDE3 (in humans and mice) and PDE8 (in mice) downstream of PI3K, and restores glucose-responsiveness of T2D islets. We implicate a role for the Class II PI3K catalytic isoform PI3K-C2β in this effect by limiting beta cell exocytosis. Conclusions: PI3K limits GSIS via PDE3 in human islets. While inhibition of p110α or PIK-C2β signalling per se, may promote nutrient-stimulated insulin release, we now suggest that this signalling pathway is perturbed in islets from T2D donors. Author Video: Author Video Watch what authors say about their articles Keywords: PI3K, cAMP, PDE, Beta cells, Insulin secretion, T2

Cannabidiol Suppresses Cytokine Storm and Protects Against Cardiac and Renal Injury Associated with Sepsis.

Cytokine release syndrome, also termed "cytokine storm," is the leading cause of morbidity and mortality among patients with various conditions such as sepsis. While cytokine storm is associated with multiple organ damage, acute cardiac and renal injury represents a hallmark of cytokine storm. Since recent reports have suggested that cannabidiol (CBD) may assist in the treatment of inflammatory diseases, our objective was to examine the effect of CBD on cytokine storm-induced cardiac and renal injury using the lipopolysaccharide (LPS)-induced sepsis mouse model. At 8 weeks of age, mice were randomly assigned to receive CBD (15 mg/kg) or vehicle one hour before a single injection of either phosphate-buffered saline or LPS (10 mg/kg) for an additional 24 h. Our results show that CBD improves cardiac function and reduces renal injury in a mouse model of cytokine storm. Moreover, our data indicate that CBD significantly reduces systemic and renal inflammation to contribute to the improvements observed in a cytokine storm-model of cardiac and renal injury. Overall, the findings of this study suggest that CBD could be repurposed to reduce morbidity in patients with cytokine storm particularly in severe infections such as sepsis

Adenylosuccinate Is an Insulin Secretagogue Derived from Glucose-Induced Purine Metabolism

SummaryPancreatic islet failure, involving loss of glucose-stimulated insulin secretion (GSIS) from islet β cells, heralds the onset of type 2 diabetes (T2D). To search for mediators of GSIS, we performed metabolomics profiling of the insulinoma cell line 832/13 and uncovered significant glucose-induced changes in purine pathway intermediates, including a decrease in inosine monophosphate (IMP) and an increase in adenylosuccinate (S-AMP), suggesting a regulatory role for the enzyme that links the two metabolites, adenylosuccinate synthase (ADSS). Inhibition of ADSS or a more proximal enzyme in the S-AMP biosynthesis pathway, adenylosuccinate lyase, lowers S-AMP levels and impairs GSIS. Addition of S-AMP to the interior of patch-clamped human β cells amplifies exocytosis, an effect dependent upon expression of sentrin/SUMO-specific protease 1 (SENP1). S-AMP also overcomes the defect in glucose-induced exocytosis in β cells from a human donor with T2D. S-AMP is, thus, an insulin secretagogue capable of reversing β cell dysfunction in T2D

Endothelin Receptor Blocker Reverses Breast Cancer–Induced Cardiac Remodeling

Background: Although some cancer therapies have overt and/or subclinical cardiotoxic effects that increase subsequent cardiovascular risk in breast cancer patients, we have recently shown that the breast tumor itself can also induce cardiac hypertrophy through the activation of the endothelin system to contribute to cardiovascular risk. However, the extent to which the suppression of the activation of the endothelin system could improve cardiac remodeling in breast cancer patients has yet to be investigated. Objectives: We aimed to retrospectively assess the cardiac morphology/function in patients with breast cancer before receiving cancer chemotherapy and to investigate if the suppression of the activation of the endothelin system improves cardiac remodeling in a mouse model of breast cancer. Methods: Our study involved 28 previously studied women with breast cancer (including 24 after tumor resection) before receiving adjuvant therapy and 17 control healthy women. In addition, we explored how the endothelin system contributed to breast cancer–induced cardiac remodeling using a mouse model of breast cancer. Results: Our results indicate that before chemotherapy, breast cancer patients already exhibit relative cardiac remodeling and subclinical cardiac dysfunction, which was associated with the activation of the endothelin system. Importantly, our mouse data also show that the endothelin receptor blocker atrasentan significantly lessened cardiac remodeling and improved cardiac function in a preclinical model of breast cancer. Conclusions: Although our findings should be further examined in other preclinical/clinical models, our data suggest that endothelin receptor blockers may play a role in cardiac health in individuals with breast cancer. (Understanding and Treating Heart Failure With Preserved Ejection Fraction: Novel Mechanisms, Diagnostics and Potential Therapeutics [Alberta HEART]; NCT02052804 and Multidisciplinary Team Intervention in Cardio-Oncology [TITAN]; NCT01621659