Glucose metabolism is a key regulator of pancreatic beta cell autophagy
AV Williams, FP Zummo, K Lanyi, JAM Shaw, PE Lovat, C Arden
Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK
Aims Macroautophagy is critical for the regulation of pancreatic beta cell mass. However, the regulation of beta cell autophagy by nutrient availability remains unclear. The aim of the current study was to determine a role for glucose sensing in regulating autophagy in beta cells.
Methods Glucose metabolism was manipulated in INS‐1E and mouse islets using a glucokinase activator (GKA), glucokinase inhibitor (5‐thioglucose) or siRNA against glucokinase (siGK). Autophagy was measured by LC3 I‐II conversion (western blotting) and p62 accumulation (immunostaining).
Results Acute (2h) stimulation of glucose metabolism using a GKA decreased LC3 II (INS‐1E: 37 ± 7%; islets: 29 ± 8%; p < 0.01), while inhibition of glucose metabolism increased LC3 II (5‐thioglucose: 1.97 ± 0.3‐fold (INS‐1E); 1.3 ± 0.1‐fold (islets). siGK: 1.8 ± 0.2‐fold. p < 0.05). This data supports an inhibitory role for glucose metabolism in regulating autophagy. These changes occur simultaneously with changes in mTOR activation suggesting an mTOR dependent mechanism. Exposure to chronic nutrient excess (0.5mM palmitate −/+ 25mM glucose, 24h) impaired autophagic flux evidenced by increased LC3 II (INS‐1E: 2.6 ± 0.1‐fold; mouse islets: 2.3 ± 0.5‐fold; p < 0.05) with p62 accumulation (INS‐1E: 2.1 ± 0.5‐fold, p < 0.05). The addition of GKA for the final 2h partially reversed the nutrient excess‐mediated increase in both LC3 II (control: 1.6 ± 0.1‐fold; GKA: 1.1 ± 0.3‐fold) and cell death (control: 13.9 ± 1.4‐fold; GKA: 11.1 ± 1.7‐fold, p < 0.05). This cytoprotective effect was prevented by downregulation of autophagy (siScr: 29 ± 5%; siATG5: 1.1 ± 0.2%, p < 0.05).
Conclusions Beta cell autophagy is regulated by acute changes in glucose metabolism which impacts on beta cell survival. Understanding these mechanisms is essential to understand the underlying pathways mediating autophagic deregulation in type 2 diabetes.
Chronic administration of a long‐acting glucagon analogue results in enhanced pancreatic islet function in a diet‐induced obesity murine model
K Suba1, Y Patel1, A Martin Alonso1, M Ukwuoma1, V Kalogianni1, I Leclerc1, B Owen1, GA Rutter2, SR Bloom1, V Salem1
1Section of Endocrinology and Investigative Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, Hammersmith Hospital Campus, London, UK, 2Section of Cell Biology, Department of Metabolism, Digestion and Reproduction, Imperial College London, Hammersmith Hospital Campus, London, UK
Aims GLP‐1 receptor (GLP‐1R) and glucagon receptor (GCGR) dual agonists are in development to treat type 2 diabetes but the contribution of the glucagon element to islet function is poorly characterised. We investigated the longitudinal in vivo effects of a long‐acting glucagon analogue G778 on pancreatic islet calcium dynamics.
Methods C57Bl/6 mice with diet‐induced obesity received daily subcutaneous injections of saline or G778 100nmol/kg for 60 days (n = 6/7). A standard intraperitoneal glucose tolerance test was performed at the start and end of the study. All mice had islets with beta cell‐specific expression of GCaMP6f implanted in the anterior chamber of the eye as a direct, longitudinal readout of function in vivo with confocal microscopy. Such islets were separately tested in vitro to measure the effects of G778 on whole‐islet calcium dynamics.
Results At 60 days, there was a non‐significant difference in body weight loss between the saline and G778‐treated groups (0.9g ± 1.1 vs 3.4g ± 0.7 p = 0.07), but a marked improvement in glucose tolerance (15min glucose 15.9mmol/l vs 9.2mmol/l p < 0.001). Islet readouts confirmed a significant increase in calcium signal and faster propagation of calcium waves in the G778‐treated group in vivo which supported in vitro findings that G778 promoted a larger number of calcium waves (a proxy marker of insulin secretion) above 7mM glucose (n = 30 islets across 5 experiments per group, p = 0.05).
Summary Our data suggest that the glucagon element of a glucagon/GLP‐1 analogue can have positive effects on insulin secretory function in the setting of obesity‐associated glucose intolerance, independent of weight loss.
Inhibition of integrin A2B1 negates collagen‐I induced changes in connexin‐mediated hemichannel activity in an in vitro model of diabetic nephropathy
JA Potter, GW Price, CL Cliff, BM Williams, CE Hills, PE Squires
School of Life Sciences, University of Lincoln, Lincoln, UK
Aims Tubulointerstitial fibrosis, the underlying pathology of diabetic nephropathy, is characterised by extracellular matrix accumulation. Aberrant connexin (Cx) expression and increased adenosine triphosphate (ATP) have been linked to inflammation and fibrosis, and we have previously reported that glucose‐evoked transforming growth factor‐beta1 (TGF‐β1) increases connexin‐mediated hemichannel ATP release. While extracellular matrix modification has been linked to altered cell phenotype, this study explores a role for collagen‐I in regulating connexin‐mediated hemichannel activity.
Methods Human kidney (HK‐2) proximal tubule cells were cultured on collagen‐I (50µg/ml) ± TGF‐β1 (10ng/ml) ± anti‐α2β1 integrin neutralising antibody (2.5µg/ml) for 48h. Uncoated plastic served as the control. The expression of candidate proteins was determined by immunoblotting. Carboxyfluorescein (200µM) dye uptake confirmed hemichannel activity.
Results TGF‐β1 decreased Cx26 to 57.0 ± 3.4% and increased collagen‐I expression to 187.0 ± 3.1% as compared to control (n‐4, p < 0.01). Co‐incubation of HK‐2 cells with TGF‐β1 + α2β1 (neutralising antibody against the principle collagen integrin), restored Cx26 expression to 76.2 ± 6.1% (n‐4, p < 0.01). In support of this data, when cultured on collagen‐I, Cx26 expression decreased to 58.7 ± 10.1%; and a further 31.0 ± 8.9% (n‐4, p < 0.01) in the presence of TGF‐β1. When cultured on collagen‐I ± TGF‐β1, HK‐2 cells demonstrated increased carboxyfluorescein uptake to 260.0 ± 10.9% and 182.5 ± 8.1%, respectively, while incubation with neutralising α2β1 antibody significantly reduced dye uptake by 27.0 ± 3.7% and by 24.0 ± 2.6%, respectively, as compared to uncoated control (n‐4, p < 0.01).
Conclusion Collagen‐I ± TGF‐β1 decreases Cx26 expression, an effect paralleled by increased hemichannel activity. Inhibition of integrin α2β1 negates collagen/TGF‐β1 induced dye uptake, suggesting a role for collagen‐I in regulation of connexin‐mediated communication.
Blindness in people with diabetes: Interleukin‐17A can mediate blood–retinal barrier disruption, which is a key mediating mechanism of vision loss
EM Byrne, M Llorián-Salvador, A Margariti, M Chen, H Xu
Wellcome‐Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, UK
Aims Blood–retinal barrier (BRB) breakdown is a key feature of diabetic eye diseases. Only approximately half of patients respond to current treatments, therefore novel therapies are required. Inflammatory cytokines are well‐established mediators of BRB breakdown. IL‐17A is expressed more highly in the eyes of diabetic people than non‐diabetic people. We aimed to understand how interleukin‐17A (IL‐17A) affects the BRB and the mechanisms responsible.
Methods In vitro: ARPE‐19 human retinal pigment epithelium cells were treated with IL‐17A. The expression of ZO‐1, claudin‐5, IL‐17RA and RC, IL‐22R, pSTAT3, STAT3 and beta‐actin were examined by western Blotting. pSTAT3, ZO‐1, beta‐catenin and claudin‐5 were visualised using immunofluorescence. Transepithelial electrical resistance (TEER) and FITC‐Dextran permeability assays functionally assessed barrier function. In vivo: Wild‐type C57BL/6 mice were injected intravitreally (50ng/eye) or intravenously (20ng/g) with IL‐17A. Retinal fundus images injection facilitated identification of retinal damage and vascular leakage. Eyes were collected for leakage assessment.
Results IL‐17A damaged tight junctions in human outer‐retinal barrier cell line, ARPE‐19. Functionally, IL‐17A reduced ARPE‐19 barrier function in terms of TEER and permeability. FITC‐Dextran extravasation was observed in IL‐17A‐injected mice. IL‐17A intravitreal and intravenous injection increased retinal albumin compared to non‐injected controls [p = 0.002 (n = 20), p = 0.0092 (n = 6)]. Alterations in STAT3 phosphorylation were observed in ARPE‐19 cells following exposure to IL‐17A, indicating this pathway is an initial responder to IL‐17A‐mediated barrier breakdown.
Conclusions This study has identified that IL‐17A activates STAT3 in RPE cells, which comprise the outer BRB. Future studies will reveal whether inhibitors of the STAT3 pathway can prevent IL‐17A‐mediated BRB breakdown in vitro and in vivo.