Abstract
ABSTRACT Macroautophagy/autophagy is critical for the regulation of pancreatic β-cell mass and its deregulation has been implicated in the pathogenesis of type 2 diabetes (T2D). We have previously shown that treatment of pancreatic β-cells with the GLP1R (glucagon like peptide 1 receptor) agonist exendin-4 stimulates autophagic flux in a setting of chronic nutrient excess. The aim of this study was to identify the underlying pathways contributing to enhanced autophagic flux. Pancreatic β-cells (INS-1E),mouse and human islets were treated with glucolipotoxic stress (0.5 mM palmitate and 25 mM glucose) in the presence of exendin-4. Consistent with our previous work, exendin-4 stimulated autophagic flux. Using chemical inhibitors and siRNA knockdown, we identified RAPGEF4/EPAC2 (Rap guanine nucleotide exchange factor 4) and downstream calcium signaling to be essential for regulation of autophagic flux by exendin-4. This pathway was independent of AMPK and MTOR signaling. Further analysis identified PPP3/calcineurin and its downstream regulator TFEB (transcription factor EB) as key proteins mediating exendin-4 induced autophagy. Importantly, inhibition of this pathway prevented exendin-4-mediated cell survival and overexpression of TFEB mimicked the cell protective effects of exendin-4 in INS-1E and human islets. Moreover, treatment of db/db mice with exendin-4 for 21 days increased the expression of lysosomal markers within the pancreatic islets. Collectively our data identify the RAPGEF4/EPAC2-calcium-PPP3/calcineurin-TFEB axis as a key mediator of autophagic flux, lysosomal function and cell survival in pancreatic β-cells. Pharmacological modulation of this axis may offer a novel therapeutic target for the treatment of T2D. Abbreviations: AKT1/protein kinase B: AKT serine/threonine kinase 1; AMPK: 5’ AMP-activated protein kinase; CAMKK: calcium/calmodulin-dependent protein kinase kinase; cAMP: cyclic adenosine monophosphate; CASP3: caspase 3; CREB: cAMP response element-binding protein; CTSD: cathepsin D; Ex4: exendin-4(1-39); GLP-1: glucagon like peptide 1; GLP1R: glucagon like peptide 1 receptor; GLT: glucolipotoxicity; INS: insulin; MTOR: mechanistic target of rapamycin kinase; NFAT: nuclear factor of activated T-cells; PPP3/calcineurin: protein phosphatase 3; PRKA/PKA: protein kinase cAMP activated; RAPGEF3/EPAC1: Rap guanine nucleotide exchange factor 3; RAPGEF4/EPAC2: Rap guanine nucleotide exchange factor 4; SQSTM1/p62: sequestosome 1; T2D: type 2 diabetes; TFEB: transcription factor EB
Highlights
Macroautophagy/autophagy is a highly conserved pathway that serves to recycle unwanted or damaged organelles to provide energy to maintain intracellular homeostasis and sus tain core metabolic functions [1,2]
glucagon like peptide 1 receptor (GLP1R) agonists are known to exert their anti-diabetic actions via the activation of numerous signaling pathways, with the PtdIns3K-AKT-mechanistic target of rapamycin kinase nuclear factor of activated T-cells (NFAT) (MTOR) axis being critical for their anti-apoptotic actions [27]
We identify the RAPGEF4-Ca2+-PPP3/calcineurin-transcription factor EB (TFEB) axis as a potential target to improve autophagic flux and β-cell survival (Figure 8E)
Summary
Macroautophagy/autophagy is a highly conserved pathway that serves to recycle unwanted or damaged organelles to provide energy to maintain intracellular homeostasis and sus tain core metabolic functions [1,2]. Classically considered a pro-survival mechanism active during nutrient deprivation, autophagy can serve to remove superfluous nutrients, damaged/dysfunctional organelles or misfolded proteins, in metabolically active tissues [4]. A role for autophagy in regulating cell survival in response to increased circulating nutrients has been proposed [5]. Pancreatic β-cells play a critical role in blood glucose homeostasis through the release of INS (insulin) in response to elevated glucose concentrations [6]. Increased circulating nutrients parti cularly lipid, due to obesity and/or high fat diet, are major drivers in this process [9]. Autophagy appears critical for basal β-cell physiology and survival
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