Combined transcriptome and proteome profiling of the pancreatic ß-cell response to palmitate unveils key pathways of ß-cell lipotoxicity.

BACKGROUND: Prolonged exposure to elevated free fatty acids induces ß-cell failure (lipotoxicity) and contributes to the pathogenesis of type 2 diabetes. In vitro exposure of ß-cells to the saturated free fatty acid palmitate is a valuable model of lipotoxicity, reproducing features of ß-cell failure observed in type 2 diabetes. In order to map the ß-cell response to lipotoxicity, we combined RNA-sequencing of palmitate-treated human islets with iTRAQ proteomics of insulin-secreting INS-1E cells following a time course exposure to palmitate. RESULTS: Crossing transcriptome and proteome of palmitate-treated ß-cells revealed 85 upregulated and 122 downregulated genes at both transcript and protein level. Pathway analysis identified lipid metabolism, oxidative stress, amino-acid metabolism and cell cycle pathways among the most enriched palmitate-modified pathways. Palmitate induced gene expression changes compatible with increased free fatty acid mitochondrial import and ß-oxidation, decreased lipogenesis and modified cholesterol transport. Palmitate modified genes regulating endoplasmic reticulum (ER) function, ER-to-Golgi transport and ER stress pathways. Furthermore, palmitate modulated cAMP/protein kinase A (PKA) signaling, inhibiting expression of PKA anchoring proteins and downregulating the GLP-1 receptor. SLC7 family amino-acid transporters were upregulated in response to palmitate but this induction did not contribute to ß-cell demise. To unravel critical mediators of lipotoxicity upstream of the palmitate-modified genes, we identified overrepresented transcription factor binding sites and performed network inference analysis. These identified LXR, PPARα, FOXO1 and BACH1 as key transcription factors orchestrating the metabolic and oxidative stress responses to palmitate. CONCLUSIONS: This is the first study to combine transcriptomic and sensitive time course proteomic profiling of palmitate-exposed ß-cells. Our results provide comprehensive insight into gene and protein expression changes, corroborating and expanding beyond previous findings. The identification of critical drivers and pathways of the ß-cell lipotoxic response points to novel therapeutic targets for type 2 diabetes.

Mitogen-activated protein kinases and protein phosphatase 5 mediate glucocorticoid-induced cytotoxicity in pancreatic islets and ß-cells.

Glucocorticoid excess is associated with glucose intolerance and diabetes. In addition to inducing insulin resistance, glucocorticoids impair ß-cell function and cause ß-cell apoptosis. In this study we show that dexamethasone activates mitogen-activated protein kinases (MAPKs) signaling in MIN6 ß-cells, as evident by enhanced phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK). In contrast, the integrated stress response pathway was inhibited by dexamethasone. A p38 MAPK inhibitor attenuated dexamethasone-induced apoptosis in ß-cells and isolated islets and decreased glucocorticoid receptor phosphorylation at S220. In contrast, a JNK inhibitor augmented DNA fragmentation and dexamethasone-induced formation of cleaved caspase 3. We also show that inhibition of protein phosphatase 5 (PP5) augments apoptosis in dexamethasone-exposed islets and ß-cells, with a concomitant activation of p38 MAPK. In conclusion, our data provide evidence that in islets and ß-cells, p38 MAPK and JNK phosphorylation work in concert with PP5 to regulate the cytotoxic effects exerted by glucocorticoids.

ß-Cell adaptation in a mouse model of glucocorticoid-induced metabolic syndrome.

Glucocorticoids (GCs) are stress hormones primarily responsible for mobilizing glucose to the circulation. Due to this effect, insulin resistance and glucose intolerance are concerns in patients with endogenous overproduction of GCs and in patients prescribed GC-based therapy. In addition, hypercortisolemic conditions share many characteristics with the metabolic syndrome. This study reports on a thorough characterization, in terms of glucose control and lipid handling, of a mouse model where corticosterone is given via the drinking water. C57BL/6J mice were treated with corticosterone (100 or 25 µg/ml) or vehicle in their drinking water for 5 weeks after which they were subjected to insulin or glucose tolerance tests. GC-treated mice displayed increased food intake, body weight gain, and central fat deposit accumulations. In addition, the GC treatment led to dyslipidemia as well as accumulation of ectopic fat in the liver and skeletal muscle, having a substantial negative effect on insulin sensitivity. Also glucose intolerance and hypertension, both part of the metabolic syndrome, were evident in the GC-treated mice. However, the observed effects of corticosterone were reversed after drug removal. Furthermore, this study reveals insights into ß-cell adaptation to the GC-induced insulin resistance. Increased pancreatic islet volume due to cell proliferation, increased insulin secretion capacity, and increased islet chaperone expression were found in GC-treated animals. This model mimics the human metabolic syndrome. It could be a valuable model for studying the complex mechanisms behind the development of the metabolic syndrome and type 2 diabetes, as well as the multifaceted relations between GC excess and disease.

Thapsigargin down-regulates protein levels of GRP78/BiP in INS-1E cells.

Pancreatic ß-cells have a well-developed endoplasmic reticulum (ER) and express large amounts of chaperones and protein disulfide isomerases (PDI) to meet the high demand for synthesis of proteins. We have observed an unexpected decrease in chaperone protein level in the ß-cell model INS-1E after exposure to the ER stress inducing agent thapsigargin. As these cells are a commonly used model for primary ß-cells and has been shown to be vulnerable to ER stress, we hypothesize these cells are incapable of mounting a chaperone defense upon activation of ER stress. To investigate the chaperone expression during an ER stress response, induced by thapsigargin in INS-1E cells, we used quantitative mass spectrometry based proteomics. The results displayed a decrease of GRP78/BiP, PDIA3 and PDIA6. Decrease of GRP78/BiP was verified by Western blot and occurred in parallel with enhanced levels of p-eIF2α and CHOP. In contrast to INS-1E cells, GRP78/BiP was not decreased in MIN6 cell or rat and mouse islets after thapsigargin exposure. Investigation of the decreased protein levels of GRP78/BiP indicates that this is not a consequence of reduced mRNA expression. Rather the reduction results from the combined effect of reduced protein synthesis and enhanced proteosomal degradation and possibly also degradation via autophagy. Induction of ER stress with thapsigargin leads to lower protein levels of GRP78/BiP, PDIA3 and PDIA6 in INS-1E cells which may contribute to the susceptibility of ER stress in this ß-cell model.