In the present study mechanism of aberrant GSIS were delineated by protein profiling of insulin-producing cells cultured at elevated levels of glucose and palmitate using SELDI-TOF-MS and 2D-PAGE.
INS-1E cells cultured at control condition of 5.5 mM glucose responded to stimulatory glucose concentration, GSIS, with approximately 10-fold enhanced insulin secretion over a 30-min period compared to basal insulin secretion, which is comparable to the secretory response of human islets [16, 17]. However, in individuals with T2DM, GSIS is severely reduced or even absent [2, 17, 18]. Such alterations in the dynamic range of insulin levels are the result of lowered insulin release at stimulatory glucose concentrations combined or not with elevated insulin secretion at non-stimulatory glucose levels, the basal insulin secretion. Whereas absent initial rise of insulin release is a hallmark of T2DM [2, 18, 19], elevated basal insulin release is characteristic of T2DM individuals with insulin resistance . The latter individuals display both hyperglycemia and hyperlipidemia, which made us investigate how elevated levels of glucose and fatty acid palmitate affected the insulin-producing cell. Palmitate was chosen based on its effects of elevating basal insulin release [3, 4]. In addition, the fatty acid is associated with particularly β-cell detrimental effects, where the underlying causes have only partly been defined [4, 6–10].
When episodes of hyperglycemia are not extended, granular exocytosis is balanced by formation of new insulin granules resulting in maintained insulin content . In the present study, insulin content was severely reduced after extended hyperglycemia similar to what was previously observed in INS-1E cells and primary islets [22, 23]. In contrast, palmitate did not affect insulin content. De-granulation has been suggested to be a main contributor to the loss of GSIS. However, cells cultured in the presence of 20 mM glucose and palmitate released similar amounts of insulin in response to a stimulatory glucose concentration as control cells, despite reduced insulin content. We concluded that other mechanism than lowered amounts of insulin granules must also be operational in the aberrant insulin release observed from β-cells cultured at elevated glucose alone or in combination with palmitate. To address mechanisms responsible for the inability to maintain hormonal stores and to delineate other processes contributing to the aberrant insulin release, glucose- and palmitate-regulated β-cell proteins were identified by protein profiling.
Profiling was conducted with SELDI-TOF-MS and 2D-PAGE to address how elevated levels of glucose and palmitate affected the β-cell proteome. The combination of the approaches was triggered by the preferential detection of proteins in the low molecular range by SELDI-TOF-MS and high molecular range by 2D-PAGE [14, 24]. Indeed, in the present study more than 80% of the peaks/spots detected by SELDI-TOF-MS or 2D-PAGE were in the 4–20 kDa or 20–150 kDa range, respectively. Among these peaks and spots differential expression was discovered.
To elucidate mechanisms by which elevated levels of the nutrients incur alterations in β-cell secretory function, the identification of the differentially expressed proteins is essential. Whereas such proteins are inherently separated by 2D-PAGE and can be subjected to PMF, differentially expressed proteins obtained by SELDI-TOF-MS need first to be separated and enriched. The higher number of identified proteins derived from the 2D-PAGE approach reflects this condition. On the other hand when changes in complex protein patterns are studied and the identities of the components are not demanded, SELDI-TOF-MS is superior to 2D-PAGE . In addition, the amount of cells required to generate mass spectra and gels by the two approaches, respectively, was about two orders of magnitude higher when utilizing 2D-PAGE. Although not a consideration when profiling cell lines, the enhanced sensitivity in SELDI-TOF-MS is crucial when analyzing protein patterns of scarce tissue e.g. human islets .
In the present study, several proteins were differentially expressed in response to elevated levels of glucose. The up-regulation of glycolytic enzymes phosphoglycerate kinase 1 and pyruvate kinase 3 together with enhanced expression of NADH dehydrogenase flavoprotein 1 of complex I of the respiratory chain indicate enhanced glucose metabolism. Under such conditions formation of reactive oxygen species (ROS) is enhanced . It was therefore not surprising that glucose-6-phosphate dehydrogenase was up-regulated. This enzyme is induced by elevated ROS and determines production of nicotineamide adenine dinucleotide phosphate hydrogen (NADPH) via shuttling of glucose through the pentose phosphate shunt. All major ROS-metabolic enzymes, directly or indirectly depend on NADPH . The glucose-induced proteins glycyl-tRNA synthetase (gars) and heterogenous nuclear ribonucleoprotein A3 (hnrnp A3) both serve regulatory functions in protein translation. Whereas gars catalyzes the synthesis of glycyl-tRNA, which is required to insert glycine into proteins , hnrnp A3 is involved in pre-mRNA processing, transcriptional regulation, recombination and telomere maintenance . Further effects on protein synthesis were indicated by the up-regulated endoplasmic reticulum (ER)-associated alpha glucosidase 2 and cytoplasmic t-complex protein 1. Both proteins take part in protein folding where the former enzyme is an important part of the calnexin/calreticulin protein synthesis quality control system  and the latter acts as a chaperone with folding capacity . The importance of glucose-induced ROS and its negative effects on proteins was also indicated by up-regulation of the lon protein, which is a mitochondrial ATP-dependent protease engaged in mitochondrial protein degradation by recognizing unfolded proteins [31, 32].
However, proteasomal degradation may also be enhanced as indicated by the rise in proteasome p45. In this context accumulation of insulin and other granular proteins in the ER may play a role as indicated by the glucose-induced up-regulation of rab2. This small GTPase decreases vesicular transport between the ER and the Golgi complex when induced . Indeed, glucose-induced rab2 with ensuing impaired anterograde transport of secretory granule protein precursors may contribute to explain the observed decrease in GSIS and insulin content. The latter findings are in contrast to when islets were exposed to elevated glucose levels for a shorter time period . When such islets were protein profiled manifestations of enhanced granular formation maturation and trafficking were obtained. The lowered hormonal levels obtained in cells exposed to prolonged elevated glucose levels indicate that protein degradation is surpassing synthesis, where the latter may even be attenuated. Such cellular reactions are observed as manifestations of the unfolded protein response (UPR), which is a cellular adaptive response systems initiated when the protein synthesis machinery is compromised . Indeed, initiation of the UPR has been observed in β-cells exposed to prolonged elevated glucose levels . In such cells, induction of lipogenic enzymes resulting in lipid accumulation was proposed to explain the impaired GSIS. When the lipogenic expression was decreased, GSIS was improved. The improvement of the stimulatory secretory levels in the present study when including the fatty acid palmitate during the acute exposure may therefore be explained by maintaining fatty acid cellular levels, which are critical for optimal GSIS .
Increased basal insulin release was observed when cells were cultured in the presence of palmitate, confirming previous studies in INS-1E cells [4, 12, 37] and isolated islets . Several β-cell proteins were regulated by palmitate. One of these proteins was identified as calmodulin. This protein is likely to undergo post-translational modifications such as phosphorylation , which may account for the three separate peaks observed. Calmodulin in INS-1E cells was lowered when palmitate was added to culture medium containing 20 mM glucose. Its role in insulin secretion is pleiotropic including regulation of the cytoplasmic Ca2+ concentration by controlling Ca2+-ATPases of the plasma membrane and the ER [39, 40], which has effects on mobilization and exocytosis of insulin granules [41, 42]. In addition, calmodulin is regulating kinase and phospahatase activities via Ca2+/calmodulin-dependent protein kinases and calcineurin, respectively [43, 44]. In a previous study, we observed that palmitate-induced rise in basal insulin secretion was counteracted in INS-1E cells over-expressing CPT1 in a regulated manner . The role of calmodulin for this partial normalization of basal insulin release appears to be minimal since calmodulin levels were not affected by over-expressing the fatty acid transporter. Instead, over-expression of CPT1 normalized a 7.14 kDa protein, which was highly elevated in cells exposed to augmented levels of palmitate or glucose. The beneficial effects of CPT1 over-expression was accompanied by alleviation of apoptosis and attenuation of UPR-markers phosphorylated eIF2α and pro-apoptotic CHOP/GADD153 . Factors contributing to elevated basal insulin secretion and palmitate-induced alterations in GSIS have instead been attributed to altered expression of enzymes of glucose metabolism including glucokinase, phosphofructokinase and pyruvate dehydrogenase, and of the fatty acid receptor GPR40 [45–47].