Ernest Sargsyan

FFAR1 Is Involved in Both the Acute and Chronic Effects of Palmitate on Insulin Secretion

Free fatty acids (FFAs) have pleiotropic effects on the pancreatic β-cell. Although acute exposure to FFAs stimulates glucose-stimulated insulin secretion (GSIS), prolonged exposure impairs GSIS and causes apoptosis. FFAs exert their effects both via intracellular metabolism and interaction with the FFA receptor 1 (FFAR1/GPR40). Here we studied the role of FFAR1 in acute and long-term effects of palmitate on GSIS and insulin content in isolated human islets by using the FFAR1 agonist TAK-875 and the antagonist ANT203. Acute palmitate exposure potentiated GSIS approximately 3-fold, whereas addition of the antagonist decreased this potentiation to approximately 2-fold. In the absence of palmitate, the agonist caused a 40% increase in GSIS. Treatment with palmitate for 7 days decreased GSIS to 70% and insulin content to 25% of control level. These negative effects of long-term exposure to palmitate were ameliorated by FFAR1 inhibition and further aggravated by additional stimulation of the receptor. In the absence of extracellularly applied palmitate, long-term treatment with the agonist caused a modest increase in GSIS. The protective effect of FFAR1 inhibition was verified by using FFAR1-deficient MIN6 cells. Improved β-cell function by the antagonist was paralleled by the decreased apoptosis and lowered oxidation of palmitate, which may represent the potential mechanisms of protection. We conclude that FFAR1 in the pancreatic β-cell plays a substantial role not only in acute potentiation of GSIS by palmitate but also in the negative long-term effects of palmitate on GSIS and insulin content.

Diazoxide-induced β-cell rest reduces endoplasmic reticulum stress in lipotoxic β-cells

Elevated levels of glucose and lipids are characteristics of individuals with type 2 diabetes mellitus (T2DM). The enhanced nutrient levels have been connected with deterioration of beta-cell function and impaired insulin secretion observed in these individuals. A strategy to improve beta-cell function in individuals with T2DM has been intermittent administration of K(ATP) channel openers. After such treatment, both the magnitude and kinetics of insulin secretion are markedly improved. In an attempt to further delineate mechanisms of how openers of K(ATP) channels improve beta-cell function, the effects of diazoxide on markers of endoplasmic reticulum (ER) stress was determined in beta-cells exposed to the fatty acid palmitate. The eukaryotic translation factor 2-alpha kinase 3 (EIF2AK3; also known as PERK) and endoplasmic reticulum to nucleus signaling 1 (ERN1; also known as IRE1) pathways, but not the activating transcription factor (ATF6) pathway of the unfolded protein response, are activated in such lipotoxic beta-cells. Inclusion of diazoxide during culture attenuated activation of the EIF2AK3 pathway but not the ERN1 pathway. This attenuation was associated with reduced levels of DNA-damage inducible transcript 3 (DDIT3; also known as CHOP) and beta-cell apoptosis was decreased. It is concluded that reduction of ER stress may be a mechanism by which diazoxide improves beta-cell function.

AMP-activated protein kinase agonist dose dependently improves function and reduces apoptosis in glucotoxic β-cells without changing triglyceride levels

Prolonged hyperglycaemia leads to impaired glucose-stimulated insulin secretion (GSIS) and apoptosis in insulin-producing beta-cells. The detrimental effects have been connected with glucose-induced lipid accumulation in the beta-cell. AMP-activated protein kinase (AMPK) agonist, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), promotes utilization of nutrient stores for energy production. It was tested how impaired GSIS and elevated apoptosis observed in insulinoma (INS)-1E cells after prolonged culture at 27 mM glucose were affected by the inclusion of 0.3 or 1 mM AICAR during culture. Glucose-induced impairment of insulin release was reverted by the inclusion of 0.3 but not 1 mM AICAR, which did not affect insulin content. The glucose-induced rise in triglyceride (TG) content observed in the cells cultured at 27 mM glucose was not altered by the inclusion of either 0.3 or 1 mM AICAR. Inclusion of 1 but not 0.3 mM AICAR during culture induced phosphorylation of AMPK and its downstream target acyl-CoA carboxylase. Phosphorylation was paralleled by reduced number of apoptotic cells and lowered expression of pro-apoptotic C/EBP homologous protein (CHOP). In conclusion, AICAR dose dependently improves beta-cell function and reduces apoptosis in beta-cells exposed to prolonged hyperglycaemia without changing TG levels.

Lipotoxicity is glucose-dependent in INS-1E cells but not in human islets and MIN6 cells.

BackgroundProlonged elevated levels of lipids have negative effects on beta-cell function and mass (lipotoxicity). To what extent exposure to high glucose concentration is important in the harmful effects of lipids (glucolipotoxicity) has been debated.MethodsWe addressed beta-cell lipotoxicity by measuring apoptosis in isolated intact control human islets and insulin-secreting cell lines MIN6 and INS-1E cultured in the presence of palmitate and low (5.5 mM) or high (25 mM) glucose for 48 hours.ResultsIn both cell lines and human islets palmitate induced apoptosis after culture at low glucose. Palmitate-induced apoptosis was not increased after culture at high compared to low glucose in human islets and MIN6 cells but glucose-induced rise in apoptosis was observed in INS-1E cells. The rise in apoptosis in INS-1E cells was partially reversed by inclusion of AMPK-agonist AICAR. When CPT1-inhibitor etomoxir was included during culture at low glucose palmitate-triggered apoptosis was accentuated both in the islets and the cell lines. Palmitate oxidation in human islets and the cell lines was comparable after culture at low glucose. At high glucose, palmitate oxidation was reduced by 30% in human islets and MIN6 cells but by 80% in INS-1E cells. In INS-1E cells, AICAR increased oxidation of palmitate. Presence of etomoxir at low glucose decreased palmitate oxidation both in the islets and the cell lines.ConclusionsIn summary, lipotoxicity is evident not only in the presence of high but also low glucose concentrations. Additional effects of glucose are prominent in INS-1E but not in MIN6 cells and intact control human islets, which are able to efficiently oxidize fatty acids at high glucose and in this way avoid glucolipotoxicity.

Glucolipotoxicity in INS-1E cells is counteracted by carnitine palmitoyltransferase 1 over-expression.

Effects of non-esterified fatty acids (FAs) are accentuated when applied together with elevated glucose through preferential use of glucose as fuel, which leads to decreased oxidation of FAs. We examined how over-expression of the mitochondrial FA transporter carnitine palmitoyltransferase 1 (CPT1) affects glucose-stimulated insulin secretion (GSIS), apoptosis and ER stress in INS-1E cells cultured in the presence of elevated levels of glucose and palmitate. INS-1E cells were infected with Tet-ON regulated adenovirus containing CPT1 and cultured for 48h in the presence of 0.5mM palmitate and 20mM glucose. Over-expressing CPT1 lowered basal insulin secretion in a dose-dependent manner thereby improving GSIS from INS-1E cells. Also, apoptosis was alleviated and ER-stress markers p-eIF2alpha and CHOP were decreased in cells over-expressing CPT1. We conclude that regulated over-expression of CPT1 is beneficial for glucolipotoxic beta-cells.

Palmitate-induced impairments of β-cell function are linked with generation of specific ceramide species via acylation of sphingosine.

Prolonged exposure to palmitate impairs β-cell function and mass. One of the proposed mechanisms is alteration in ceramide (Cer) generation. In the present study, exposure to palmitate induced the level of palmitoyl transferase and Cer synthases, enzymes of the Cer de novo and salvage pathways, and doubled total Cer levels, which was associated with decreased insulin secretion and augmented apoptosis in MIN6 cells and human islets. By inhibiting enzymes of the pathways pharmacologically with myriocin (ISP-1) or fumonisin B1 or by small interfering RNA (siRNA), we showed that Cer(14:0), Cer(16:0), Cer(20:1), and Cer(24:0) species, generated by the salvage pathway, are linked to the harmful effect of palmitate on β-cells. Oleate attenuates negative effects of palmitate on β-cells. When oleate was included during culture of MIN6 cells with palmitate, the palmitate-induced up-regulation of the enzymes of the de novo and salvage pathways was prevented resulting in normalized levels of all Cer species except Cer(20:1). Our data suggest that enhanced Cer generation in response to elevated palmitate levels involves both de novo and salvage pathways. However, the negative effects of palmitate on β-cells are attributed to generation of Cer species Cer(14:0), Cer(16:0), and Cer(24:0) via acylation of sphingosine.

Free fatty acid receptor 1 (FFAR1/GPR40) signaling affects insulin secretion by enhancing mitochondrial respiration during palmitate exposure.

Fatty acids affect insulin secretion via metabolism and FFAR1-mediated signaling. Recent reports indicate that these two pathways act synergistically. Still it remains unclear how they interrelate. Taking into account the key role of mitochondria in insulin secretion, we attempted to dissect the metabolic and FFAR1-mediated effects of fatty acids on mitochondrial function. One-hour culture of MIN6 cells with palmitate significantly enhanced mitochondrial respiration. Antagonism or silencing of FFAR1 prevented the palmitate-induced rise in respiration. On the other hand, in the absence of extracellular palmitate FFAR1 agonists caused a modest increase in respiration. Using an agonist of the M3 muscarinic acetylcholine receptor and PKC inhibitor we found that in the presence of the fatty acid mitochondrial respiration is regulated via Gαq protein-coupled receptor signaling. The increase in respiration in palmitate-treated cells was largely due to increased glucose utilization and oxidation. However, glucose utilization was not dependent on FFAR1 signaling. Collectively, these results indicate that mitochondrial respiration in palmitate-treated cells is enhanced via combined action of intracellular metabolism of the fatty acid and the Gαq-coupled FFAR1 signaling. Long-term palmitate exposure reduced ATP-coupling efficiency of mitochondria and deteriorated insulin secretion. The presence of the FFAR1 antagonist during culture did not improve ATP-coupling efficiency, however, it resulted in enhanced mitochondrial respiration and improved insulin secretion after culture. Taken together, our study demonstrates that during palmitate exposure, integrated actions of fatty acid metabolism and fatty acid-induced FFAR1 signaling on mitochondrial respiration underlie the synergistic action of the two pathways on insulin secretion.

Palmitate-induced changes in protein expression of insulin secreting INS-1E cells.

Elevated blood levels of glucose and lipids in individuals with type 2 diabetes mellitus have been observed to cause impairment of insulin secretion from pancreatic beta-cells. Chronic exposure to either of the circulating fatty acid oleate or palmitate has different effects on the beta-cell. Whereas palmitate causes functional impairment of the beta-cell and apoptosis, oleate has only minor negative effects on beta-cell function and mass. The aim of the present study was to delineate mechanisms by which the fatty acids affect the beta-cell differently. In particular, the aim was to identify beta-cell proteins exclusively regulated by palmitate. INS-1E cells were cultured for 24h in medium supplemented with palmitate or oleate. After culture, cells were lysed and subjected to two-dimensional gel electrophoresis. Proteins specifically regulated by palmitate were excised from the gel and identified by peptide mass fingerprinting using MALDI-TOF MS. Proteins exclusively regulated by palmitate were classified into proteins of carbohydrate or protein metabolism and Ca(2+) or mRNA binding proteins. The specific palmitate-induced down-regulation of enzymes of glycolysis, proteins of protein turnover and anti-apoptotic protein may contribute to explain the different effects exerted by palmitate and oleate on beta-cell function and mass.

Initial hyperinsulinemia and subsequent [beta]-cell dysfunction is associated with elevated palmitate levels

Background:The prevalence of obesity-related diabetes in childhood is increasing and circulating levels of nonesterified fatty acids may constitute a link. Here, the association between palmitate and insulin secretion was investigated in vivo and in vitro.Methods:Obese and lean children and adolescents (n = 80) were included. Palmitate was measured at fasting; insulin and glucose during an oral glucose tolerance test (OGTT). Human islets were cultured for 0 to 7 d in presence of 0.5 mmol/l palmitate. Glucose-stimulated insulin secretion (GSIS), insulin content and apoptosis were measured.Results:Obese subjects had fasting palmitate levels between 0.10 and 0.33 mmol/l, with higher average levels compared to lean subjects. While obese children with elevated palmitate (>0.20 mmol/l) had accentuated insulin levels during OGTT, obese adolescents with high palmitate had delayed first-phase insulin response. In human islets exposed to palmitate for 2 d GSIS was twofold enhanced, but after 7 d attenuated. Intracellular insulin content decreased time-dependently in islets cultured in the presence of palmitate and cleaved caspase 3 increased.Conclusion:The rapid accentuated and delayed insulin secretory responses observed in obese children and adolescents, respectively, with high palmitate levels may reflect changes in islet secretory activity and integrity induced by extended exposure to the fatty acid.

Oleate protects beta-cells from the toxic effect of palmitate by activating pro-survival pathways of the ER stress response.

Long-term exposure of beta cells to saturated fatty acids impairs insulin secretion and increases apoptosis. In contrast, unsaturated fatty acids protect beta-cells from the long-term negative effects of saturated fatty acids. We aimed to identify the mechanisms underlying this protective action of unsaturated fatty acids. To address the aim, insulin-secreting MIN6 cells were exposed to palmitate in the absence or presence of oleate and analyzed by using nano-LC MS/MS based proteomic approach. Important findings were validated by using alternative approaches. Proteomic analysis identified 34 proteins differentially expressed in the presence of palmitate compared to control samples. These proteins play a role in insulin processing, mitochondrial function, metabolism of biomolecules, calcium homeostasis, exocytosis, receptor signaling, ER protein folding, antioxidant activity and anti-apoptotic function. When oleate was also present during culture, expression of 15 proteins was different from the expression in the presence of palmitate alone. Most of the proteins affected by oleate are targets of the ER stress response and play a pro-survival role in beta cells such as protein folding and antioxidative defence. We conclude that restoration of pro-survival pathways of the ER stress response is a major mechanism underlying the protective effect of unsaturated fatty acids in beta-cells treated with saturated fatty acids.