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Type 2 diabetes, which features β-cell failure, is caused by the decrease of β-cell mass and insulin secretory function. Current treatments fail to halt the decrease of functional β-cell mass. Strategies to prevent β-cell apoptosis and dysfunction are highly desirable. Recently, our group and others have reported that blockade of N-methyl-d-aspartate receptors (NMDARs) in the islets has been proposed to prevent the progress of type 2 diabetes through improving β-cell function. It suggests that a sustained activation of the NMDARs may exhibit deleterious effect on β-cells. However, the exact functional impact and mechanism of the sustained NMDAR stimulation on islet β-cells remains unclear. Here, we identify a sustained activation of pancreatic NMDARs as a novel factor of apoptotic β-cell death and function. The sustained treatment with NMDA results in an increase of intracellular [Ca2+] and reactive oxygen species, subsequently induces mitochondrial membrane potential depolarization and a decrease of oxidative phosphorylation expression, and then impairs the mitochondrial function of β-cells. NMDA specifically induces the mitochondrial-dependent pathway of apoptosis in β-cells through upregulation of the proapoptotic Bim and Bax, and downregulation of antiapoptotic Bcl-2. Furthermore, a sustained stimulation of NMDARs impairs β-cell insulin secretion through decrease of pancreatic duodenal homeobox-1 (Pdx-1) and adenosine triphosphate synthesis. The activation of nuclear factor-κB partly contributes to the reduction of Pdx-1 expression induced by overstimulation of NMDARs. In conclusion, we show that the sustained stimulation of NMDARs is a novel mediator of apoptotic signaling and β-cell dysfunction, providing a mechanistic insight into the pathological role of NMDARs activation in diabetes., (Copyright © 2017 Endocrine Society.)

In the nervous system, excessive activation of NMDA receptors causes neuronal injury. Although activation of NMDARs has been proposed to contribute to the progress of diabetes, little is known about the effect of excessive long-term activation of NMDARs on β-cells, especially under the challenge of hyperglycemia. Here we thoroughly investigated whether endogenous glutamate aggravated β-cell dysfunction under chronic exposure to high-glucose via activation of NMDARs. The glutamate level was increased in plasma of diabetic mice or patients and in the supernatant of β-cell lines after treatment with high-glucose for 72 h. Decomposing the released glutamate improved GSIS of β-cells under chronic high-glucose exposure. Long-term treatment of β-cells with NMDA inhibited cell viability and decreased GSIS. These effects were eliminated by GluN1 knockout. The NMDAR antagonist MK-801 or GluN1 knockout prevented high-glucose-induced dysfunction in β-cells. MK-801 also decreased the expression of pro-inflammatory cytokines, and inhibited I-κB degradation, ROS generation and NLRP3 inflammasome expression in β-cells exposed to high-glucose. Furthermore, another NMDAR antagonist, Memantine, improved β-cells function in diabetic mice. Taken together, these findings indicate that an increase of glutamate may contribute to the development of diabetes through excessive activation of NMDARs in β-cells, accelerating β-cells dysfunction and apoptosis induced by hyperglycemia.

G-protein coupled receptor 40 (GPR40) is a membrane-bound G-protein-coupled receptor with high binding affinity to medium- and long-chain free fatty acids (FFAs). Acute activation of GPR40 on pancreatic β-cells causes insulin secretion, whereas prolonged activation may contribute to a deterioration of the effect of saturated FFAs on β-cells. It has been documented that different types of FFAs produce various effects on insulin secretion; however, little information is available regarding the expression of GPR40 and its function after long-term exposure of β-cells to unsaturated FFAs. In the present study, GPR40 expression and function were assessed in INS-1 β-cells after 48 h exposure to different types of unsaturated FFAs. The mRNA and protein expression of GPR40 was increased significantly by long-term exposure of cells to polyunsaturated α-linolenic acid, but not to either oleic acid or linoleic acid. Immunocytochemistry revealed a reduction in the number of insulin-containing granules in cells treated with α-linolenic acid, which was correlated with an increase in cellular expression of GPR40. Basal and glucose-stimulated insulin secretion were markedly suppressed by 48 h treatment of cells with saturated palmitic acid, but not unsaturated α-linolenic acid. By testing various FFAs, it was found that FFA-induced suppression of basal and glucose-stimulated insulin secretion was attenuated by an increase in the degree of unsaturation of the FFAs and GPR40 expression in response to FFA treatment in INS-1 cells. The results of the present study indicate that long-term in vitro treatment of INS-1 rat pancreatic β-cells by unsaturated FFAs protects the cells against from gluco- and lipotoxicities and that this coincides with an increase in GPR40 expression., (© 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Blackwell Publishing Asia Pty Ltd.)

Free fatty acids (FFAs) regulate insulin secretion in a complex pattern and induce pancreatic beta-cell dysfunction in type 2 diabetes. Voltage-dependent Ca2+ channels (VDCC) in beta-cells play a major role in regulating insulin secretion. The aim of present study is to clarify the action of the FFA, linoleic acid, on VDCC in beta-cells. The VDCC current in primary cultured rat beta-cells were recorded under nystatin-perforated whole-cell recording configuration. The VDCC was identified as high-voltage-gated Ca2+ channels due to there being no difference in current amplitude under holding potential between -70 and -40 mV. Linoleic acid (10 microM) significantly inhibited VDCC currents in beta-cells, an effect which was fully reversible upon washout. Methyl-linoleic acid, which does not activate G protein coupled receptor (GPR)40, neither did alter VDCC current in rat beta-cells nor did influence linoleic acid-induced inhibition of VDCC currents. Linoleic acid-induced inhibition of VDCC current was not blocked by preincubation of beta-cells with either the specific protein kinase A (PKA) inhibitor, H89, or the PKC inhibitor, chelerythrine. However, pretreatment of beta-cells with thapsigargin, which depletes intracellular Ca2+ stores, completely abolished linoleic acid-induced decrease in VDCC current. Measurement of intracellular Ca2+ concentration ([Ca2+](i)) illustrated that linoleic acid induced an increase in [Ca2+](i) and that thapsigargin pretreatment inhibited this increase. Methyl-linoleic acid neither did induce increase in [Ca2+](i) nor did it block linoleic acid-induced increase in [Ca2+](i). These results suggest that linoleic acid stimulates Ca2+ release from intracellular Ca2+ stores and inhibits VDCC currents in rat pancreatic beta-cells via Ca2+-induced inactivation of VDCC.

Pancreatic beta-cell dysfunction is an important pathological change in type 2 diabetes, which is tightly related to obesity. However, the direct role of adipose tissue in beta-cell dysfunction has not been well understood. In this study, we examined the effects of 3T3-L1 adipocytes on MIN6 insulin-secreting cells in a co-culture system. MIN6 cells used here kept most of beta-cell functions but less sensitive to glucose stimulation. Tolbutamide, the KATP channel blocker, was therefore used to stimulate insulin secretion in this report. MIN6 cells co-cultured with 3T3-L1 adipocytes had significantly reduced intracellular calcium concentration ([Ca2+]i) and lost the ability to secrete insulin in response to tolbutamide, compared to the control cells. 3T3-L1 adipocytes significantly decreased the expression of insulin, glucokinase and Kir6.2 genes but increased the expression of uncoupling protein-2 (UCP-2) in MIN6 cells after one week of co-culture, as measured by semi-quantitative RT-PCR. 3T3-L1 adipocyte-conditioned medium also significantly decreased insulin secretion and the expression of insulin, glucokinase and Kir6.2 genes in MIN6 cells. The conditioned medium also reduced tyrosine kinase activity in MIN6 cells. The inhibitor of protein tyrosine kinase, genistein, decreased the expression of glucokinase and Kir6.2 in MIN6 cells, while two free fatty acids, oleic acid and linoleic acids, were found to increase UCP-2 expression. The present study demonstrates that 3T3-L1 adipocytes directly impair insulin secretion and the expression of important genes in MIN6 cells. The effects of T3-L1 adipocytes on MIN6 cells are ascribed to secreted bioactive factors and may be mediated via multiple pathways, which include the upregulation of UCP-2 expression via free fatty acids, and downregulation of glucokinase and Kir6.2 expression via decreasing protein tyrosine kinase activity.

Free fatty acids (FFAs), in addition to glucose, have been shown to stimulate insulin release through the G protein-coupled receptor (GPCR)40 receptor in pancreatic beta-cells. Intracellular free calcium concentration ([Ca(2+)](i)) in beta-cells is elevated by FFAs, although the mechanism underlying the [Ca(2+)](i) increase is still unknown. In this study, we investigated the action of linoleic acid on voltage-gated K(+) currents. Nystatin-perforated recordings were performed on identified rat beta-cells. In the presence of nifedipine, tetrodotoxin, and tolbutamide, voltage-gated K(+) currents were observed. The transient current represents less than 5%, whereas the delayed rectifier current comprises more than 95%, of the total K(+) currents. A long-chain unsaturated FFA, linoleic acid (10 microm), reversibly decreased the amplitude of K(+) currents (to less than 10%). This reduction was abolished by the cAMP/protein kinase A system inhibitors H89 (1 microm) and Rp-cAMP (10 microm) but was not affected by protein kinase C inhibitor. In addition, forskolin and 8'-bromo-cAMP induced a similar reduction in the K(+) current as that evoked by linoleic acid. Insulin secretion and cAMP accumulation in beta-cells were also increased by linoleic acid. Methyl linoleate, which has a similar structure to linoleic acid but no binding affinity to GPR40, did not change K(+) currents. Treatment of cultured cells with GPR40-specific small interfering RNA significantly reduced the decrease in K(+) current induced by linoleic acid, whereas the cAMP-induced reduction of K(+) current was not affected. We conclude that linoleic acid reduces the voltage-gated K(+) current in rat beta-cells through GPR40 and the cAMP-protein kinase A system, leading to an increase in [Ca(2+)](i) and insulin secretion.

In addition to serving as an energy reservoir, the adipocyte has been characterized as an endocrine cell, secreting many bioactive factors which influence energy homeostasis. Being overweight, with excessive adipose tissue, is considered to be part of the pathogenesis of type 2 diabetes. Insulin resistance and beta-cell dysfunction are two major pathophysiological changes seen in type 2 diabetes. In addition to inducing insulin resistance in insulin-responsive tissues, adipocyte-derived factors play an important role in the pathogenesis of beta-cell dysfunction. Leptin, free fatty acids, adiponectin, tumor necrosis factor-alpha and interleukin-6 are all produced and secreted by adipocytes, and may directly influence aspects of beta-cell function, including insulin synthesis and secretion, insulin cell survival and apoptosis. During the progression from normal weight to obesity and on to overt diabetes, the adipocyte-derived factors contribute to the occurrence and development of beta-cell dysfunction and type 2 diabetes.