Inhibition of cholinergic potentiation of insulin secretion from pancreatic islets by chronic elevation of glucose and fatty acids: Protection by casein kinase 2 inhibitor

Abstract

Objectives: Chronic hyperlipidemia and hyperglycemia are characteristic features of type 2 diabetes (T2DM) that are thought to cause or contribute to β-cell dysfunction by "glucolipotoxicity." Previously we have shown that acute treatment of pancreatic islets with fatty acids (FA) decreases acetylcholine-potentiated insulin secretion. This acetylcholine response is mediated by M3 muscarinic receptors, which play a key role in regulating β-cell function. Here we examine whether chronic FA exposure also inhibits acetylcholine-potentiated insulin secretion using mouse and human islets.

Methods: Islets were cultured for 3 or 4 days at different glucose concentration with 0.5 mM palmitic acid (PA) or a 2:1 mixture of PA and oleic acid (OA) at 1% albumin (PA/BSA molar ratio 3.3). Afterwards, the response to glucose and acetylcholine were studied in perifusion experiments.

Results: FA-induced impairment of insulin secretion and Ca²⁺ signaling depended strongly on the glucose concentrations of the culture medium. PA and OA in combination reduced acetylcholine potentiation of insulin secretion more than PA alone, both in mouse and human islets, with no evidence of a protective role of OA. In contrast, lipotoxicity was not observed with islets cultured for 3 days in medium containing less than 1 mM glucose and a mixture of glutamine and leucine (7 mM each). High glucose and FAs reduced endoplasmic reticulum (ER) Ca²⁺ storage capacity; however, preserving ER Ca²⁺ by blocking the IP3 receptor with xestospongin C did not protect islets from glucolipotoxic effects on insulin secretion. In contrast, an inhibitor of casein kinase 2 (CK2) protected the glucose dependent acetylcholine potentiation of insulin secretion in mouse and human islets against glucolipotoxicity.

Conclusions: These results show that chronic FA treatment decreases acetylcholine potentiation of insulin secretion and that this effect is strictly glucose dependent and might involve CK2 phosphorylation of β-cell M3 muscarinic receptors.

Keywords: Acetylcholine; Fatty acids; Glucolipotoxicity; Insulin secretion; Pancreatic islets.

Figure 1

Effects of palmitic acid on glucose- and acetylcholine-stimulated insulin release in mouse islets. Islets were cultured for 3 days with or without 0.5 mM palmitate (bound to 1% BSA) and increasing concentrations of glucose: 10 mM (Panel A); 16 mM (Panel D); 25 mM (Panel G). Panels B and C, E and F, H and I: a magnified view of selected sections of the perfusion experiments from Panels A, D and G is presented to clearly show the inhibiting effect of fatty acids on glucose and acetylcholine stimulation of insulin secretion. An acetylcholine (Ach) ramp from 0 to 1 μM (12.5 nM increment/min) was applied after a 90 min islet preperfusion with 0, 4, and 8 mM glucose (G) (30 min for each intervention). 8 mM glucose was present during the acetylcholine ramp. Open circles: cultured with glucose alone; filled circles: glucose plus 0.5 mM palmitate. Effect of glucolipotoxicity on insulin secretion in mouse islets strongly depends on glucose concentration. Note: Here and in all other experiments that include acetylcholine, 10 μM neostigmine, a cholinesterase inhibitor, was used to prevent its breakdown. The sign “+” indicates that acetylcholine was added on top of 8 mM glucose. FAs are not present during the test. Each curve represents the mean ± SE of 3–4 perfusions.

Figure 2

Oleic acid does not protect acetylcholine potentiation of insulin secretion against glucolipotoxicity; CK2 inhibitor completely protected mouse islets from mild and partially from severe glucolipotoxicity. Panel A: Mouse islets were cultured for 3 days with 10 mM glucose and ±0.5 mM of a mixture of PA and OA (2:1 ratio) and then subjected to perifusion experiments as described in Figure 1. The insert is a magnified view of a selected section of the perfusion experiment to clearly show glucose-stimulated insulin release. Panel B: Islets were cultured as described in Panel A except inhibitor of casein kinase 2 (CK2; 10 μM) was added to culture medium. Panel C: Experimental design was same as in Panel A except glucose concentration in culture medium was increased to 16 mM. The insert is a magnified view of a selected section of the perfusion experiment to clearly show the inhibiting effect of fatty acids on glucose-stimulated insulin secretion. Panel D: Parallel to Panel C, experimental condition was used except 10 μM of CK2 was added to culture medium. CK2 inhibitor completely preserved islets function cultured at 10 mM and partially protected islets from glucolipotoxicity at 16 mM glucose. Each curve represents the mean ± SE of 3–4 perfusions.

Figure 3

Glucolipotoxicity reduces second phase of insulin secretion. Mouse islets were cultured for 3 days with 16 mM glucose and ±0.5 mM of a mixture of PA and OA (2:1 ratio) and then subjected to perifusion experiments. A glyburide ramp from 0 to 1 μM (12.5 nM increment/min) was applied after 30 min of islet preperfusion with 3 mM glucose. Afterwards, 16 mM glucose was added at saturated 1 μM glyburide concentration for additional 20 min. Then all stimuli were removed for 30 min and 30 mM of KCl was added for the additional 20 min. Stimulation of insulin release by 16 mM glucose in islets exposed to glucolipotoxicity in culture was greatly reduced in the presence of glyburide.

Figure 4

Absolute requirement for glucose presence in culture or perfusion medium to demonstrate the glucolipotoxicity effects in mouse islets. Panel A: Glucose in culture medium was replaced by 7 mM glutamine plus 7 mM leucine ± 0.5 mM of a mixture PA/OA. Following 3 day culture, islets were subjected to perfusion experiments to test the response to acetylcholine in the presence of 4 mM glutamine plus 10 mM leucine. Panel B: Islets were cultured for 3 days with 16 mM glucose ± 0.5 mM PA/OA and then the acetylcholine-stimulated insulin secretion was tested in the presence of 4 mM of amino acid mixture. Panel C: Experimental conditions were the same as in Panel B except 8 mM of α-ketoisocaproic acid (KIC) was used as substrate to support acetylcholine effect. These experiments strongly suggest that glucose metabolism is crucial to demonstrate the glucolipotoxic effect in culture or test system.

Figure 5

Gene expression profile of mouse islets. Panel A: Comparison of relative mRNA expression of CK2α1 (Csnk2a1), Caspase3, Bad and Bcl-XL in islets exposed for 3 days to 10 and 16 mM glucose (n = 4). Panel B: Effect of fatty acids (0.5 mM PA/OA) on gene expression at 16 mM glucose (n = 10).

Figure 6

Effects of glucose and acetylcholine on free intracellular Ca²⁺in mouse islets after 4 days exposure to 16 mM glucose and 0.5 mM mixture of palmitic and oleic acid. Panel A: Glucose and acetylcholine-induced Ca²⁺ rise was reduced in islets cultured for 4 days with 16 mM glucose and 0.5 mM PA/OA mixture. Islets were stimulated initially with 10 mM glucose and then 10 μM of nifedipine was added to block the voltage-dependent calcium channels. Afterwards, 1 μM of acetylcholine was added to stimulate Ca²⁺ release from endoplasmic reticulum. At the end, all stimuli and inhibitors were removed. Panels B and C Present area under curve for glucose- and acetylcholine-stimulated Ca²⁺ rise, respectively. Data presented from 3 experiments. Note: Decrease in glucose-stimulated Ca²⁺ signaling was stronger after 4 days of islet exposure to glucolipotoxicity when compared to 3 day culture (see Figure 7).

Figure 7

IP₃R inhibitor Xestospongin C did not prevent the inhibitory effect of palmitic acid on insulin release (Panel A) and on the glucose-dependent Ca²⁺rise (Panel B). Panel A: Effects of islet culture for 3 days at different glucose concentrations on glucose- and thapsigargin-stimulated Ca²⁺ rise. Typical experiment is presented (n = 4). Panel B: A magnified view of selected section of the perfusion experiment from Panel A to clearly show the left shift and decrease maximum of the glucose-stimulated calcium rise. Panel C: Intracellular Ca²⁺ signaling after islet culturing for 3 days with 0.5 mM PA and 10 mM glucose. Typical experiment is presented (n = 4). Panel D: Islets were cultured for 3 days with 16 mM glucose ± PA (0.5 mM) and in the presence of IP₃R inhibitor Xestospongin C (1 μM). Panel E: Insulin secretion profile for the experiments presented on panel D. The perfusion was done in the absence of the inhibitor (compare with panel D of Figure 1). Data presented as means ± SE of 4 experiments.

Figure 8

Chronic effects of FAs on isolated human islets in culture. Panel A: Islets were cultured for 3 days in the presence of 16 mM glucose ± 0.5 mM of PA. Glucose-stimulated insulin release was reduced in all experiments. Acetylcholine-potentiation of insulin secretion was unchanged in experiments with 3 isolates but was reduced in another 4 islet preparations resulting in statistically insignificant changes (n = 7). Panel B: Islets were cultured for 3 days with 16 mM glucose ± 2:1 mixture of PA and OA (n = 3). Panel C: Experimental condition was the same as in Panel B except acetylcholine ramp was applied at 6 mM glucose (n = 3). Panel D: Experimental condition was the same as in Panel B except inhibitor of casein kinase 2 was added to culture medium (n = 3).