Search

It was reported previously that isolated human islets from individuals with type 2 diabetes mellitus (T2DM) show reduced glucose-stimulated insulin release. To assess the possibility that impaired bioenergetics may contribute to this defect, glucose-stimulated respiration (O2), glucose usage and oxidation, intracellular Ca2+, and insulin secretion (IS) were measured in pancreatic islets isolated from three healthy and three type 2 diabetic organ donors. Isolated mouse and rat islets were studied for comparison. Islets were exposed to a "staircase" glucose stimulus, whereas IR and O2 were measured. O2 of human islets from normals and diabetics increased sigmoidally from equal baselines of 0.25 nmol/100 islets/min as a function of glucose concentration. Maximal VO2 of normal islets at 24 mM glucose was 0.40 ± 0.02 nmol·min-1·100 islets-1, and the glucose S0.5 was 4.39 ± 0.10 mM. The glucose stimulation of respiration of islets from diabetics was lower, Vmax of 0.32 ± 0.01 nmol·min-1·100 islets-1, and the S0.5 shifted to 5.43 ± 0.13 mM. Glucose-stimulated IS and the rise of intracellular Ca(2+) were also reduced in diabetic islets. A clinically effective glucokinase activator normalized the defective O2, IR, and free calcium responses during glucose stimulation in islets from type 2 diabetics. The body of data shows that there is a clear relationship between the pancreatic islet energy (ATP) production rate and IS. This relationship was similar for normal human, mouse, and rat islets and the data for all species fitted a single sigmoidal curve. The shared threshold rate for IS was ∼13 pmol·min-1·islet-1. Exendin-4, a GLP-1 analog, shifted the ATP production-IS curve to the left and greatly potentiated IS with an ATP production rate threshold of ∼10 pmol·min-1·islet-1. Our data suggest that impaired β-cell bioenergetics resulting in greatly reduced ATP production is critical in the molecular pathogenesis of type 2 diabetes mellitus. [ABSTRACT FROM AUTHOR]

Doliba NM, Qin W, Vinogradov SA, Wilson DF, Matschinsky FM. Palmitic acid acutely inhibits acetylcholine- but not GLP-1-stimulated insulin secretion in mouse pancreatic islets. Am J Physiol Endocrinol Metab 299: E475-E485, 2010. First published July 6, 2010: doi: 10.1152/ajpendo.00072.2010.--Fatty acids, acetylcholine, and GLP-1 enhance insulin secretion in a glucose-dependent manner. However, the interplay between glucose, fatty acids, and the neuroendocrine regulators of insulin secretion is not well understood. Therefore, we studied the acute effects of PA (alone or in combination with glucose, acetylcholine, or GLP-1) on isolated cultured mouse islets. Two different sets of experiments were designed. In one, a fixed concentration of 0.5 mM of PA bound to 0.15 mM BSA was used; in the other, a PA ramp from 0 to 0.5 mM was applied at a fixed albumin concentration of 0.15 mM so that the molar PA/BSA ratio changed within the physiological range. At a fixed concentration of 0.5 mM, PA markedly inhibited acetylcholine-stimulated insulin release, the rise of intracellular Ca2+, and enhancement of cAMP production but did not influence the effects of GLP-1 on these parameters of islet cell function. 2-ADB, an IP3 receptor inhibitor, reduced the effect of acetylcholine on insulin secretion and reversed the effect of PA on acetylcholine-stimulated insulin release. Islet perfusion for 35-40 min with 0.5 mM PA significantly reduced the calcium storage capacity of ER measured by the thapsigargin-induced Ca2+ release. Oxygen consumption due to low but not high glucose was reduced by PA. When a PA ramp from 0 to 0.5 mM was applied in the presence of 8 mM glucose, PA at concentrations as low as 50 µM significantly augmented glucose-stimulated insulin release and markedly reduced acetylcholine's effects on hormone secretion. We thus demonstrate that PA acutely reduces the total oxygen consumption response to glucose, glucose-dependent acetylcholine stimulation of insulin release, Ca2+, and cAMP metabolism, whereas GLP-1's actions on these parameters remain unaffected or potentiated. We speculate that acute emptying of the ER calcium by PA results in decreased glucose stimulation of respiration and acetylcholine potentiation of insulin secretion. [ABSTRACT FROM AUTHOR]

Fuel stimulation of insulin secretion from pancreatic β-cells is thought to be mediated by metabolic coupling factors that are generated by energized mitochondria, including protons, adenine nucleotides, and perhaps certain amino acids (AA), as for instance aspartate, glutamate, or glutamine (Q). The goal of the present study was to evaluate the role of such factors when insulin release (IR) is stimulated by glucose or AA, alone or combined, using 31P, 23Na and ¹H NMR technology, respirometry, and biochemical analysis to study the metabolic events that occur in continuously superfused mouse β-HC9 cells contained in agarose beads and enhanced by the phosphodiesterase inhibitor IBMX. Exposing β-HC9 cells to high glucose or 3.5 mM of a physiological mixture of 18 AA (AAM) plus 2 mM glutamine caused a marked stimulation of insulin secretion associated with increased oxygen consumption, cAMP release, and phosphorylation potential as evidenced by higher phosphocreatine and lower Pi peak areas of 31P NMR spectra. Diazoxide blocked stimulation of IR completely, suggesting involvement of ATP-dependent potassium (KATP) channels in this process. However, levels of MgATP and MgADP concentrations, which regulate channel activity, changed only slowly and little, whereas the rate of insulin release increased fast and very markedly. The involvement of other candidate coupling factors was therefore considered. High glucose or AAM + Q increased pHi. The availability of temporal pH profiles allowed the precise computation of the phosphate potential (ATP/Pi × ADP) in fuel-stimulated IR. Intracellular Na+ levels were greatly elevated by AAM + Q. However, glutamine alone or together with 2-amino-2-norbornanecarboxylic acid (which activates glutamate dehydrogenase) decreased β-cell Na levels. Stimulation of β-cells by glucose in the presence of AAM + Q (0.5 mM) was associated with rising cellular concentrations of glutamate and glutamine and strikingly lower aspartate levels. Methionine sulfoximine, an inhibitor of glutamine synthetase, blocked the glucose enhancement of AMM + Q-induced JR and associated changes in glutamine and aspartate but did not prevent the accumulation of glutamate. The results of this study demonstrate again that an increased phosphate potential and a functional KATP channel are essential for metabolic coupling during fuel-stimulated insulin release but illustrate that determining the identity and relative importance of all participating coupling factors and second messengers remains a challenge largely unmet. [ABSTRACT FROM AUTHOR]

Neural and endocrine factors (i.e., Ach and GLP-1) restore defective glucose-stimulated insulin release in pancreatic islets lacking sulfonylurea type 1 receptors (SUR1-/-) (Doliba NM, Qin w, Vatamaniuk MZ, Li C, Zelent D, Najafi H, Buettger CW, Collins HW, Carr RD, Magnuson MA, and Matschinsky FM. Am J Physiol Endocrinol Metab 286: E834–E843, 2004). The goal of the present study was to assess fuel-induced respiration in SUR1-/- islets and to correlate it with changes in intracellular Ca2+, insulin, and glucagon secretion. By use of a method based on O2 quenching of phosphorescence, the O2 consumption rate (OCR) of isolated islets was measured online in a perifusion system. Basal insulin release (IR) was 7–10 times higher in SUR1-/- compared with control (CON) islets, but the OCR was comparable. The effect of high glucose (16.7 mM) on IR and OCR was markedly reduced in SUR1-/- islets compared with CON. Ach (0.5 μM) in the presence of 16.7 mM glucose caused a large burst of IR in CON and SUR1-/- islets with minor changes in OCR in both groups of islets. In SUR1-/- islets, high glucose failed to inhibit glucagon secretion during stimulation with amino acids or Ach. We conclude that 1) reduced glucose responsiveness of SUR1-/- islets may be in part due to impaired energetics, as evidenced by significant decrease in glucose-stimulated OCR; 2) elevated intracellular Ca2+ levels may contribute to altered insulin and glucagon secretion in SUR1-/- islets; and 3) The amplitudes of the changes in OCR during glucose and Ach stimulation do not correlate with IR in normal and SUR1-/- islets suggesting that the energy requirements for exocytosis are minor compared with other ATP-consuming reactions. [ABSTRACT FROM AUTHOR]

Inhibition of ATP-sensitive K+ (KATP) channels by an increase in the ATP/ADP ratio and the resultant membrane depolarization are considered essential in the process leading to insulin release (IR) from pancreatic β-cells stimulated by glucose. It is therefore surprising that mice lacking the sulfonylurea type 1 receptor (SURI-/-) in [3-cells remain euglycemic even though the knockout is expected to cause hypoglycemia. To complicate matters, isolated islets of SURI-/- mice secrete little insulin in response to high glucose, which extrapolates to hyperglycemia in the intact animal. It remains thus unexplained how euglycemia is maintained. In recognition of the essential role of neural and endocrine regulation of IR, we evaluated the effects of acetylcholine (ACh) and glucagon-like peptide-l (GLP-1) on IR and free intracellular Ca2+ concentration ([Ca2+]i) of freshly isolated or cultured islets of SURI-/- mice and B6D2F1 controls (SURI+/+). IBMX, a phosphodiesterase inhibitor, was also used to explore cAMP-dependent signaling in IR. Most striking, and in contrast to controls, SUR1-/- islets are hypersensitive to ACh and IBMX, as demonstrated by a marked increase of IR even in the absence of glucose. The hypersensitivity to ACh was reproduced in control islets by depolarization with the SUR1 inhibitor glyburide. Pretreatment of perifused SUR1-/- islets with ACh or IBMX restored glucose stimulation of IR, an effect expectedly insensitive to diazoxide. The calcium channel blocker verapamil reduced but did not abolish ACh-stimulated IR, supporting a role for intracellular Ca2+ stores in stimulus-secretion coupling. The effect of ACh on IR was greatly potentiated by GLP-1 (10 nM). ACh caused a dose-dependent increase in [Ca2+]i at 0.l–1 μM or biphasic changes (an initial sharp increase in [Ca2+]i followed by a sustained phase of low [Ca2+]i) at 1–100 μM. The latter effects were observed in substrate-free medium or in the presence of 16.7 mM glucose. We conclude that SUR1 deletion depolarizes the β-cells and markedly elevates basal [Ca2+]i. Elevated [Ca2+]i in turn sensitizes the β-cells to the secretory effects of ACh and IBMX. Priming by the combination of high [Ca2+]i, ACh, and GLP-1 restores the defective glucose responsiveness, precluding the development of diabetes but not effectively enough to cause hyperinsulinemic hypoglycemia. [ABSTRACT FROM AUTHOR]