Acute metabolic amplification of insulin secretion in mouse islets is mediated by mitochondrial export of metabolites, but not by mitochondrial energy generation.

OBJECTIVE: The ß-cell metabolism of glucose and of some other fuels (e.g. α-ketoisocaproate) generates signals triggering and acutely amplifying insulin secretion. As the pathway coupling metabolism with amplification is largely unknown, we aimed to narrow down the putative amplifying signals. MATERIALS/METHODS: An experimental design was used which previously prevented glucose-induced, but not α-ketoisocaproate-induced insulin secretion. Isolated mouse islets were pretreated for one hour with medium devoid of fuels and containing the sulfonylurea glipizide in high concentration which closed all ATP-sensitive K(+) channels. This concentration was also applied during the subsequent examination of fuel-induced effects. In perifused or incubated islets, insulin secretion and metabolic parameters were measured. RESULTS: The pretreatment decreased the islet ATP/ADP ratio. Whereas glucose and α-ketoisovalerate were ineffective or weakly effective, respectively, when tested separately, their combination strongly enhanced the insulin secretion. Compared with glucose, the strong amplifier α-ketoisocaproate caused less increase in NAD(P)H-fluorescence and less mitochondrial hyperpolarization. Compared with α-ketoisovalerate, α-ketoisocaproate caused greater increase in NAD(P)H-fluorescence and greater mitochondrial hyperpolarization. Neither α-ketoacid anion enhanced the islet ATP/ADP ratio during onset of the insulin secretion. α-Ketoisocaproate induced a higher pyruvate content than glucose, slowly elevated the citrate content which was not changed by glucose and generated a much higher acetoacetate content than other fuels. α-Ketoisovalerate alone or in combination with glucose did not increase the citrate content. CONCLUSIONS: In ß-cells, mitochondrial energy generation does not mediate acute metabolic amplification, but mitochondrial production of acetyl-CoA and supplemental acetoacetate supplies cytosolic metabolites which induce the generation of specific amplifying signals.

Determination of beta-cell function: insulin secretion of isolated islets.

The kinetics of insulin secretion, not just the total amount, is of decisive relevance for the physiological regulation of glucose homeostasis. Thus to characterize the relevant features of the secretory response to an insulinotropic stimulus a method is needed which is able to resolve the temporal response pattern, in particular to distinguish the first phase from the second phase response. The perifusion of collagenase-isolated islets is a method which permits to register responses of near-physiological complexity with a preparation that can also be used for cell physiological and biochemical investigations on stimulus--secretion oupling.

Studies of first phase insulin secretion using imposed plasma membrane depolarization.

The first phase of glucose-induced insulin secretion is generally regarded to represent the release of a finite pool of secretion-ready granules, triggered by the depolarization-induced influx of Ca2+ through L-type Ca2+ channels. However, the experimental induction of insulin secretion by imposed plasma membrane depolarization may be more complicated than currently appreciated. A comparison of the effects of high K+ concentrations with those of KATP channel closure, which initiates the electrical activity of the beta cell, suggests that 40 mM K+, which is a popular tool to produce a first phase-like secretion, is of supraphysiological strength, whereas the 20 mV depolarization by 15 mM K+ is nearly inefficient. A major conceptual problem consists in the occurrence of action potentials during KATP channel closure, but not during K+ depolarization, which leaves the K+ channel conductance unchanged. Recent observations suggest that the signal function of the endogenously generated depolarization is not homogeneous, but may rather differ between the component mainly determined by KATP channel closure (slow waves) and that mainly determined by Ca2+ influx (action potentials).

Selective enhancement of nutrient-induced insulin secretion by ATP-sensitive K+ channel-blocking imidazolines.

The contribution of ATP-sensitive K(+) channel (K(ATP) channel)-dependent and -independent signaling to the insulinotropic characteristics of imidazolines was explored using perifused mouse islets and beta-cells. Up to a concentration of 100 muM efaroxan had no insulinotropic effect in the presence of a basal glucose concentration, but enhanced the effect of a stimulatory concentration of glucose or nonglucidic nutrients (ketoisocaproate plus glutamine). The secretion by a non-nutrient (40 mM KCl) was not enhanced. At 500 microM, efaroxan stimulated insulin secretion when glucose was basal. Likewise, at 0.1 to 10 microM RX871024 [2-(imidazolin-2-yl)-1-phenylindole] showed a purely enhancing effect, but at 100 microM it elicited a strong KCl-like secretory response in the presence of basal glucose. At 0.1 and 1 microM RX871024 did not significantly depolarize the beta-cell membrane. However, at a purely enhancing drug concentration (10 microM RX871024 or 100 microM efaroxan) K(ATP) channel activity was strongly reduced, the membrane was depolarized, and the cytosolic Ca(2+) concentration was elevated in the presence of basal glucose. Insulin secretion by sulfonylurea receptor (SUR)1 knockout (KO) islets, which have no functional K(ATP) channels, was not increased by efaroxan (100 or 500 microM) or by 10 microM RX871024 but was increased by 100 microM RX871024. The imidazolines phentolamine and alinidine (100 microM) were also ineffective on SUR1 KO islets. It is concluded that a significant K(ATP) channel block is compatible with a purely enhancing effect of the imidazolines on nutrient-induced insulin secretion. Only RX871024 has an additional, nondepolarizing effect, which at a high drug concentration is able to elicit a K(ATP) channel-independent secretion.

Triggering and amplification of insulin secretion by dimethyl alpha-ketoglutarate, a membrane permeable alpha-ketoglutarate analogue.

Cytosolic alpha-ketoglutarate is a potential signalling compound at late steps of stimulus-secretion-coupling in the course of insulin secretion induced by glucose and other fuels. This hypothesis is mainly based on the insulin-releasing effect of the membrane permeable ester dimethyl alpha-ketoglutarate which enters the beta-cell and is cleaved to produce cytosolic monomethyl alpha-ketoglutarate and eventually alpha-ketoglutarate. The present study tested this hypothesis. Insulin release, K(ATP) channel currents, membrane potential, ATP/ADP ratio and fluorescence of NAD(P)H (reduced pyridine nucleotides) were measured in mouse pancreatic islets and beta-cells. At a substimulatory glucose concentration (5 mM), dimethyl alpha-ketoglutarate (15 mM) produced a sustained insulin release, but no change of the islet ATP/ADP ratio and NAD(P)H fluorescence. In the absence of glucose, however, dimethyl alpha-ketoglutarate (15 mM) did not stimulate insulin release although it increased the ATP/ADP ratio and NAD(P)H fluorescence. Insulin secretion induced by a maximally effective concentration of the K(ATP) channel-blocking sulfonylurea glipizide was strongly amplified by dimethyl alpha-ketoglutarate in the presence of 5 mM glucose, but only moderately in the absence of glucose. Dimethyl alpha-ketoglutarate directly inhibited K(ATP) channels in inside-out membrane patches, depolarized the plasma membrane of intact beta-cells and generated action potentials. In conclusion, the stimulation of insulin secretion by extracellularly applied dimethyl alpha-ketoglutarate depends on inhibition of beta-cell K(ATP) channels by direct action of dimethyl alpha-ketoglutarate. The metabolism of alpha-ketoglutarate generated intracellularly by ester cleavage contributes to stimulation of insulin secretion both by indirect K(ATP) channel inhibition (via activation of ATP production) and by an amplifying effect.

Technique for iliopsoas ultrasound-guided active electromyography-directed botulinum a toxin injection in cerebral palsy.

Symptomatic hip flexion deformity secondary to iliopsoas spasticity may interfere with gait, impair sitting balance, or contribute to hip subluxation or dislocation. A nonsurgical, minimally invasive technique to ameliorate iliopsoas spasticity is presented. The technique uses intramuscular injections of botulinum A toxin to provide selective neuromuscular blockade of the iliacus or psoas muscles or both. Because of the anatomic location of the target muscles, this technique uses ultrasound guidance for needle placement. Active electromyographic stimulation is used to verify the needle position adjacent to active myoneural interfaces. The authors' experience to date includes the treatment of 28 patients (53 hips). Use of this technique has resulted in improved hip range of motion. No intraoperative or postoperative adverse events or complications have been observed.