Biomedical subjects

Nicolai M Doliba

Publications and source records attributed to Nicolai M Doliba.

9 recordsLinked to original sources

PubMed2006

Functional genomics of the beta-cell: short-chain 3-hydroxyacyl-coenzyme A dehydrogenase regulates insulin secretion independent of K+ currents.

Recent advances in functional genomics afford the opportunity to interrogate the expression profiles of thousands of genes simultaneously and examine the function of these genes in a high-throughput manner. In this study, we describe a rational and efficient approach to identifying novel regulators of insulin secretion by the pancreatic beta-cell. Computational analysis of expression profiles of several mouse and cellular models of impaired insulin secretion identified 373 candidate genes involved in regulation of insulin secretion. Using RNA interference, we assessed the requirements of 10 of these candidates and identified four genes (40%) as being essential for normal insulin secretion. Among the genes identified was Hadhsc, which encodes short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD), an enzyme of mitochondrial beta-oxidation of fatty acids whose mutation results in congenital hyperinsulinism. RNA interference-mediated gene suppression of Hadhsc in insulinoma cells and primary rodent islets revealed enhanced basal but normal glucose-stimulated insulin secretion. This increase in basal insulin secretion was not attenuated by the opening of the KATP channel with diazoxide, suggesting that SCHAD regulates insulin secretion through a KATP channel-independent mechanism. Our results suggest a molecular explanation for the hyperinsulinemia hypoglycemic seen in patients with SCHAD deficiency.

Animals↗

PubMed2006

Cholinergic regulation of fuel-induced hormone secretion and respiration of SUR1-/- mouse islets.

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 Ca(2+), insulin, and glucagon secretion. By use of a method based on O(2) quenching of phosphorescence, the O(2) 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 microM) 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 Ca(2+) 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.

ATP-Binding Cassette Transporters↗

PubMed2006

Green tea polyphenols modulate insulin secretion by inhibiting glutamate dehydrogenase.

Insulin secretion by pancreatic beta-cells is stimulated by glucose, amino acids, and other metabolic fuels. Glutamate dehydrogenase (GDH) has been shown to play a regulatory role in this process. The importance of GDH was underscored by features of hyperinsulinemia/hyperammonemia syndrome, where a dominant mutation causes the loss of inhibition by GTP and ATP. Here we report the effects of green tea polyphenols on GDH and insulin secretion. Of the four compounds tested, epigallocatechin gallate (EGCG) and epicatechin gallate were found to inhibit GDH with nanomolar ED(50) values and were therefore found to be as potent as the physiologically important inhibitor GTP. Furthermore, we have demonstrated that EGCG inhibits BCH-stimulated insulin secretion, a process that is mediated by GDH, under conditions where GDH is no longer inhibited by high energy metabolites. EGCG does not affect glucose-stimulated insulin secretion under high energy conditions where GDH is probably fully inhibited. We have further shown that these compounds act in an allosteric manner independent of their antioxidant activity and that the beta-cell stimulatory effects are directly correlated with glutamine oxidation. These results demonstrate that EGCG, much like the activator of GDH (BCH), can facilitate dissecting the complex regulation of insulin secretion by pharmacologically modulating the effects of GDH.

Adenosine Diphosphate↗

PubMed2006

Foxa1-deficient mice exhibit impaired insulin secretion due to uncoupled oxidative phosphorylation.

Foxa1 (formerly hepatic nuclear factor 3alpha) belongs to the family of Foxa genes that are expressed in early development and takes part in the differentiation of endoderm-derived organs and the regulation of glucose homeostasis. Foxa1-/- pups are growth retarded and hypoglycemic but glucose intolerant in response to an intraperitoneal glucose challenge. However, the mechanism of glucose intolerance in this model has not been investigated. Here, we show that Foxa1-/- islets exhibit decreased glucose-stimulated insulin release in islet perifusion experiments and have significantly reduced pancreatic insulin and glucagon content. Moreover, Foxa1-/- beta-cells exhibit attenuated calcium influx in response to glucose and glyburide, suggesting an insulin secretion defect either at the level or upstream of the ATP-sensitive K+ channel. Intracellular ATP levels after incubation with 10 mmol/l glucose were about 2.5 times lower in Foxa1-/- islets compared with controls. This diminished ATP synthesis could be explained by increased expression of the mitochondrial uncoupling protein uncoupling protein 2 (UCP2) in Foxa1-deficient islets, resulting in partially uncoupled mitochondria. Chromatin immunoprecipitation assays indicate that UCP2 is a direct transcriptional target of Foxa1 in vivo. Thus, we have identified a novel function for Foxa1 in the regulation of oxidative phosphorylation in pancreatic beta-cells.

Animals↗

PubMed2004

Restitution of defective glucose-stimulated insulin release of sulfonylurea type 1 receptor knockout mice by acetylcholine.

Inhibition of ATP-sensitive K+ (K(ATP)) 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 beta-cells stimulated by glucose. It is therefore surprising that mice lacking the sulfonylurea type 1 receptor (SUR1-/-) in beta-cells remain euglycemic even though the knockout is expected to cause hypoglycemia. To complicate matters, isolated islets of SUR1-/- 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-1 (GLP-1) on IR and free intracellular Ca2+ concentration ([Ca2+]i) of freshly isolated or cultured islets of SUR1-/- mice and B6D2F1 controls (SUR1+/+). 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 0.1-1 microM or biphasic changes (an initial sharp increase in [Ca2+]i followed by a sustained phase of low [Ca2+]i) at 1-100 microM. 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 beta-cells and markedly elevates basal [Ca2+]i. Elevated [Ca2+]i in turn sensitizes the beta-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.

1-Methyl-3-isobutylxanthine↗

PubMed2003

Differential effects of glucose and glyburide on energetics and Na+ levels of betaHC9 cells: nuclear magnetic resonance spectroscopy and respirometry studies.

In the present study, noninvasive (31)P and (23)Na(+)-nuclear magnetic resonance (NMR) technology and respirometry were used to compare the effect of high glucose (30 mmol/l) with the effect of the antidiabetic sulfonylurea (SU) compound glyburide (GLY) on energy metabolism, Na(+) flux, insulin, and cAMP release of continuously superfused beta-HC9 cells encapsulated in microscopic agarose beads. Both high glucose and GLY increased oxygen consumption in beta-HC9 cells (15-30%) with a maximal effect at 8 mmol/l for glucose and at 250 nmol/l for GLY. At the same time, insulin release from beta-cells increased by 15- and 25-fold with high glucose or GLY, respectively. The P-creatine (PCr) level was greatly increased and inorganic phosphate (P(i)) was decreased with 30 mmol/l glucose in contrast to the decreased level of PCr and increased P(i) with GLY. ATP levels remained unchanged during both interventions. Studies on isolated mitochondria of beta-HC9 cells showed that GLY added to mitochondria oxidizing glutamine or glutamate abolished the stimulation of respiration by ADP (state 3) meanwhile leaving state 3 respiration unchanged during oxidation of other substrates. Exposure of beta-HC9 cells to 5 mmol/l glucose decreased intracellular Na(+) levels monitored by (23)Na(+)-NMR spectroscopy and 30 mmol/l glucose resulted in a further decrease in cytosolic Na(+). In contrast, Na(+) increased when 1 micro mol/l GLY was added to the perfusate containing 5 mmol/l glucose. These data support the hypothesis that glucose activates the beta-cell through a "push mechanism" due to substrate pressure enhancing fuel flux, energy production, and extrusion of Na(+) from the cells in contrast to SU receptor (SUR)-1 inhibitors, which may modify intermediary and energy metabolism secondarily through a "pull mechanism" due to higher energy demand resulting from increased ion fluxes and the exocytotic work load.

Adenosine Diphosphate↗

PubMed2002

Effect of coenzyme Q10 supplementation on mitochondrial function after myocardial ischemia reperfusion.

BACKGROUND: Coenzyme Q10 (CoQ10) protects myocardium from ischemia-reperfusion (IR) injury as evidenced by improved recovery of mechanical function, ATP, and phosphocreatine during reperfusion. This protection may result from CoQ10's bioenergetic effects on the mitochondria, from its antioxidant properties, or both. The purpose of this study was to elucidate the effects of CoQ10 supplementation on mitochondrial function during myocardial ischemia-reperfusion using an isolated mitochondrial preparation. METHODS: Isolated hearts (n = 6/group) from rats pretreated with liposomal CoQ10 (10 mg/kg iv, CoQ10), vehicle (liposomal only, Vehicle), or saline (Saline) 30 min before the experiments were subjected to 15 min of equilibration (EQ), 25 min of ischemia (I), and 40 min of reperfusion (RP). Left ventricular-developed pressure (DP) was measured. Mitochondria were isolated at end-equilibration (end-EQ), at end-ischemia (end-I), and at end-reperfusion (end-RP). Mitochondrial respiratory function (State 2, 3, and 4, respiratory control index (RCI, ratio of State 3 to 4), and ADP:O ratio) was measured by polarography using NADH (alpha-ketoglutarate, alpha-KG)- or FADH (succinate, SA)-dependent substrates. RESULTS: CoQ10 improved recovery of DP at end-RP (67 +/- 11% in CoQ10 vs 47 +/- 5% in Vehicle and 50 +/- 11% in Saline, P < 0.05 vs Vehicle and Saline). CoQ10 did not change preischemic mitochondrial function. IR decreased State 3 and RCI in all groups using either substrate. CoQ10 had no effect in the mitochondrial oxidation of alpha-KG at end-I. CoQ10 improved State 3 at end-I when SA was used (167 +/- 21 in CoQ10 vs 120 +/- 10 in Saline and 111 +/- 10 ng-atoms O/min/mg protein in Vehicle, P < 0.05). Using alpha-KG as a substrate, CoQ10 improved RCI at end-RP (4.2 +/- 0.2 in CoQ10 vs 3.2 +/- 0.2 in Saline and 3.0 +/- 0.3 in Vehicle, P < 0.05). Using SA, CoQ10 improved State 3 (181 +/- 10 in CoQ10 vs 142 +/- 9 in Saline and 140 +/- 12 ng-atoms O/min/mg protein in Vehicle, P < 0.05) and RCI (2.21 +/- 0.06 in CoQ10 vs 1.85 +/- 0.11 in Saline and 1.72 +/- 0.08 in Vehicle, P < 0.05) at end-RP. CONCLUSIONS: The cardioprotective effects of CoQ10 can be attributed to the preservation of mitochondrial function during reperfusion as evidenced by improved FADH-dependent oxidation.

Animals↗

PubMed2002

Ischemic preconditioning improves mitochondrial tolerance to experimental calcium overload.

BACKGROUND: Ca(2+) overload leads to mitochondrial uncoupling, decreased ATP synthesis, and myocardial dysfunction. Pharmacologically opening of mitochondrial K(ATP) channels decreases mitochondrial Ca(2+) uptake, improving mitochondrial function during Ca(2+) overload. Ischemic preconditioning (IPC), by activating mitochondrial K(ATP) channels, may attenuate mitochondrial Ca(2+) overload and improve mitochondrial function during reperfusion. The purpose of these experiments was to study the effect of IPC (1) on mitochondrial function and (2) on mitochondrial tolerance to experimental Ca(2+) overload. METHODS: Rat hearts (n = 6/group) were subjected to (a) 30 min of equilibration, 25 min of ischemia, and 30 min of reperfusion (Control) or (b) two 5-min episodes of ischemic preconditioning, 25 min of ischemia, and 30 min of reperfusion (IPC). Developed pressure (DP) was measured. Heart mitochondria were isolated at end-Equilibration (end-EQ) and at end-Reperfusion (end-RP). Mitochondrial respiratory function (state 2, oxygen consumption with substrate only; state 3, oxygen consumption stimulated by ADP; state 4, oxygen consumption after cessation of ADP phosphorylation; respiratory control index (RCI, state 3/state 4); rate of oxidative phosphorylation (ADP/Deltat), and ADP:O ratio) was measured with polarography using alpha-ketoglutarate as a substrate in the presence of different Ca(2+) concentrations (0 to 5 x 10(-7) M) to simulate Ca(2+) overload. RESULTS: IPC improved DP at end-RP. IPC did not improve preischemic mitochondrial respiratory function or preischemic mitochondrial response to Ca(2+) loading. IPC improved state 3, ADP/Deltat, and RCI during RP. Low Ca(2+) levels (0.5 and 1 x 10(-7) M) stimulated mitochondrial function in both groups predominantly in IPC. The Control group showed evidence of mitochondrial uncoupling at lower Ca(2+) concentrations (1 x 10(-7) M). IPC preserved state 3 at high Ca(2+) concentrations. CONCLUSIONS: The cardioprotective effect of IPC results, in part, from preserving mitochondrial function during reperfusion and increasing mitochondrial tolerance to Ca(2+) loading at end-RP. Activation of mitochondrial K(ATP) channels by IPC and their improvement in Ca(2+) homeostasis during RP may be the mechanism underlying this protection.

Adenosine Diphosphate↗

PubMed2002

Mitochondrial function during ischemic preconditioning.

Background. Ischemic preconditioning (IPC) protects the myocardium from ischemia reperfusion injury. The effect of IPC on the mitochondria is not well known. However, one of the mechanisms postulated in IPC (the opening of the mitochondrial K(ATP) channels) is likely to result in changes in mitochondrial function. Therefore, the purpose of this study was to determine the effect of IPC on mitochondrial function during ischemia reperfusion. Methods. Isolated rat hearts (n = 6/group) were subjected to (1) 30 minutes of equilibration, 25 minutes of ischemia, and 30 minutes of reperfusion (RP) (control group) or (2) 10 minutes of equilibration, two-5 minute episodes of IPC (each followed by 5 minutes of re-equilibration), 25 minutes of ischemia, and 30 minutes of RP (IPC group). Left ventricular rate pressure product (RPP) was measured. At end-equilibration (end-EQ) and at end-reperfusion (end-RP) mitochondria were isolated. Mitochondrial respiratory function (state 2, 3, and 4), respiratory control index (RCI), rate of oxidative phosphorylation (ADP/Delta t), and ADP:O ratio were measured by polarography with the use of NADH- or FADH-dependent substrates. Results. IPC improved recovery of RPP at end-RP (72% +/- 5% in IPC vs 30% +/- 4% in control, P <.05). Ischemia reperfusion (IR) decreased state 3, ADP/Delta t, and RCI in both groups compared with end-EQ. IPC improved state 3 (47 +/- 3 in IPC vs 37 +/- 2 ng-atoms O/min/mg protein in control), ADP/Delta t (17 +/- 1 in IPC vs 13 +/- 1 nmol/s/mg protein in control), and RCI (3.7 +/- 0.1 in IPC vs 2.1 +/- 0.2 in control) at end-RP compared with control with the use of NADH-dependent substrate (P <.05 vs control). IPC also improved state 3 (85 +/- 6 in IPC vs 71 +/- 4 ng-atoms O/min/mg protein in control), ADP/Delta t (18 +/- 2 in IPC vs 12 +/- 1 nmol/s/mg protein in control), RCI (2 +/- 0.1 in IPC vs 1.5 +/- 0.1 in control), and ADP:O ratios (1.4 +/- 0.04 in IPC vs 1.7 +/- 0.09 in control) at end-RP compared with control with the use of FADH-dependent substrate (P <.05 vs control). Conclusions. The cardioprotective effects of IPC can be attributed at least in part to the preservation of mitochondrial function during reperfusion.

Adenosine Triphosphate↗