Abstract
Fuel stimulation of insulin secretion from pancreatic p-cells is thought to be me-diated by metabolic coupling factors that are generated by energized mitochondria, including protons, adenine nucleotides and perhaps certain amino acids, as for instance aspartate, glutamate or glutamine. The goal of the present study was to evaluate the role of such factors when insulin release (IR) is stimulated by glucose or amino acids (AA), alone or combined, using 31 P-, 23 Na- and 1 H-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 plus 2 mM glutamine (Q) caused a marked stimulation of insulin secretion associated with increased oxygen consumption (V 02 ), c-AMP release and phosphorylation potential (P-potential) as evidenced by higher Phosphocreatine (PCr) and lower P i peak areas of 31 P NMR spectra. Diazoxide blocked stimulation of IR completely suggesting involvement of K ATP channels in this process. However, levels of [MgATP] and [MgADP] which regulate channel activity changed only slowly and little while 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 intracellular pH. The availability of temporal pH profiles allowed the precise computation of the phosphate potential (ATP/P × ADP) in fuel stimulated IR. Intracellular Na + levels were greatly elevated by AAM + Q. However, glutamine alone or together with 2-amino-2-norbornane-carboxylic acid (BCH, 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, glutamine and strikingly lower aspartate levels. Methionine sulfoximine (MSO), an inhibitor of glutamine synthetase, blocked the glucose enhancement of AMM + Q induced IR 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 K ATP 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. Keywords: amino acids, energy metabolism, oxygen consumption, sodium, mitochondria.
Highlights
It is considered to be that the metabolism of amino acids contributes to the maintainance of citric acid cycle intermediates in the β-cell, a process termed anaplerosis, and considered to be important for insulin release (IR)
The uncoupling of respiration and oxidative phosphorylation by FCCP (5 μM) blocked insulin and c-AMP release and markedly increased oxygen consumption indicating that respiration and oxidative phosphorylation are well coupled in this preparation and that both c-AMP and insulin release require enhanced ATP production
There are, certain features and drawbacks which have to be kept in mind when interpreting results obtained with this β-cell model system: 1) The phosphodiesterase inhibitor IBMX is required to permit optimal studies of fuel stimulated insulin release; 2) Cells are cultured and perifused in the presence of 25 mM creatine, a measure taken to facilitate studying the P-potential by NMR but creating perhaps an artificially high intracellular P-creatine pool as energy trap; 3) The β-HC9 cells are highly responsive to an amino acid mixture independent of glucose, in striking contrast to normal β-cells; and 4) The temporal resolution of phosphorous-NMR is relatively low compared to the rapidity of onset of fuel stimulated respiration and insulin release
Summary
It is considered to be that the metabolism of amino acids contributes to the maintainance of citric acid cycle intermediates in the β-cell, a process termed anaplerosis, and considered to be important for insulin release (IR). One hypothesis postulates that the flux through GDH may be directed toward glutamate formation and that glutamate, released from mitochondria, acts as a metabolic coupling factor in glucose-induced insulin exocytosis [36]. The authors [35] failed to observe an increase in the glutamate levels during stimulation of insulin release from pancreatic islets by glucose, leucine, succinic acid methyl ester and α-ketoisocaproic acid and suggest the traditional idea that insulin release is associated with flux through GDH in the direction of α-ketoglutarate. The results suggest that glutamine is the major factor in the amino acid-induced elevation of the phosphorylation potential, oxygen consumption and insulin release of β-HC9 cells. The results suggest that glutamine plays an important, but not an independent primary triggering role in the stimulus-secretion coupling
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